Working toward the Future: Insights into Francisella tularensis Pathogenesis and Vaccine Development

Abstract

Summary: Francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. Recent advances in the field of Francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. In this review, we summarize the major properties of several Francisella species, including F. tularensis and F. novicida, and provide an up-to-date synopsis of the genes necessary for pathogenesis by these organisms and the determinants that are currently being targeted for vaccine development.

INTRODUCTION

Francisella tularensis is a gram-negative coccobacillus that has an exceedingly low infectious dose. It is a category A select agent and is one of the most infectious bacteria known. Following the terrorist attacks of 2001 and subsequent anthrax mailings in the fall of that year, there has been a renewed interest in the study of this organism. Advancements in the field of F. tularensis genetics have lead to a dramatic expansion in the generation of mutant strains of various F. tularensis subspecies. Collectively, this has led to an improved understanding of F. tularensis biology, host responses to infection, and virulence factors required for infection and/or disease elicitation. Many investigators in this field have focused on the development of a vaccine capable of protecting against the most virulent biovars of F. tularensis. Of particular interest are those providing substantive protection against type A strains delivered by the respiratory route. Here, we review the major characteristics of F. tularensis and provide an update regarding genes required for pathogenesis and determinants being targeted for vaccine development.

FRANCISELLA TULARENSIS AND TULAREMIA

Classification

Francisella tularensis is one of the most infectious and pathogenic bacteria known. It is the etiological agent of the debilitating febrile illness tularemia. The bacterium is a gram-negative, capsulated, facultative intracellular pathogen and is one of the members of the genus Francisella of the Gammaproteobacteria class. Francisella has no close pathogenic relatives but exists in a sister clade with the arthropod endosymbiont Wolbachia persica. It is also distantly related to human pathogens Coxiella burnetii and Legionella pneumophila (109). F. tularensis is commonly classified into three subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. tularensis subsp. mediasiatica, based on genetic makeup, virulence, ability to produce acid from glycerol, and citrulline ureidase activity (49) (Table 1). Francisella novicida is also often considered a subspecies of F. tularensis; however, recent whole-genome single-nucleotide polymorphism analysis indicates that it is likely an independent species (104). F. tularensis subsp. tularensis and F. tularensis subsp. holarctica are the primary biovars associated with disease in humans. F. tularensis subsp. tularensis, also known as type A Francisella, is found primarily in North America and is highly virulent in humans. This subspecies is responsible for roughly 70% of Francisella disease cases in North America (186). Type A strains have an infectious dose of <10 CFU in humans (174, 175) and can lead to life-threatening illness, particularly when infection occurs via the respiratory route. Molecular subtyping techniques indicate that F. tularensis subsp. tularensis can be further divided into two genetically distinct clades (A.I and A.II) that differ with respect to disease outcome, transmission, and geographic location (59, 98, 104, 187, 194). F. tularensis subsp. holarctica, or type B strains, is found throughout much of the Northern Hemisphere and is the primary cause of tularemia in Europe (141). These organisms have an infectious dose of <10³ CFU and cause a milder form of tularemia in humans. The live vaccine strain (LVS) that was developed in the former Soviet Union and gifted to the United States in the 1950s is a human-attenuated type B derivative. F. tularensis subsp. mediasiatica and F. novicida are focally distributed and are rarely associated with disease in humans. F. tularensis subsp. mediasiatica is primarily isolated to central Asian regions of the former USSR, while F. novicida is found in North America and Australia (57, 141, 147). F. novicida has been extensively studied as a model organism in the laboratory setting due to its enhanced genetic tractability relative to other subspecies and its relative avirulence in humans. All F. tularensis subspecies are highly pathogenic in animal models, particularly in rabbits and mice. F. novicida is also highly pathogenic in mice, but its virulence remains less characterized outside this model system.

TABLE 1.

Characteristics of Francisella species^a

Species or subspecies	LD50 (CFU) in:		Straind	Accession no.	Length (bp)	No. of:
	Miceb	Humansc				Genes	Pseudogenese
F. tularensis subsp. tularensis	<10	<10	Schu S4	NC 006570	1,892,819	1,852	201
			FSC198	NC 008245	1,892,616	1,852	199
			WY96-3418	NC 009257	1,898,476	1,872	0
F. tularensis subsp. holarctica	<10	<103	LVS	NC 007880	1,895,994	2,020	213
			OSU18	NC 008369	1,895,727	1,932	328
			FTNF002-00	NC 009749	1,890,909	1,887	2
F. tularensis subsp. mediasiatica	NRf	NR	FSC147	NC 010677	1,893,886	1,750	297
F. novicida	<10	>103	U112	NC 008601	1,910,031	1,781	14

^aAdapted from Table 1 in reference 140 with kind permission from Springer Science + Business Media (© Springer 2005) and from reference 203 with kind permission of Wiley-Blackwell.

^bDoses delivered subcutaneously.

^cEstimated LD based on virulence in animal models and case studies.

^dSeveral additional Francisella isolates are currently being sequenced, including F. tularensis subsp. tularensis (strains MA00-2987 [NZ ABRI00000000] and FSC033 [NZ AAYE00000000]), F. tularensis subsp. holarctica (strains FSC200 [NZ AASP00000000], FSC022 [NZ AAYD00000000], and 257 [NZ AAUD00000000], and F. novicida (strains FTG [NZ ABXZ00000000], FTE [NZABSS00000000], GA99-3548 [NZ ABAH00000000], and GA99-3549 [NZ AAYF00000000]).

^eRefers to sequences resembling functional genes but thought to have lost protein-coding capability.

^fNR, doses have not been reported.

Comparative Genomics

There are currently eight completely sequenced Francisella genomes (Table 1) and an additional nine genomes for which shotgun sequencing is currently under way. Sequence analysis of F. tularensis subsp. tularensis (FSC198 [NC 008245], Schu S4 [NC 006570], and WY96-3418 [NC 009257]), F. tularensis subsp. holarctica (FTNF002-00 [NC 009749], OSU18 [NC 008369], and LVS [NC 067880]), F. tularensis subsp. mediasiatica (FSC147 [NC 010677]), and F. novicida (U112 [NC 008601]) indicates that these strains are highly similar at the genetic level. The genome of each strain is roughly 1.8 Mb, with F. novicida U112 having the largest genome at 1.91 Mb. All genomes have a G+C content of approximately 32%, with between 1,800 and 2,000 putative coding sequences depending on the subspecies and strain. Between 70 and 90% of open reading frames within these isolates are predicted to code for functional proteins. Interestingly, the more virulent subspecies, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, contain roughly 200 to 300 pseudogenes, depending on the strain, while the less pathogenic F. novicida U112 contains only 14 pseudogenes (109, 203). Additionally, nearly 30% of annotated genes within an F. tularensis isolate are characterized as hypothetical proteins with unknown function, suggesting that Francisella is likely to encode novel virulence determinants. A 30-kb region with low G+C content (27.5%) that is unique to Francisella among the 17 gammaproteobacterial genomes exists in duplicate in type A and B strains of F. tularensis but is present in single copy in F. novicida. This locus has been identified as a pathogenicity island and is required for aspects of F. tularensis survival within host cells.

Comparative genomic studies have indicated that there is a high level of nucleotide identity between and within F. tularensis subspecies, ranging from roughly 97% to 99%. Despite this, there are numerous DNA rearrangements present between subspecies, particularly between type A and type B strains, and among different type A strains (148). These rearranged sequences are flanked by repeated DNA insertion sequence elements, indicating that they likely evolved from homologous recombination events. In contrast, little genomic reorganization is observed in type B strains. Though the precise impact of these rearrangements remains unclear, it is of note that they exist primarily in the more virulent type A strains.

Comparisons of deletion events, repeat sequences, and single-nucleotide polymorphisms between sequenced Francisella genomes have provided important insights into the evolution of these organisms. The presence of conserved genomic deletion events and single-nucleotide variations in F. tularensis and F. novicida isolates suggest that these species have evolved vertically, with F. novicida being the most ancestral. Additionally, the highly virulent type A strains appeared before the less virulent type B strains (59). The reduced genomic heterogeneity of type B strains compared with type A strains and the recovery of type B strains from around the world indicate that F. tularensis subsp. holarctica has evolved recently and spread rapidly (59). The evolution of F. tularensis from a common ancestor appears to have resulted from both a loss and a gain of genetic information over time, as type A strains have undergone a reduction in their genomic content relative to F. novicida, but type B strains contain additional genomic content that is otherwise absent from their type A counterparts. These observations indicate either that rearrangements occurred in type A Francisella after type A and B strains diverged evolutionarily or that type B strains were derived from one type A strain that lost the ability to undergo such rearrangements (148).

Epidemiology of F. tularensis

Though its primary environmental niche remains unknown, F. tularensis has a broad and complex host distribution, infecting a number of wildlife species, including lagomorphs, rodents, insectivores, carnivores, ungulates, marsupials, birds, amphibians, fish, and invertebrates (14, 18, 136, 196). F. tularensis is most frequently found in rodents, hares, and rabbits; however, these are unlikely reservoirs for F. tularensis, considering that infection often leads to acute disease in these animals. It has also been suggested that protozoa may play a role as hosts in aquatic cycles, which is supported by the demonstration of F. tularensis in amoebal cysts (2, 111, 141, 202). Finally, although most arthropod vectors serve only as transient hosts, F. tularensis may be transmitted by ticks throughout their life cycle, raising the possibility that a single tick may infect multiple hosts (90, 149).

A primary route of F. tularensis transmission to humans and other animals is through arthropod vectors such as ticks, biting flies, and possibly mosquitoes. Infection by F. tularensis can also occur through direct contact with contaminated water, food supplies, or infected animals (49). F. tularensis is occasionally acquired by inhalation of organisms that have been aerosolized through disruption of contaminated materials. For these reasons, high-risk groups include hunters or trappers, who might come into contact with infected animals, and landscapers, who may encounter aerosolized organisms through mechanical disruption of contaminated soil or animal carcasses. Though F. tularensis organisms are readily aerosolized, transmission via human-to-human contact has yet to be reported.

F. tularensis-mediated disease was first recognized as a plague-like illness in rodents during an outbreak in Tulare County, CA, in 1911, resulting in the first isolation of the bacterium (70). Three years later, human disease caused by F. tularensis in two patients in Ohio who had recent contact with wild rabbits was described (215). In 1919, Edward Francis established that a number of clinical symptoms were specifically caused by “Bacterium tularense,” named for the county in which the disease was found to be endemic, and the name “tularemia” was subsequently used to describe them (68, 70). Tularemia has been referred to as “rabbit fever,” “market men's disease,” and “meat-cutter's disease,” all named for the frequent incidence of disease associated with dressing rabbits for meat. The terms “deer-fly fever” and “glandular type of tick fever” have also been used to describe tularemia in the context of symptoms arising from a tick or fly bite resulting in a noted enlargement of lymph nodes. Identification of symptoms and potential sources led to the subsequent accumulation of tularemia reports in the United States, with roughly 14,000 cases reported by 1945 (96) and a peak incidence of 2,291 cases in 1939 (60, 183). Concurrently, reports of a similar disease were emerging from Japan and Russia. Large waterborne outbreaks in the 1930s and 1940s further solidified the epidemic potential of this organism and prompted further investigation into the characteristics of F. tularensis. The largest recorded tularemia outbreak occurred via airborne transmission of the European biovar F. tularensis subsp. holarctica and involved more than 600 individuals between 1966 and 1967 in a farming area of Sweden (45). In this case, most individuals acquired tularemia while doing farm work that created aerosols, such as sorting hay.

Today, the worldwide occurrence of human tularemia is likely underestimated and underreported due to the generic nature of the disease symptoms. It is well established that natural tularemia outbreaks are typically highly localized, with areas of endemicity often encompassing only a few hundred square kilometers. Outbreaks of tularemia often occur in parallel with outbreaks in rodents, hares, rabbits, and other small mammals (196). Tularemia is known to be dispersed throughout much of the Northern Hemisphere, particularly in parts of North America, Europe, and northern Asia (57, 91). There have been reports of human tularemia in every state in the United States except Hawaii, with a localization of most cases to south-central and western states (18, 24a, 48). Overall, reported cases of tularemia have dropped from several thousand per year prior to 1950 to fewer than 200 in the 1990s (18, 24a, 48). Cases are typically sporadic or occur in small clusters during June through September, correlating with the incidence of arthropod-borne transmission (18, 48, 58). A summary of a number of tularemia reports during the 1980s in the United States revealed that 63% of infected individuals reported an attached tick, and 23% reported contact with wild rabbits (197). The most recent major incident involving F. tularensis in the United States occurred on Martha's Vineyard in 2000 and involved 15 patients with one fatality; 11 of the patients had acquired pneumonic tularemia (61). Many of those infected were landscapers, and it is speculated that lawn mowing or brush cutting was a major risk factor (61). Though less numerous, reports of tularemia continue to arise from Martha's Vineyard annually, with landscapers representing a majority of the infected. Most tularemia reports in Europe are from the northern and central countries, particularly Scandinavian countries (196). Disease in many of these countries occurs in an uneven geographical distribution, with high percentages of reports coming from localized rural regions. Furthermore, a strain similar to Francisella novicida was recently isolated from a patient in Australia, indicating that the geographic distribution of Francisella is likely more widespread than previously reported (216).

Tularemia

Tularemia is an acute febrile illness, the type and severity of which depend on the route of infection and the infecting biovar. F. tularensis can infect humans through the skin, mucous membranes, gastrointestinal tract, and respiratory tract. Major target organs include the lymph nodes, lungs, spleen, liver, and kidneys (58, 69, 116, 154, 190). Infection acquired through the skin or mucous membranes results in ulceroglandular tularemia, which comprises up to 90% of all cases (195). Ulceroglandular tularemia results from direct contact of the organism with the skin, often while handling infected animals or animal tissues or as a result of vector-borne transmission. A primary ulcer develops at the infection site, followed by painful swelling of the nearby lymph nodes. After an incubation period that can last up to 21 days, there is a rapid onset of high fever accompanied by flu-like symptoms. F. tularensis may further disseminate to and replicate in other organs in the body, particularly the lungs, liver, and spleen. Ulceroglandular tularemia has a mortality rate of less than 5% (58), though dissemination and replication within the lung may lead to a more serious respiratory disease. Inhalation of live organisms or accumulation of organisms in the lung following dissemination from other infection routes often leads to respiratory tularemia, the most severe form of the disease. Outbreaks resulting from respiratory transmission are rare but can involve a large number of cases, depending on the mechanism of dispersion. Symptoms for respiratory tularemia can be somewhat variable. While inhalation of F. tularensis subsp. holarctica results in a mild and generally non-life-threatening respiratory infection, inhalation of F. tularensis subsp. tularensis results in an acute, serious infection that presents with a high fever, chills, malaise, and cough. Organisms deposited into the lung readily spread to the draining lymph nodes and further disseminate to the liver and spleen, where severe inflammation and tissue damage can occur. Tularemia resulting from respiratory infection of type A Francisella has mortality rates approaching 30% to 60% if untreated (50, 174, 175). However, the fatality rate is reduced to less than 2% when antibiotics are administered in a timely fashion (58). Other, less common forms of the disease include oculoglandular tularemia, which results from direct contact of organisms with the eye, accounting for 1% to 4% of all cases (141). Ingestion of food or water contaminated with F. tularensis may also cause oropharyngeal and/or gastrointestinal tularemia, which is the least common form of the disease. Typhoidal tularemia is a term used to describe infection with severe systemic symptoms without regional ulcerations or swollen lymph nodes indicative of a site of inoculation (49). Though less common, these additional disease forms highlight the ability of Francisella to infect humans via multiple routes.

Potential as a Biological Weapon

F. tularensis has long been considered a potential biological weapon based on its ability to cause severe disease upon inhalation of doses as low as 10 CFU (174). The biological weapons programs in several countries, including Japan, the former Soviet Union, and the United States, developed weapons containing F. tularensis (49, 87). In the 1960s, F. tularensis was one of a number of agents stockpiled by the United States military as part of a biological weapons development program that was eventually terminated by executive order in 1970 (30). Despite efforts to disengage biological weapons programs around the world, former Soviet Union biological weapons senior scientist Ken Alibeck reported that weaponization efforts occurred in the Soviet Union well into the 1990s (3). In light of recent world events, the extreme infectivity and the ability to potentially disseminate aerosolized organisms over an urban area continue to drive concerns regarding Francisella weaponization and/or intentional release. In 1969, a report from a World Health Organization committee assessed the bioweapon threat of F. tularensis. It estimated that an aerosol release of 50 kg of F. tularensis over an urban area with a population of nearly 5 million individuals would result in 250,000 incapacitating casualties and 19,000 deaths (220a). More recently, the Centers for Disease Control and Prevention (CDC) estimated that the total base costs to society resulting from such an attack would approach $5.4 billion for every 100,000 persons exposed (102). In the event of an intentional release of F. tularensis, it is likely that prompt treatment of at-risk individuals would dramatically reduce the impact of the event.

F. TULARENSIS PATHOGENESIS

The success of Francisella as a pathogen is intimately associated with its ability to survive and replicate within a wide variety of host cell types. Upon entering a mammalian host, Francisella is known to target macrophages. However, it has become increasingly clear that these organisms can infect and survive in a number of additional cell types, including dendritic cells, neutrophils, hepatocytes, and lung epithelial cells. While the importance of these cell types to infection is not completely understood, it is well documented that Francisella replicates within mononuclear phagocytes in vivo and exhibits a disease cycle within these cells that appears to differ little between strains or subspecies.

Intracellular Life Cycle of F. tularensis in Phagocytic Cells

Francisella enters cells through the process of phagocytosis. It has been reported that F. tularensis may utilize an unusual mechanism involving the formation of spacious asymmetric pseudopod loops. This process, termed “looping phagocytosis” (33), involves actin rearrangement through phosphatidylinositol 3-kinase signaling and is strongly dependent on the presence of complement factor C3 and complement receptor CR3 (32, 33). Francisella may also enter cells via the mannose receptor, type I and II class A scavenge receptors, and the Fcγ receptor (11, 151, 178). Following internalization into host cells, F. tularensis is able to alter normal bactericidal processes. It prevents induction of the respiratory burst (66), limiting its exposure to superoxide or other reactive oxygen by-products. It alters phagosome maturation and as a result only transiently interacts with components of the endocytic trafficking network (Fig. 1). The organism initially resides in a membrane-bound compartment that acquires limited amounts of early endosomal and late endosomal-lysosomal markers, including EEA1, CD63, LAMP1, and LAMP2 (35). The F. tularensis-containing vacuole (FCV) fails to acquire the acid hydrolase cathepsin D and does not fuse with lysosomes (35). In addition, F. tularensis alters host cell trafficking by escaping from the phagosome and entering the host cell cytosol, where it undergoes extensive replication (27, 35, 82, 173). While the relative timing of these events appears to differ between the various Francisella species and the host cell types infected (27, 35, 82, 173), mutants that fail to prevent fusion with the lysosome and/or are unable to escape from the phagosome are highly attenuated in virulence in vitro and in vivo (19, 117, 133, 135).

FIG. 1.

Illustration of Francisella survival inside macrophages. Francisella is taken up by macrophages through looping phagocytosis (33) into an endosomal compartment that transiently acquires late endosome-associated markers (29, 32). Francisella then exits the phagosomal compartment and replicates to high numbers in the cellular cytosol. Prior to lysis of the cell, Francisella has been shown to reside in an autophagy-like compartment (27).

There are conflicting reports regarding the extent to which the FCV acidifies as it transiently interacts with components of the endocytic pathway. It also remains controversial whether exposure to acidic pH is necessary and/or sufficient for F. tularensis egress from the phagosome. Studies conducted by Clemens et al. using THP1 cells (a human macrophage-like cell line) or primary macrophages derived from peripheral blood monocytes have demonstrated that FCVs harboring LVS or type A F. tularensis become only minimally acidified (pH of 6.7) and acquire limited amounts of the proton vacuolar ATPase (34, 35). Additionally, use of the proton pump inhibitor bafilomycin A prior to infection of these macrophage types with F. tularensis strains does not alter the efficiency of F. tularensis phagosomal escape (34). In contrast, studies published by Santic et al. and Chong et al. have reported significant levels of FCV acidification and vacuolar ATPase acquisition in primary human and murine macrophages infected with F. tularensis Schu S4, LVS, and F. novicida (29, 173). Treatment of these macrophages with bafilomycin A prior to infection significantly reduced the efficiency with which these F. tularensis derivatives were able to escape from the phagosome (29, 173).

Phagosomal escape requires viable F. tularensis and occurs via an unknown mechanism that involves degradation of the surrounding lipid bilayers (27, 34, 35, 78, 173). At roughly 12 h postinfection, Francisella begins to replicate to high numbers within the host cell cyotosol, eventually leading to cell death, egress of Francisella, and presumably infection of nearby cells. Escape of F. tularensis from the phagosome and replication within the host cell cytosol is dependent on genes present in the Francisella pathogenicity island (FPI) (29, 78, 83, 110, 117, 138, 171, 173, 176). Francisella has also been shown to reside in vesicles similar to autolysosomes at ≥24 h postinfection, prior to host cell death, indicating that Francisella may reenter the endocytic pathway through host cell autophagy (27). The significance of this process for either Francisella infection or the immune response to infection remains unclear. Francisella may also exhibit an extracellular phase, as both LVS and Schu S4 have been found in the plasma following infection of mice via various inoculation routes (63). Whether this observation correlates to humans or plays a significant role in the ability of the organism to cause disease awaits further investigation.

IMMUNITY AND HOST RESPONSE TO INFECTION

Successful development of a Francisella vaccine will ultimately rely on a comprehensive understanding of the host immune response to infection. Many of the details regarding the host response to F. tularensis infection have come from studies using the less virulent F. novicida or the F. tularensis subsp. holarctica LVS, both of which are thought to differ from the more virulent type A strains in certain aspects of infection. Studies carried out with various murine infection models have shown that low doses of the attenuated LVS strain can be cleared by innate host defense mechanisms, while the fully virulent type A and B strains are able to rapidly kill mice prior to generation of a cell-mediated immune response. The precise mechanisms by which virulent strains avoid and overcome murine immune responses remain unknown. Differences in the host response to these subspecies and the route of infection highlight the complexities of this issue and suggest that the correlates of immunity need to be evaluated for each potential infection scenario.

Innate Immunity

The innate immune responses to F. tularensis infection share much in common with the responses seen with other intracellular pathogens. Francisella infection results in an early pronounced inflammatory response, with initial induction of proinflammatory and Th1-type cytokines, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ) (38, 81, 189, 217). Tumor necrosis factor alpha and IFN-γ are essential for control of infection, as depletion of either converts typically sublethal infections into lethal ones (55, 56, 112). Macrophages, dendritic cells, and natural killer (NK) cells are likely responsible for the cytokine induction seen almost immediately postinfection (17, 118). Activation of proinflammatory cytokines in murine macrophages occurs in a Toll-like receptor 2 (TLR2)-dependent manner, indicating that TLR2-mediated signaling may be crucial for early pathogen recognition (37). IFN-γ activation of macrophages and other professional phagocytes is also particularly important for initial containment of Francisella, as these cells are a primary target of the organism for infection and replication. In addition to macrophages, neutrophils have been shown to be important in the initial control of infection, but their importance may differ with respect to the different tissues infected. In mouse infection models, depletion of neutrophils increases sensitivity to systemic infection but has little effect on respiratory infection with Francisella (41, 56, 184).

There is evidence that Francisella evades and modulates the host immune response beyond its ability to inhibit maturation of the host phagosome and escape lysosomal degradation. Francisella diminishes the capacity of macrophages to respond to engagement of TLRs with secondary stimuli such as lipopolysaccharide (LPS) (198). Similar effects have also been demonstrated in dendritic cells (81). A recent report by Bosio et al. has indicated that when delivered via the respiratory route, the type A strain Schu S4 actively suppresses early inflammatory responses in the lung (16). In particular, Schu S4 fails to activate pulmonary macrophages and dendritic cells and actively interferes with induction of proinflammatory cytokines, in part through the induction of transforming growth factor β (16). In addition, Woolard et al. have recently demonstrated that Francisella infection of bone marrow-derived macrophages results in secretion of prostaglandin E₂, which inhibits interleukin-2 production and promotes a Th2-type response, a T-cell response that is ineffective against the clearing of intracellular organisms (220). This increase in prostaglandin E₂ has also been confirmed in the lung in vivo (219). Finally, Francisella has been shown to infect and replicate within neutrophils and inhibit the respiratory burst, thus evading neutrophil killing mechanisms (127, 179). Though the precise contributions of these findings to infection remain unclear, it is likely that immune evasion and/or suppression is essential to the highly virulent nature of Francisella and differences between subspecies.

Adaptive Immunity

Exposure to sublethal concentrations of Francisella induces strong protective immunity against secondary exposure in humans and in experimental animal models (51, 188). Though specific antibodies are readily detectable in sera upon F. tularensis infection, their importance to immunity remains unclear. Passive antibody transfer studies carried out in animals suggest that antibodies may play a role in combating infection with lower-virulence strains while playing a lesser role against the more virulent subspecies (51, 54, 188). Although Francisella antibodies may prove beneficial in some situations, they are likely not essential. Rather, they must be coupled with an effective cellular immune response to fully control infection. Adaptive immunity to F. tularensis infection is largely dependent on T-cell-mediated immunity, particularly that mediated by CD4⁺ and CD8⁺ T cells (56). In mice, either CD4⁺ or CD8⁺ cells are able to control infection by F. novicida or LVS, while both cell types seem to be required for successful defense against the highly virulent type A strains (43, 74, 222). Similar to the case for mice, CD4⁺ and CD8⁺ T-cell responses are prominent in humans vaccinated with LVS (107, 192). The T-cell effector functions are likely very closely linked to the ability to activate macrophage intracellular killing mechanisms. Despite the known requirement of CD4⁺ and CD8⁺ T cells for resolving F. tularensis infection, little is known regarding the T-cell receptors, coreceptors, memory profiles, or major histocompatibility complex restriction of T-cell responses to infection.

Immunity to Infection by Different Routes

An effective vaccine against F. tularensis will require generation of an immune response that is protective against pulmonary infection. However, much of the work examining and characterizing the immune responses to Francisella infection have involved infection by the systemic route. Though there are many general consistencies, correlates of immunity to F. tularensis infection differ in certain aspects depending on the route of infection. A number of recent reports highlight potentially key differences in the host immune response to respiratory versus systemic infection (41). In addition to the diminished role of neutrophils and reactive nitrogen species, there exists disparity in the timing of initial host inflammation when comparing respiratory versus systemic infection. During murine infection initiated via the intradermal or subcutaneous route, there is an immediate onset of inflammation within the first 2 days postinfection that includes the rapid induction of IFN-γ (36, 54). During respiratory infection this response is delayed, not occurring until 3 to 5 days postinfection. By this time, significant bacterial burdens have begun to accumulate in the livers and spleens of infected mice, and it has been speculated that systemic disease contributes to the morbidity observed in these animals (40). The delay in inflammation onset is consistent with what has been seen in human disease (6). The precise reasons for this delay remain unclear, but it may play a contributing factor in the general difficulty in vaccinating against respiratory forms of the disease. Other recent reports also highlight potential differences in T-cell responses between respiratory and systemic infection. Woolard et al. have demonstrated that intranasal infection of mice produces much lower levels of IFN-γ-secreting T cells than systemic infection (219). Furthermore, intranasal inoculation results in a delayed accumulation of T cells in the spleen and lung, along with a significant increase in the amounts of prostaglandin E₂. Collectively, these observations suggest that virulent F. tularensis subspecies alter T-cell responses to the detriment of the host (220).

Though our understanding of Francisella host/pathogen interactions is advancing, there is still a great deal that remains unclear. Of particular interest, the host immune response to infection by highly virulent type A strains has only now been investigated in any great detail. Further evaluation of the host immune response to infection, as well as identification of key Francisella virulence mediators, will be necessary to gain a more complete understanding of the interplay between Francisella and the host immune system, particularly for the development of novel prophylactic treatments.

FRANCISELLA GENETICS AND VIRULENCE FACTORS

The ability to effectively colonize or parasitize a diverse array of hosts suggests that F. tularensis is capable of adapting to a wide variety of growth environments. Despite its extreme virulence and fairly well-characterized intracellular life cycle, very little is known about the mechanisms of F. tularensis pathogenesis or the virulence factors encoded by this organism. Initial assessments of the completed genomic sequences from different F. tularensis subspecies have indicated that F. tularensis does not encode any toxins or secretion systems that are commonly present in other intracellular pathogens. In addition, F. tularensis does not encode homologs of genes that mediate phagosomal escape in other organisms, such as Listeria and Shigella. Due in large part to an increase in the development and efficiency of genetic tools, recent studies have begun to shed light on the specific virulence genes necessary for successful infection by F. tularensis.

Genetic Tools

Shuttle and integration vectors.

The field of Francisella genetics has undergone an extensive expansion over the past 10 years (71). Until recently, few vectors or selectable markers were amenable for use in Francisella, and the available methods for introducing DNA were generally inefficient. In 1994, identification of a 3,990-bp cryptic plasmid (pFNL10) from an F. novicida-like strain designated F6168 helped to usher in the first generation of useful genetic vectors for this organism (144). pFNL10 could be introduced by standard procedures and maintained in various subspecies of F. tularensis, although it was not capable of replicating in Escherichia coli and lacked any antibiotic resistance markers (144). Further modifications to pFNL10 led to the construction of second-generation vectors that carried replication origins for E. coli and selectable antibiotic resistance markers. pFNL100 included sequences from both pFNL10 and cloning vector pBR328 (143). pFNL200 was a deletion derivative of pFNL100 and expressed tetracycline and chloramphenicol resistance; however, it suffered from stability issues (143). pKK202 was a more stable derivative of pFNL200 that carried the p15A origin of replication from E. coli (139). Finally, the generation of pKK214 and its variants expanded the utility of pKK202 by incorporating a promoterless chloramphenicol acetyltransferase or green fluorescent protein reporter gene in place of the chloramphenicol acetyltransferase gene (2, 108).

More recently, Maier et al. have constructed a series of E. coli-Francisella shuttle vectors, termed pFNLTP, that are hybrids between pFNL10 and cloning vector pCR2.1-TOPO (125). These vectors can be efficiently transformed into F. tularensis subspecies by electroporation, are stably maintained even in the absence of antibiotic selection, and do not alter virulence characteristics of F. tularensis in vitro or in vivo (124, 125, 145, 146). A variety of pFNLTP1 variants have been generated, and these include derivatives that carry antibiotic resistance elements amenable for use in type A strains of F. tularensis, multiple cloning sites, reporter genes and counterselectable markers, and temperature-sensitive origins of replication (93, 125). In addition to their use as complementation and reporter gene platforms, pFNLTP1-based vectors (or vectors that have been derived from them) have been used as delivery vehicles to carry out transposon mutagenesis, targeted allelic exchange, and promoter-trap library construction (22, 125, 128).

LoVullo et al. have recently developed a series of shuttle vectors, pMP, that are based on the minimal regions of pFNL10 required for replication and regions from E. coli-mycobacterial shuttle vector pMV261 carrying the aphA1 antibiotic resistance determinant and ColE1 replication origin (121). While the original plasmid, pMP393, could be efficiently introduced by electroporation and was stable in various F. tularensis subspecies, it was frequently lost in the absence of selection (121). Second-generation variants of pMP393 have corrected maintenance issues and expanded the choice and utility of antibiotic resistance determinants for selection within F. tularensis (120). Third-generation pMP-based vectors have also been developed, in which stability has been further enhanced, useful multiple cloning sites introduced, and heterologous promoters added for gene expression studies (120). Finally, a single-copy chromosomal integration system for Francisella has been developed by that group (119). Vectors designed for this system include plasmids allowing integration at the attachment site for the Tn7 transposon (located downstream of the glmS gene) or within the blaB gene, encoding resistance to the antibiotic ampicillin (119). Development of an integration system for F. tularensis represents a major advancement for the field, as it alleviates some of the previous issues inherent with use of multicopy shuttle vectors, including lack of stability, use of heterologous antibiotic resistance determinants, and multicopy expression artifacts.

Gene disruption vectors.

Much of the lack of understanding of Francisella virulence can be directly attributed to the difficulty in generating defined genetic lesions within this family. While genetic tools and methodologies have been available for some time to disrupt genes in F. novicida, construction of mutant derivatives in the type A or B genetic background was not reported until 2004 (82). Gene disruptions in F. novicida have been generated using a variety of approaches, including allelic exchange of linear substrates (Table 2) (110). Initial efforts to disrupt genes in type A or type B strains were based largely on utilization of pUC19-derived suicide vectors (82). Optimization of these vectors, along with the development of additional vectors, has allowed the list of Francisella mutants to expand significantly (Tables 3 and 4). This list includes mutants that are defective for putative virulence factors as well as metabolic genes that may be utilized for the construction of live attenuated vaccine candidates. More recently, the TargeTron group II intron mutagenesis system has been adapted for use with various F. tularensis subspecies (162, 163). This system has proven efficient, and it is advantageous as it allows simultaneous disruption of genes that are present in more than one copy (162, 163).

TABLE 2.

F. novicida genes involved in pathogenesis

Locus tag	Name	Function	Method(s)a	Cells/animals in which mutant strain is attenuatedb	Reference(s)
FTN_0008	FTN_0008	Ten-transmembrane-spanning drug/metabolite exporter protein	Tp	C57BL/6	106
FTN_0012	FTN_0012	Hypothetical protein	Tp	C57BL/6	106
FTN_0019	pyrB	Aspartate carbamoyltransferase	Tp	C57BL/6	213
FTN_0020	carB	Carbamoylphosphate synthase large chain	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_0021	carA	Carbamoylphosphate synthase small chain	Tp	C57BL/6	213
FTN_0023	tmpT	Thiopurine S-methyltransferase	Tp	C57BL/6	106
FTN_0028	FTN_0028	Conserved hypothetical membrane protein	Tp	C57BL/6	106
FTN_0031	FTN_0031	Transcriptional regulator, LysR family	Tp	C57BL/6	106
FTN_0035	pyrF	Orotidine 5′-phosphate decarboxylase	Tp	C57BL/6	213
FTN_0036	pyrD	Diyroorotate dehydrogenase	Tp	C57BL/6	213
FTN_0045	FTN_0045	Protein of unknown function	Tp	C57BL/6	106
FTN_0055	tyrA	Prephenate dehydrogenase	Tp	C57BL/6	106
FTN_0090, FTN_1556, FTN_1061, FTN_0954	acpABC, hap	Acid phosphatasesc	Ar	J774A.1, activated THP-1, BALB/c	13, 133, 135
FTN_0096	FTN_0096	Conserved hypothetical membrane protein	Tp	C57BL/6	213
FTN_0097	FTN_0097	Aromatic amino acid transporter of the hydroxy/aromatic amino acid permease family	Tp	C57BL/6	213
FTN_0098	gidB	Methyltransferase, glucose-inhibited cell division protein	Tp	C57BL/6	213
FTN_0111	ribH	Riboflavin synthase beta-chain	Tp	C57BL/6	106
FTN_0112	ribA	RibA/GTP-cyclohydrolase II	Tp	C57BL/6	213
FTN_0113	ribB	Riboflavin synthase a-subunit 3,4-digydroxy-2-butanone 4-phosphate synthetase	Tp	C57BL/6	213
FTN_0132	FTN_0132	Protein of unknown function	Tp	C57BL/6	106, 213
FTN_0133	FTN_0133	RNase II family protein	Tp	C57BL/6	106
FTN_0143	FTN_0143	Monovalent cation:proton antiporter	Tp	C57BL/6	106
FTN_0169	FTN_0169	Conserved hypothetical membrane protein	Tp	C57BL/6	106
FTN_0177	purH	AICAR transformylase/IMP cyclohydrolase	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_0178	purA	Adenylosuccinate synthetase	Ar/In	J774A.1, BALB/c	158
FTN_0196	cyoB	Cytochrome o ubiquinol oxidase subunit I	Tp	C57BL/6	213
FTN_0197	cyoC	Cytochrome o ubiquinol oxidase subunit III	Tp	C57BL/6	213
FTN_0198	cyoD	Cytochrome o ubiquinol oxidase subunit IV	Tp	C57BL/6	213
FTN_0199	cyoE	Heme O synthase	Tp	C57BL/6	106
FTN_0202	pdxY	Pyridoxal/pyridoxine/pyridoxamine kinase	Tp	C57BL/6	213
FTN_0205	FTN_0205	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_0208	FTN_0208	Hypothetical membrane protein	Tp	C57BL/6	106
FTN_0210	FTN_0210	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_0211	pcp	Pyrrolidone-carboxylate peptidase	Tp	C57BL/6	213
FTN_0217	FTN_0217	l-Lactate dehydrogenase	Tp	C57BL/6	106
FTN_0229	pyrH	Uridylate kinase	Tp	C57BL/6	213
FTN_0240	rplD	50S ribosomal protein L4	Tp	C57BL/6	213
FTN_0241	rplW	50S ribosomal protein L23	Tp	C57BL/6	213
FTN_0248	rpsQ	30S ribosomal protein S17	Tp	RAW	199
FTN_0265	rplQ	50S ribosomal protein L17	Tp	C57BL/6	213
FTN_0266	htpG	Chaperone Hsp90, heat shock protein HtpG	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_0280	FTN_0280	Hypothetical protein	Tp	C57BL/6	106
FTN_0281	FTN_0281	Protein of unknown function	Tp	C57BL/6	106
FTN_0289	FTN_0289	ProP osmoprotectant transporter, fragment	Tp	C57BL/6	213
FTN_0292	FTN_0292	Protein of unknown function	Tp	C57BL/6	106
FTN_0295	lpxF	Phosphatidic acid phosphatase, PAP2 superfamily	Ar	C57BL/6	209
FTN_0296	FTN_0296	Amino acid transporter	Tp	C57BL/6	213
FTN_0297	FTN_0297	Conserved protein of unknown function	Tp	C57BL/6	106, 213
FTN_0298	gplX	Fructose 1,6-bisphosphatase II	Tp	C57BL/6	106, 213
FTN_0299	putP	Proline:Na+ symporter	Tp	C57BL/6	106
FTN_0300	FTT1629c	Glycosyl transferase, group 2	Tp	RAW	199
FTN_0302	FTN_0302	Hypothetical protein	Tp	C57BL/6	106
FTN_0326	FTN_0326	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_0330c	minD	Septum formation inhibitor-activating ATPase	In	PM, BMDM, C57BL/6NCrIBR	8
FTN_0337	fumA	Fumarate hydratase, class I	Tp	J774A.1, RAW, BMDM, BALB/c	199
FTN_0340	FTN_0340	Protein of unknown function	Tp	C57BL/6	106
FTN_0344	FTN_0344	Aspartate:alanine exchanger family	Tp	C57BL/6	106
FTN_0347	fkpB	FK506 binding protein-type peptidyl-prolyl, cis-trans	Tp	C57BL/6	106
FTN_0351	FTN_0351	Hypothetical protein, novel liver	Tp	C57BL/6	106
FTN_0365	FTN_0365	Conserved hypothetical membrane protein	Tp	C57BL/6	106
FTN_0393	FTN_0393	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_0395	FTN_0395	Transcriptional regulator, ArsR family	Tp	C57BL/6	106
FTN_0397	gpsA	Glycerol-3-phosphate-dehydrogenase	Tp	C57BL/6	106
FTN_0400	FTN_0400	Protein of unknown function	Tp	C57BL/6	106
FTN_0402	aroC	Chorismate synthase	Tp	C57BL/6	106
FTN_0416	FTN_0416	Conserved hypothetical membrane protein	Tp	C57BL/6	213
FTN_0417	folD	Methylene tetrahydrofolate	Tp	C57BL/6	106, 213
FTN_0419	purM	Phosphoribosylformylglycinamide cyclo-ligase	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_0420	purCD	SAICAR synthetase/phosphoribosylamine-glycine ligase	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_0422	purE	Phosphoribosylaminoimidazole carboxylase, catalytic subunit	Tp	C57BL/6	213
FTN_0426	FTN_0426	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_0427	tul4	Lipoprotein of unknown function	Ar/In	J774A.1c	110
FTN_0429	FTN_0429	Hypothetical protein	Tp	C57BL/6	213
FTN_0430	lpnB	Protein of unknown function	Tp	C57BL/6	106, 213
FTN_0431	FTN0431	Type IV pilus glycosylation protein	Tp	C57BL/6	213
FTN_0436	FTN_0436	Hypothetical protein	Tp	C57BL/6	213
FTN_0437	FTN_0437	Hydrolase, HD superfamily	Tp	C57BL/6	106
FTN_0438	rrmJ	23S rRNA methylase	Tp	C57BL/6	106
FTN_0443	maeA	NAD-dependent malic enzyme	Tp	RAW	199
FTN_0444	FTT0918	Membrane protein of unknown function	Tp	C57BL/6	181
FTN_0480	fevR	Protein of unknown function	Ar	BMDM, C57BL/6	20
FTN_0488	cspC	Cold shock protein,	Tp	C57BL/6	106
FTN_0493	mtn	Adenosylhomocysteine nucleosidase	Tp	C57BL/6	213
FTN_0494	FTN_0494	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_0494	FTN_0494	BNR/Asp box repeat protein	Tp	C57BL/6	213
FTN_0503	lolD	Lipoprotein releasing system, subunit D, ABC transporter, ATP-binding protein	Tp	C57BL/6	213
FTN_0504	cadA	Lysine decarboxylase, inducible	Tp	C57BL/6	213
FTN_0505	gcvT	Glycine cleavage complex T protein (aminomethyltransferase)	Tp	C57BL/6	213
FTN_0506	gcvH	Glycine cleavage system H protein	Tp	C57BL/6	213
FTN_0507	gcvP1	Glycine cleavage system P protein, subunit 1	Tp	C57BL/6	213
FTN_0514	pgm	Phosphoglucomutase	Tp	C57BL/6	213
FTN_0536	yjjK	ABC transporter, ATP binding protein	Tp	C57BL/6	213
FTN_0537	FTN_0537	Proton-dependent oligopeptide	Tp	C57BL/6	106
FTN_0544	FTN_0544	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_0545	flmF2	Glycosyl transferase, group 2	Tp	BALB/c, C57BL/6	101, 213
FTN_0546	flmK	Dolichyl-phosphate-mannose-protein, mannosyltransferase family protein	Tp	BALB/c, C57BL/6	101, 213
FTN_0554	FTN_0554	RNA methyltransferase,	Tp	C57BL/6	106
FTN_0559	surA	Peptidyl-prolyl cis-trans isomerase	Tp	C57BL/6	213
FTN_0560	ksgA	Dimethyladenosine transferase, kasugamycin resistance	Tp	C57BL/6	213
FTN_0571	xasA	Amino acid-polyamine-organocation	Tp	C57BL/6	106, 213
FTN_0578	FTN_0578	Major facilitator superfamily transport	Tp	C57BL/6	106
FTN_0582	gph	Phosphoglycolate phosphatase	Tp	C57BL/6	106
FTN_0594	sucC	Succinyl-coenzyme A synthetase, beta chain	Tp	RAW	199
FTN_0599	FTN_0599	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_0609	pnp	Polyribonucleotide nucleotidyltransferase	Tp	C57BL/6	106
FTN_0624	sdaC1	Serine transporter	Tp	C57BL/6	213
FTN_0643	FTN_0643	Hypothetical protein	Tp	C57BL/6	213
FTN_0646	cscK	ROK family protein	Tp	C57BL/6	106
FTN_0651	cdd	Cytidine deaminase	Tp	C57BL/6	213
FTN_0669	deoD	Purine nucleoside phosphorylase	Tp	C57BL/6	213
FTN_0678	FTN_0678	Drug:H+ antiporter-1 (DHA1) family	Tp	C57BL/6	106
FTN_0689	ppiC	Parvulin-like peptidyl-prolyl isomerase	Tp	C57BL/6	106
FTN_0690	deaD	DEAD box subfamily, ATP-dependent	Tp	C57BL/6	106
FTN_0710	FTN_0710	Type I restriction-modification system	Tp	C57BL/6	106
FTN_0713	ostA2	Organic solvent tolerance protein OstA	Tp	C57BL/6	106
FTN_0714	FTT0742	Protein of unknown function	Tp	J774A.1, RAW, BMDM, BALB/c	199
FTN_0720	FTN_0720	Transcriptional regulator, LclR family	Tp	C57BL/6	106, 213
FTN_0728	FTN_0728	Cation efflux family protein	Tp	C57BL/6	213
FTN_0737	potI	ATP binding cassette putrescine uptake	Tp	C57BL/6	106
FTN_0740	FTN_0740	Protein of unknown function	Tp	C57BL/6	106
FTN_0756	fopA	OmpA family protein	Tp	RAW	199
FTN_0757	FTN_0757	Hypothetical protein	Tp	C57BL/6	213
FTN_0768	tspO	Tryptophan-rich sensory protein	Tp	C57BL/6	106
FTN_0787	rep	UvrD/REP superfamily I	Tp	C57BL/6	106
FTN_0806	FTN_0806	β-Glucosidase-related glycosidase	Tp	C57BL/6	213
FTN_0810	FTN_0810	ROK family protein	Tp	C57BL/6	106
FTN_0814	bioF	8-Amino-7-oxononanoate synthase	Tp	C57BL/6	213
FTN_0815	bioB	Biotin synthase	Tp	C57BL/6	213
FTN_0816	bioA	Oxononanoate aminotransferase	Tp	C57BL/6	213
FTN_0817	FTN_0817	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_0818	FTN_0818	Lipase/esterase	Tp	C57BL/6	213
FTN_0821	FTN_0821	AMP binding family protein	Tp	C57BL/6	213
FTN_0822	FTN_0822	Chorismate binding family protein	Tp	C57BL/6	213
FTN_0823	trpG	Anthranilate synthase component II	Tp	C57BL/6	213
FTN_0824	FTN_0824	Major facilitator superfamily	Tp	C57BL/6	106
FTN_0825	FTN_0825	Aldo/keto reductase family protein	Tp	C57BL/6	106
FTN_0840	mdaB	NADPH-quinone reductase	Tp	C57BL/6	106
FTN_0848	FTN_0848	Amino acid antiporter	Tp	C57BL/6	213
FTN_0853	sufD	SufS activator complex, SufD subunit	Tp	C57BL/6	106
FTN_0857	FTN_0857	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_0869	FTT0989	Conserved protein of unknown function	Ar/In	BMDM, C57BL/6	21
FTN_0873	dcd	dCTP deaminase	Tp	C57BL/6	106
FTN_0877	cls	Cardiolipin synthetase	Tp	C57BL/6	106
FTN_0881	FTN_0881	Fe2/Zn2 uptake regulator protein	Tp	C57BL/6	106
FTN_0891	ruvB	Holliday junction DNA helicase, subunit B	Tp	C57BL/6	213
FTN_0918	FTN_0918	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_0925	FTN_0925	Hypothetical protein	Tp	C57BL/6	213
FTN_0933	FTN_0933	Protein of unknown function	Tp	C57BL/6	106
FTN_0938	FTN_0938	Hypothetical protein	Tp	C57BL/6	106
FTN_0998	FTN_0998	Potassium channel protein	Tp	C57BL/6	213
FTN_1006	FTN_1006	Transporter-associated protein	Tp	C57BL/6	106
FTN_1015	FTN_1015	Isochorismatase family protein	Tp	C57BL/6	106
FTN_1016	FTN_1016	Conserved protein of unknown function	Tp	C57BL/6	106, 213
FTN_1029	FTN_1029	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_1030	lipA	Lipoic acid synthetase	Tp	C57BL/6	106
FTN_1038	FTN_1038	Conserved hypothetical membrane protein	Tp	C57BL/6	213
FTN_1055	lon	DNA binding, ATP dependent	Tp	C57BL/6	106
FTN_1063	FTN_1063	tRNA-methylthiotransferase MiaB protein	Tp	C57BL/6	106
FTN_1064	phoH	PhoH-like protein	Tp	C57BL/6	213
FTN_1066	FTN_1066	Metal ion transporter protein	Tp	C57BL/6	213
FTN_1090	FTN_1090	Hypothetical protein	Tp	C57BL/6	213
FTN_1098	FTN_1098	Conserved hypothetical membrane protein	Tp	C57BL/6	106
FTN_1107	metIQ	Methionine uptake transporter family	Tp	C57BL/6	106
FTN_1111	FTN_1111	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1112	cphA	Cyanophycin synthetase	Tp	C57BL/6	213
FTN_1131	putA	Multifunctional protein, transcriptional repressor of proline utilization, proline dehydrogenase, pyrroline-5-carboxylate dehydrogenase	Tp	C57BL/6	213
FTN_1132	FTN_1132	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1133	FTN_1133	Protein of unknown function, novel lung, spleen	Tp	C57BL/6	106
FTN_1146	aspC2	Aspartate aminotransferase	Tp	RAW	199
FTN_1148	FTN_1148	Glycoprotease family protein	Tp	C57BL/6	106
FTN_1157	FTN_1157	GTP binding translational elongation	Tp	C57BL/6	106
FTN_1165	FTN_1165	Predicted ATPase of the PP loop	Tp	C57BL/6	106
FTN_1177	sbcB	Exodeoxyribonuclease I	Tp	C57BL/6	106
FTN_1186	pepO	M13 family metallopeptidase	Ar/In	C57BL/6	21
FTN_1196	FTN_1196	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_1199	FTN_1199	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_1200	capC	Capsule biosynthesis protein CapC	Tp	C57BL/6	106
FTN_1201	capB	Capsule biosynthesis protein CapB	Tp	C57BL/6	213
FTN_1209	cphB	Cyanophycinase	Tp	C57BL/6	106, 213
FTN_1211	FTN_1211	Haloacid dehalogenase-like hydrolase family protein	Tp	C57BL/6	213
FTN_1212	FTN_1212	Glycosyltransferase group 1 family protein	Tp	C57BL/6	213
FTN_1213	FTN_1213	Glycosyltransferase family protein	Tp	C57BL/6	213
FTN_1214	FTN_1214	Glycosyltransferase family protein	Tp	C57BL/6	213
FTN_1217	FTN_1217	ABC transporter, ATP binding and membrane protein	Tp	C57BL/6	213
FTN_1218	FTN_1218	Glycosyltransferase, group 1	Tp	C57BL/6	106, 213
FTN_1219	gale	UDP-glucose 4-epimerase	Tp	C57BL/6	213
FTN_1220	FTN_1220	Bacterial sugar transferase family protein	Tp	C57BL/6	213
FTN_1232	FTN_1232	Conserved hypothetical membrane protein	Tp	C57BL/6	106
FTN_1241	dedA2	DedA family protein	Tp	RAW	199
FTN_1242	dedA1	DedA family protein	Tp	C57BL/6	213
FTN_1243	recO	RecFOR complex, RecO component	Tp	C57BL/6	106
FTN_1254	FTN_1254	Hypothetical protein	Tp	C57BL/6	213
FTN_1255	FTN_1255	Glycosyl transferase family 8 protein	Tp	C57BL/6	213
FTN_1256	FTN_1256	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_1257	FTN_1257	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_1259	glyA	Serine hydroxymethyltransferase	Tp	RAW	199
FTN_1272	FTN_1272	Proton-dependent oligopeptide	Tp	C57BL/6	106
FTN_1273	fadD1	Acyl coenzyme A synthetase	Tp	C57BL/6	213
FTN_1276	emrA1	HlyD family secretion protein	Tp	C57BL/6	213
FTN_1277	FTN_1277	Outer membrane efflux protein	Tp	C57BL/6	213
FTN_1284	dnaK	Chaperone, heat shock protein, HSP 70 family	Tp	J774A.1, RAW, BMDM, BALB/c	199
FTN_1290	mglA	Macrophage growth locus, protein A	Ar/In	J774A.1, Acanthamoeba castellanii, BALB/cAnNHsd, BALB/c	110, 111, 214
FTN_1292	FTN_1292	Solute:sodium symporter	Tp	C57BL/6	106
FTN_1298	FTN_1298	GTPase of unknown function	Tp	C57BL/6	106
FTN_1309	pdpA	Protein of unknown function	Ar	CE, BALB/cByJ	176, 177, 213
FTN_1310	pdpB	Protein of unknown function	Ar/In, Tp	BMDM, C57BL/6, J774A.1, RAW, BALB/c	21, 199, 213
FTN_1311	FTN_0311	Hypothetical protein	Tp	C57BL/6	213
FTN_1312	FTN_1312	Conserved hypothetical protein	Tp	C57BL/6	106, 213
FTN_1313	FTN_1313	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1314	FTN_1314	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1315	FTN_1315	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1316	FTN_1316	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1317	FTN_1317	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1318	cds2	Hypothetical protein	Ar/In, Tp	BMDM, C57BL/6	21, 213
FTN_1319	FTN_1319	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1320	FTN_1320	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1321	iglD	Intracellular growth locus protein D	Ar/In, Tp	BALB/c, hMDMs, C57BL/6	106, 172, 213
FTN_1322	iglC	Intracellular growth locus, protein C	Ar/In	J774A.1, Acanthamoeba castellanii, BALB/cAnNHsd, C57BL/6	110, 111, 142, 213
FTN_1323	iglB	Intracellular growth locus, protein B	In	J774, BALB/c	39, 213
FTN_1324	iglA	Intracellular growth locus protein A	Ar	J774A.1, CE	46, 213
FTN_1325c	pdpD	Protein of unknown function	Ar/In	BMDM, BALB/cByJ	138, 213
FTN_1326	FTN_1326	Conserved hypothetical protein	Tp	C57BL/6	213
FTN_1333	tktA	Transketolase I	Tp	J774A.1, RAW, BMDM, BALB/c	199
FTN_1349	FTN_1349	Hypothetical protein	Tp	C57BL/6	106
FTN_1351	FTN_1351	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_1355	FTN_1355	Regulatory factor, Bvg	Tp	C57BL/6	106
FTN_1357	recB	Exodeoxyribonuclease V b-chain	Tp	C57BL/6	213
FTN_1362	FTN_1362	Hypothetical protein	Tp	C57BL/6	106
FTN_1400	FTN_1400	S-Adenosylmethionine dependent	Tp	C57BL/6	106
FTN_1403	flmF1	Glycosyltransferase	Tp	BALB/c, C57BL/6	101
FTN_1415	FTN_1415	Thioredoxin	Tp	C57BL/6	106
FTN_1417	manB	Phosphomannomutase	Tp	C57BL/6	213
FTN_1421	wbtH	Asparagine synthase	Tp	C57BL/6	213
FTN_1423	wbtG	Glycosyl transferase	Tp	C57BL/6	213
FTN_1425c-FTN_1427c	wbtDEF	LPS O-antigen biosynthetic gene cluster	Ar/In	BALB/c	200, 213
FTN_1432	wbtA	dTDP-glucose 4,6-dehydratase	Tp	C57BL/6	213
FTN_1439	fadA	Acetyl coenzyme A acetyltransferase	Tp	C57BL/6	106
FTN_1440	FTN_1440	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_1444	ocd	Ornithine cyclodeaminase, mu-crystallin homolog	Tp	RAW	199
FTN_1455	FTN_1455	Conserved hypothetical protein	Tp	C57BL/6	106
FTN_1459	FTN_1459	Short-chain dehydrogenase	Tp	C57BL/6	106
FTN_1464	lepB	Signal peptidase I	Tp	C57BL/6	106
FTN_1465	pmrA	Two-component response regulator, orphan	Ar	J774A.1, activated THP-1, HeLa, BALB/c	134
FTN_1467	proC	Pyrroline-5-carboxylate reductase	Tp	C57BL/6	106
FTN_1471	pcs	(CDP-alcohol) phosphatidyltransferase	Tp	C57BL/6	106
FTN_1500	FTN_1500	Hypothetical protein	Tp	C57BL/6	213
FTN_1501	FTN_1501	Na+/H+ antiporter	Tp	C57BL/6	213
FTN_1507	FTN_1507	Pilus assembly protein	Tp	C57BL/6	106
FTN_1513	xerC	Integrase/recombinase XerC	Tp	C57BL/6	213
FTN_1518	relA	GTP pyrophosphokinase	Tp	C57BL/6	213
FTN_1433	FTN_1433	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_1438	fadB-acbP	Fusion product of 3-hydroxacyl-coenzyme A dehydrogenase and acyl coenzyme A binding protein	Tp	C57BL/6	213
FTN_1470	ispA	Geranyltransferase	Tp	C57BL/6	213
FTN_1471	pcs	Phosphatidylcholine synthase	Tp	C57BL/6	213
FTN_1534	FTN_1534	Conserved protein of unknown function	Tp	C57BL/6	106
FTN_1536	FTN_1536	Amino acid-polyamine-organocation	Tp	C57BL/6	106
FTN_1538	groL	Chaperone protein, GroEL	Tp	C57BL/6	213
FTN_1548	FTN_1548	Conserved hypothetical lipoprotein	Tp	C57BL/6	213
FTN_1551	ampD	N-Acetylmuramoyl-l-alanine amidase	Tp	C57BL/6	213
FTN_1558	xerD	Integrase/recombinase	Tp	C57BL/6	213
FTN_1567	rpoC	DNA-directed RNA polymerase, beta′ subunit/160-kDa subunit	Tp	RAW	199
FTN_1582	FTN_1582	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_1587	FTN_1587	Protein of unknown function	Tp	C57BL/6	106
FTN_1588	FTN_1588	Major facilitator superfamily transport	Tp	C57BL/6	106
FTN_1592	oppB	Peptide/opine/nickel uptake transporter (PepT) family protein	Ar/In	C57BL/6	21
FTN_1597	prfC	Peptide chain release factor 3	Tp	C57BL/6	213
FTN_1602	deoB	Phosphopentomutase	Tp	C57BL/6	213
FTN_1607	cca	tRNA nucleotidyltransferase	Tp	C57BL/6	213
FTN_1608	dsbB	Disulfide bond formation protein	Tp	J774A.1, RAW, BMDM, BALB/c, C57BL/6	199, 213
FTN_1611	FTN_1611	Major facilitator superfamily transport	Tp	C57BL/6	106
FTN_1617	qseC	Sensor histidine kinase	Tp	C57BL/6	213
FTN_1633	apt	Adenine phosphoribosyltransferase	Tp	C57BL/6	213
FTN_1634	sucB	Dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex	Tp	C57BL/6	213
FTN_1639	sdhC	Succinate dehydrogenase, cytochrome b556	Tp	C57BL/6	213
FTN_1645	FTN_1645	AtpC ATP synthase, F1 sector, subunit epsilon	Tp	C57BL/6	106
FTN_1653	FTN_1653	Hypothetical membrane protein	Tp	C57BL/6	213
FTN_1654	FTN_1654	Transport protein	Tp	C57BL/6	213
FTN_1655	rluC	Ribosomal large subunit pseudouridine synthase C	Tp	C57BL/6	213
FTN_1656	FTN_1656	ATPase	Tp	C57BL/6	213
FTN_1657	FTN_1657	Major facilitator superfamily transport protein	Tp	C57BL/6	213
FTN_1658	hisS	Histidyl-tRNA synthetase	Tp	C57BL/6	213
FTN_1659	rbfA	Ribosome binding factor A	Tp	C57BL/6	213
FTN_1665	FTN_1665	Magnesium chelatase	Tp	C57BL/6	106
FTN_1673	nuoH	NADPH dehydrogenase I, H subunit	Tp	C57BL/6	106
FTN_1682	figA (fslA)	Siderophore biosynthesis protein	Ar/In	J774A.1	47, 213
FTN_1683	FTN_1683	Conserved membrane protein	Tp	C57BL/6	213
FTN_1684	lysA	Diaminopimelate decarboxylase	Tp	C57BL/6	213
FTN_1686	figE	Hypothetical membrane protein involved in siderophore uptake	Ar	BALB/c	132
FTN_1699	purL	Phosphoribosylformylglycinamide synthase	Tp	J774A.1, RAW, BMDM, BALB/c	199, 213
FTN_1700	purF	Amidophosphoribosyltransferase	Ar/In	J774A.1, BALB/c	158, 213
FTN_1715	kdpD	Two-component sensor protein KdpD	Tp	C57BL/6	213
FTN_1726	FTN_1726	Pyridoxal-dependent decarboxylase	Tp	C57BL/6	106
FTN_1731	pip	Proline iminopeptidase	Tp	C57BL/6	106
FTN_1743	clpB	ClpB protein	Tp	RAW	199, 213
FTN_1744	FTN_1744	Chitinase	Tp	C57BL/6	213
FTN_1745	purT	Phosphoribosylglycinamide formyltransferase 2	Tp	C57BL/6	213
FTN_1753	FTN_1753	Rieske (2Fe-2S) domain protein	Tp	C57BL/6	106
FTN_1756	bcp	Bacterioferritin comigratory protein	Tp	C57BL/6	106
FTN_1760	FTN_1760	Zinc binding alcohol dehydrogenase	Tp	C57BL/6	106
FTN_1778	trpE	Anthranilate synthase component I	Tp	C57BL/6	106

^aAr, allelic replacement; In, insertion; Tp, transposon insertion.

^bBMDM, bone marrow-derived macrophages; PM, peritoneal macrophages; CE, chicken embryos; hMDMs, human monocyte-derived macrophages; RAW, RAW264.7 murine macrophages.

^cAttenuated phenotype more pronounced with accumulated mutations.

TABLE 3.

F. tularensis subsp. holarctica genes involved in pathogenesis

Locus tag	Name	Function	Method(s)a	Cells/animals in which mutant strain is attenuatedb	Reference(s)
FTL_0009	FTT1747	Outer membrane protein	STM	BALB/c	191
FTL_0010	glpE	Thiosulfate sulfurtransferase	STM	BALB/c	191
FTL_0012	recA	Recombinase A protein	STM	BALB/c	191
FTL_0028-FTL_0030	carA, carB, pyrB	Uracil biosynthesis	Tp	MDM	179
FTL_0050	FTT1645	Hypothetical protein	Tp	J774A.1, BALB/c	124
FTL_0058	FTT1688	Aromatic amino acid transporter of the hydroxy/aromatic amino acid permease family	Tp	J774A.1	124
FTL_0073	FTT1676	Membrane protein	STM	BALB/c	191
FTL_0094	clpB	Caseinolytic protease	Ar, Tp, STM	BMM, J774A.1c, BALB/c	124, 129, 191
FTL_0111	iglA	Intracellular growth	STM	BALB/c	191
FTL_0112	iglB	Intracellular growth	STM	BALB/c	191
FTL_0113	iglC	Intracellular growth	STM	BALB/c	191
FTL_0113, FTL_1159	iglC1, iglC2	Intracellular growth locus C	Ar	J774A.1, PM, PEC, BALB/c	82, 117
FTL_0133	feoB	Ferrous iron transport protein	STM	BALB/c	191
FTL_0193	cyoC	Cytochrome o-ubiquinol oxidase subunit III	STM	BALB/c	191
FTL_0257	rpmJ	50S ribosomal protein L36	STM	BALB/c	191
FTL_0304	FTT1490	Na+/H+ antiporter	Tp	J774A.1	124
FTL_0337	FTT0843	Unknown	STM	BALB/c	191
FTL_0382	rocE	Amino acid permease	STM	BALB/c	191
FTL_0387	aspC1	Aspartate aminotransferase	STM	BALB/c	191
FTL_0395-FTL_0396	purMCD	Purine biosynthesis	Ar/In, Ar	J774A.1, THP-1, PM, A549, BALB/c	145, 146
FTL_0421	tul4	Lipoprotein, T-cell-stimulating antigen	STM	BALB/c	191
FTL_0428	parB	Chromosome partition protein B	STM	BALB/c	191
FTL_0430	FTT0910	Unknown	STM	BALB/c	191
FTL_0439	FTT0919	Hypothetical outer membrane protein	Tp	J774A.1, BALB/c	124
FTL_0439	FTT0918	Unknown	STM	BALB/c	191
FTL_0440	FTT0920	Transposase	STM	BALB/c	191
FTL_0456	rpsU1	30S ribosomal protein S21	STM	BALB/c	191
FTL_0483	glgB	Glycogen branching enzyme, GlgB; polysaccharide metabolism	Tp, STM	J774A.1, BALB/c	124, 191
FTL_0485	glgC	Glucose-1-phosphate adenylyltransferase	STM	BALB/c	191
FTL_0514	FTT1611	Unknown	STM	BALB/c	191
FTL_0519	minD	Division inhibitor ATPase	STM	BALB/c	191
FTL_0520	minC	Septum site-determining protein	STM	BALB/c	191
FTL_0525	fumA	Fumarate hydratase	STM	BALB/c	191
FTL_0540	lpxB	Lipid A-disaccharide synthase	STM	BALB/c	191
FTL_0544	FTT1564	Hypothetical protein	Tp	J774A.1, BALB/c	191
FTL_0552	pmrA	Two-component response regulator, orphan	Ar/In	J774A.1, PM, MH-S, BALB/c, C57BL/6	165
FTL_0584	fadB	Acyl coenzyme A binding protein	STM	BALB/c	191
FTL_0589	FTT1525c	Hypothetical membrane protein	STM	BALB/c	191
FTL_0592	wbtA	dTDP-glucose 4,6-dehydratase	Ar, Tp	J774A.1, BALB/c, BALB/cByJ	124, 160, 180, 191
FTL_0594	wbtC	UDP-glucose-4-epimerase	Tp	J774A.1	124
FTL_0597	wbtF	NAD-dependent epimerase, LPS modification	STM	BALB/c	191
FTL_0601	wbtI	Sugar transaminase/perosamine synthetase	Pm	BALB/c	115
FTL_0606	wbtM	dTDP-glucose 4,6-dehydratase	Tp	J774A.1	124
FTL_0616	rpoA2	DNA-directed RNA polymerase, subunit	STM	BALB/c	191
FTL_0617	bfr	Bacterioferritin	STM	BALB/c	191
FTL_0645	FTT1416c	Lipoprotein	STM	BALB/c	191
FTL_0663	FTT1400c	Unknown	STM	BALB/c	191
FTL_0706	FTT1238c	Hypothetical membrane protein	Tp	J774A.1, BALB/c	124
FTL_0723	FTT1221	Unknown	STM	BALB/c	191
FTL_0766	ggt	Gamma-glutamyltranspeptidase	Tp	J774A.1, RAW, THP-1, BMM, BALB/c	4, 124
FTL_0768	bipA	GTP-binding translational elongation factor Tu and G family protein	STM	BALB/c	191
FTL_0789	aspC2	Aspartate aminotransferase, amino acid biosynthesis	Tp, STM	J774A.1, BALB/c	124, 191
FTL_0803	FTT1152	Unknown	STM	BALB/c	191
FTL_0837	metIQ	d-Methionine transport protein (ABC transporter)	Tp, STM	J774A.1, BALB/c	124, 191
FTL_0838	metN	d-Methionine transport protein (ABC transporter)	Tp	J774A.1	124
FTL_0846	FTT1117c	Isochorismatase hydrolase family protein	STM	BALB/c	191
FTL_0878	FTT0610	DNA/RNA endonuclease family	Tp	J774A.1	124
FTL_0886	FTT0618c	Conserved hypothetical protein YleA	Tp	J774A.1, BALB/c	124
FTL_0891	tig	Molecular chaperone	STM	BALB/c	191
FTL_0892	clpP	ATP-dependent Clp protease subunit P	STM	BALB/c	191
FTL_0893	clpX	ATP-dependent Clp protease subunit X	STM	BALB/c	191
FTL_0894	lon	ATP-dependent protease Lon	STM	BALB/c	191
FTL_0899	hflX	Protease, GTP binding subunit	STM	BALB/c	191
FTL_0903	hflK	Protease modulator	STM	BALB/c	191
FTL_0928	elbB	DJ-1/PfpI family protein	STM	BALB/c	191
FTL_0950	rplY	50S ribosomal protein L25	STM	BALB/c	191
FTL_0960	sthA	Soluble pyridine nucleotide transhydrogenase	STM	BALB/c	191
FTL_1029	pilF	Type IV pilus lipoprotein	Ar	C3H/HeN	25
FTL_1030	rluB	Ribosomal large subunit pseudouridine synthase B	STM	BALB/c	191
FTL_1071, FTL_1478	guaA, guaB	Guanine biosynthesis	Ar/In	J774, BALB/c	170
FTL_1075	FTT1015	Unknown	STM	BALB/c	191
FTL_1096	FTT1103	Hypothetical lipoprotein	Tp	J774A.1, BALB/c	124, 191
FTL_1096	FTT1103	Lipoprotein	STM	BALB/c	191
FTL_1097	FTT1102	Macrophage infectivity potentiator	STM	BALB/c	191
FTL_1134	NA	Membrane protein	STM	BALB/c	191
FTL_1158, FTL_0112	iglB	Intracellular growth locus B	Ar	J774A.1	19
FTL_1225	FTT0975	Unknown	STM	BALB/c	191
FTL_1233	FTT0968c	Amino acid antiporter	STM	BALB/c	191
FTL_1240	aroG	Phospho-2-dehydro-3-deoxyheptonate aldolase	STM	BALB/c	191
FTL_1262	FTT0945	Chorismate family binding protein, aromatic amino acid and folate biosynthesis	Tp	J774A.1	124
FTL_1266	lipP	Lipase/esterase	STM	BALB/c	191
FTL_1273	bioF	8-Amino-7-oxononanoate synthase	STM	BALB/c	191
FTL_1274	bioC	Biotin synthesis	STM	BALB/c	191
FTL_1275	bioD	Dethiobiotin synthetase	STM	BALB/c	191
FTL_1328	fopA	Outer membrane-associated protein	STM	BALB/c	191
FTL_1354	FTT0759	Membrane protein	STM	BALB/c	191
FTL_1392	deaD	Cold shock DEAD box protein A	STM	BALB/c	191
FTL_1393	ppiC	Peptidyl-prolyl cis-trans isomerase or parvulin	STM	BALB/c	191
FTL_1404	rplT	50S ribosomal protein L20	STM	BALB/c	191
FTL_1414	capA	Transmembrane HSP60 family protein	STM	BALB/c	191
FTL_1414-FTL_1416	capACB	Capsule biosynthesis	Ar, Tp	J774A.1, BALB/c	124, 191
FTL_1415	capC	Capsular polyglutamate biosynthesis protein CapC	STM	BALB/c	191
FTL_1416	capB	Capsular polyglutamate biosynthesis protein CapB	STM	BALB/c	191
FTL_1419	cphB	Cyanophycinase	STM	BALB/c	191
FTL_1452	rpmA	50S ribosomal protein L27	STM	BALB/c	191
FTL_1458	secA	Preprotein translocase, subunit A	STM	BALB/c	191
FTL_1461	deoD	Purine nucleoside phosphorylase	STM	BALB/c	191
FTL_1473	uvrA	DNA excision repair enzyme, subunit A	STM	BALB/c	191
FTL_1474	greA	Transcription elongation factor	STM	BALB/c	191
FTL_1475	FTT1314c	Type IV pilus fiber building block protein	STM	BALB/c	191
FTL_1504	katG	Catalase	STM	BALB/c	191
FTL_1528	FTT0708	Major facilitator superfamily transport protein	STM	BALB/c	191
FTL_1542	migR	Transcriptional regulator	Ar	MDM	22
FTL_1553	sucC	Succinyl coenzyme A synthetase beta chain	STM	BALB/c	191
FTL_1554	sucD	Succinyl coenzyme A synthetase alpha chain	STM	BALB/c	191
FTL_1581	tivA	Hypothetical lipoprotein	Ar/In	CE, MDM	93
FTL_1583	xasA	Glutamate-aminobutyric acid antiporter, XasA; amino acid transport	Tp	J774A.1	124
FTL_1601	yibK	tRNA/rRNA methyltransferase	STM	BALB/c	191
FTL_1622	FTT0444	Multidrug transporter	STM	BALB/c	191
FTL_1623	FTT0443	Unknown	STM	BALB/c	191
FTL_1664	deoB	Phosphopentomutase	Ar/In	CE, MDM, DC, HEK-293	93
FTL_1670	dsbB	Disulfide bond formation protein, DsbB	Tp	J774A.1	124
FTL_1672	acrB	RND efflux pump	In, STM	BALB/c	15, 191
FTL_1678	FTT0101	Membrane protein	STM	BALB/c	191
FTL_1701	gplX	Fructose-1,6-bisphosphatase	Tp, STM	J774A.1, BALB/c	124, 191
FTL_1750	secE	Preprotein translocase, subunit E	STM	BALB/c	191
FTL_1771	pilT	Twitching motility protein PilT	Tp	C3H/HeN	25
FTL_1793	sodB	Fe-superoxide dismutase	Ar	BALB/c, C57BL/6, MH-S	9
FTL_1806	FTT0053	Major facilitator superfamily transporter	Tp	J774A.1	124
FTL_1832	FTT0029c	Unknown	STM	BALB/c	191
FTL_1865	tolC	Glutamate decarboxylase	Ar	C3H/HeN	77
FTL_1867	yegQ	Protease	STM	BALB/c	191
FTL_1912	rpsA	30S ribosomal protein S1	STM	BALB/c	191
FTL_1914	ripA	Hypothetical protein	Ar	J774A.1, TC-1, C57BL/6	72
FTL_1936	FTT0209c	Periplasmic solute binding family protein	STM	BALB/c	191
FTL_1947	yjjk	ABC transporter ATP binding protein	STM	BALB/c	191
FTL_R0003	16S rRNA	16S rRNA	STM	BALB/c	191
FTL_R0004	tRNA-Ile	tRNA-isoleucine	STM	BALB/c	191
FTT0890	pilA	Type IV pilus fiber building block protein	Recomb.	C57BL/6	65

^aAr, allelic replacement; In, insertion; Tp, transposon insertion; STM, signature-tagged mutagenesis; Recomb., direct repeat-mediated deletion.

^bMDM, monocyte-derived macrophages; PM, peritoneal macrophages; PEC, peritoneal exudate cells; CE, chicken embryos; DC, dendritic cells.

^cIntermediate attenuation phenotype.

TABLE 4.

F. tularensis subsp. tularensis genes involved in pathogenesis

Locus tag	Name	Function	Method(s)a	Cells/animals in which mutant strain is attenuatedb	Reference(s)
FTT0026c	fslE	Siderophore uptake	Tp, Ar	BALB/c	100, 159
FTT0056c	FTT0056c	Major facilitator superfamily transport protein	Tp	Hep G2	155
FTT0069c	FTT0069	Unannotated	Tp	BALB/c	100
FTT0107c	dsbB	Transposon mutant	Tp, Ar	C57BL/6	157
FTT0107c	dsbB	Disulfide bond formation protein	Tp	Hep G2, J774A.1	155
FTT0118	prfC	Peptide chain release factor 3	Tp	BALB/c	100
FTT0129	FTT0129	Major facilitator superfamily transport protein	Tp	Hep G2	155
FTT0132	glpA	Glycerol-3-phosphate dehydrogenase	Tp	BALB/c	100
FTT0141	rplA	50S ribosomal protein L1	Tp	BALB/c	100
FTT0156	FTT0156	Acid phosphatase	Tp	BALB/c	100
FTT0203c	purH	Bifunctional purine biosynthesis protein	Tp	Hep G2	155
FTT0204	purA	Adenylosuccinate synthetase	Tp	Hep G2	155
FTT0245	usp	Universal stress protein	Tp	BALB/c	100
FTT0250	ppdK	Phosphoenolpyruvate synthase/pyruvate phosphate dikinase	Tp	BALB/c	100
FTT0290	moxR	MoxR-like ATPase	Tp	BALB/c	100
FTT0425	asd	Aspartate semialdehyde dehydrogenase	Tp	BALB/c	100
FTT0435	ctv	Carbon-nitrogen hydrolase	Ar	BMDM, MH-S, BALB/c	123
FTT0444	tet	Drug:H+ antiporter-1 (DNA1) family protein	Tp	BALB/c	100
FTT0486	mutL	DNA mismatch repair enzyme with ATPase activity	Tp	BALB/c	100
FTT0588	aroA	3-Phosphoshikimate I-carboxyvinyl transferase	Tp	Hep G2	155
FTT0609	FTT0609	Unannotated	Tp	BALB/c	100
FTT0623	tig	Trigger factor protein (peptidyl-prolyl cis/trans isomerase), chaperone	Tp	BALB/c	100
FTT0626	lon	DNA binding, ATP-dependent protease LA	Tp	BALB/c	100
FTT0654	elbB	DJ-1/PfpI family protein	Tp	BALB/c	100
FTT0687c	hslU	ATP-dependent protease HslVU, ATPase subunit	Tp	BALB/c	100
FTT0862c	htpX	Zn-dependent protease with chaperone function	Tp	BALB/c	100
FTT0876c	aroC	Chorismate synthase	Tp	BALB/c	100
FTT0891	FTT0891	Conserved hypothetical membrane protein	Tp	BALB/c	100
FTT0893-FTT0894	purMCD	Purine biosynthesis	Ar	MDM, J774A.1, A549, BALB/c	146
FTT0918	FTT0918	Hypothetical protein	Ar	BALB/c	204
FTT1087c	rep	UvrD/REP superfamily I DNA and RNA helicases	Tp	BALB/c	100
FTT1103	FTT1103	Conserved hypothetical lipoprotein	Tp/Ar	Hep G2, BALB/c, C57BL/6	155, 157
FTT1120c	tgt	Queuine tRNA-ribosyltransferase	Tp	BALB/c	100
FTT1130c	cphA	Cyanophycin synthetase	Tp	BALB/c	100
FTT1158c	pilO	Type IV pilus glycosylation protein	Tp	BALB/c	100
FTT1179	bipA	GTP binding translational elongation, factor Tu and G family protein	Tp	BALB/c	100
FTT11802c	trpE	Anthranilate synthase component I	Tp	BALB/c	100
FTT1181c	ggt	Gamma-glutamyltranspeptidase	Ar, Tp	BALB/c, HepG2	100, 155
FTT1234	FTT1234	Choloylglycine hydrolase family protein	Tp	Hep G2	155
FTT1236	FTT1236	Hypothetical protein	Tp	Hep G2, J774A.1	155
FTT1244c	yfiO	Conserved hypothetical lipoprotein	Tp	Hep G2	155
FTT1278c	rnhB	RNase HII	Tp	BALB/c	100
FTT1310c	hflB	ATP-dependent metalloprotease	Tp	Hep G2	155
FTT1312c	uvrA	DNA excision repair enzyme, subunit A	Tp	BALB/c	100
FTT1356, FTT1711	idlD	Intracellular growth locus D	Ar	BALB/c	100
FTT1357c, FTT1712c	iglC2, iglC1	Intracellular growth locus C	Ar	BALB/c	204
FTT1358, FTT1713	iglB	Intracellular growth locus B	Ar	BALB/c	100
FTT1455	wbtI	Sugar transamine/perosamine synthetase	Ar	BALB/c	100
FTT1455	wbtI	LPS O-antigen synthesis	Tp	BALB/c	100
FTT1459c-FTN1461c	wbtDEF	LPS O-antigen biosynthetic cluster	Ar/In	J774A.1, BALB/c	200
FTT1490	FTT1490	Na+/H+ antiporter	Tp	Hep G2	155
FTT1542c	omp26	Protein of unknown function	Tp	BALB/c	100
FTT1561	kdtA	3-Deoxy-d-manno-octulosonic-acid transferase	Ar	BALB/c	100
FTT1631	glpX	Fructose 1,6-bisphosphatase II	Ar	BALB/c	100
FTT1640c	FTT1640	Activator of osmoprotectant transporter ProP, fragment	Tp	BALB/c	100
FTT1663	carA	Carbamoylphosphate synthase small chain	Tp	Hep G2	155
FTT1664	carB	Carbamoylphosphate synthase large chain	Tp	Hep G2	155
FTT1665	pyrB	Aspartate carbamoyltransferase	Tp	Hep G2	155
FTT1720c	purL	Phosphoribosylformylglycinamide synthase	Tp	BALB/c, Hep G2	100, 155
FTT1721	purF	Amidophosphoribosyltransferase	Tp	BALB/c, Hep G2	100, 155
FTT1724c	tolC	Outer membrane efflux protein	Tp	BALB/c	100
FTT1744c	ipdC	Indolepyruvate decarboxylase	Tp	BALB/c	100
FTT1762c	FTT1762c	Acetyltransferase protein	Tp	Hep G2	155
FTT1773c	trpB	Tryptophan synthase beta chain	Tp	BALB/c	100
FTT1782c	yjjK	ABC transporter ATP binding protein	Tp	BALB/c	100

^aAr, allelic replacement; In, insertion; Tp, transposon insertion.

^bBMDM, bone marrow-derived macrophages; MDM, moncyte-derived macrophages; HepG2, human hepatocellularcarcinoma cells; MH-S, murine alveolar macrophages.

The development of Francisella genetic tools has also allowed for the implementation of global mutagenesis strategies. These include transposon mutagenesis of F. novicida, the LVS, and type A strain Schu S4. In 2004, Kawula et al. described the use of Tn5 transposon-transposase complexes to create random, stable insertion mutations in the LVS chromosome (103). Another strategy, employed by Maier et al., involved the utilization of either a temperature-sensitive version of pFNLTP1 or a derivative of pFNLTP1 in which the origin of replication for Francisella had been removed as a delivery vehicle for Himar transposon mutagenesis (124, 126). This library has since been used to identify mutants of LVS defective for intracellular growth within macrophages (124). More recently, a number of negative selection strategies including transposon site hybridization and signature-tagged mutagenesis have been optimized for use with Francisella and employed to identify genes required for growth of F. novicida or LVS in mice (106, 191, 213). In 2007, Gallagher et al. completed a full-scale genomic mutagenesis of the F. novicida U112 genome in which each putative nonessential open reading frame had been interrupted, thereby contributing a comprehensive mutagenesis library available for analyses (76). In addition, a Tn5-based transposon mutagenesis system has also been developed (23). This system expands upon the utility of current transposon tools by allowing unmarking of the antibiotic resistance determinant following transposition into the genome and inclusion of promoterless lux and lacZ alleles for analyzing gene expression using reporter gene technology (23). Mutagenesis using this transposon system has allowed identification of genes from F. tularensis LVS that are required for inhibition of the respiratory burst in neutrophils and for intracellular growth or survival within this cell type (179). Additionally, this system was used to identify FevR, a novel regulator of iglB (22). Finally, useful genetic surrogates of F. novicida which lack all of the resident restriction-modification systems that otherwise act as a barrier to gene transfer have now been developed (75). Clearly, the ongoing genetic work with Francisella is rapidly expanding our understanding of Francisella pathogenesis, and it will no doubt lead to identification of key virulence mediators that can be exploited for the development of potential vaccines and therapeutics.

Virulence Factors

Developments in the field of F. tularensis genetics have played an important role in identifying the repertoire of virulence genes utilized by this organism during infection within the host. While the characterization of these determinants is still in its infancy, the large numbers of mutants that exhibit defects in virulence indicate that the pathogenic lifestyle of F. tularensis is both complex and multifaceted. Further characterization of these genes will be necessary to fully understand F. tularensis infection and the type of immunity required to control infection.

Surface structures. (i) Capsule.

F. tularensis is thought to encode a carbohydrate-rich capsule that may assist the bacterium in mediating resistance to various environmental and host-generated stresses. Capsule-negative mutants were first reported for F. tularensis LVS following mutagenesis with acridine orange (166). Mutants exhibited a rough rather than smooth colony phenotype on agar medium and failed to produce an electron-dense surface layer, which was readily observable on the wild-type parent by electron microscopy (166). Rough mutants of LVS were found to be more sensitive to killing by serum complement, and they exhibited a diminished capacity to cause disease in mice (166). Consistent with the potential importance of capsule production in virulence, removal of capsule-like material by exposure to hypertonic solutions of sodium chloride was also found to attenuate virulence of F. tularensis following infection of guinea pigs (89). A locus in Francisella (capBCA) containing genes with similarity to determinants encoding the poly-γ-d-glutamic capsule in Bacillus anthracis has recently been described (191). This locus has been shown to be essential for virulence of F. tularensis strains LVS and U112 in a murine model of tularemia using two independent negative-selection screens (191, 213). However, definitive demonstration that these determinants encode components of the capsule has not yet been realized.

(ii) LPS.

LPS is a major structural component of most gram-negative bacteria and is a predominant eptiope recognized by components of the innate immune system. LPS is comprised of a hydrophobic lipid A portion that anchors the structure to the outer membrane, an oligosaccharide core, and an O-polysaccharide chain that specifies antigenicity. Compared to other gram-negative bacteria such as E. coli, the LPS produced by F. tularensis, particularly by the more virulent subspecies including F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, elicits relatively low immunobiological activity (5, 167). While the exact reasons for this are unclear, the lipid A moiety of F. tularensis differs from that present in most other organisms (131, 150, 206). In particular, lipid A from Francisella lacks the 4′-phosphate moiety commonly found on the nonreducing glucosamine dimer in E. coli LPS and is hypoacylated compared to other LPS species. Wang et al. have shown that the 4′-phosphate is removed postproduction by the lipid A 4′-phosphatase LpxF (208). Deletion of lpxF leads to decreased virulence following intradermal inoculation in the footpads of mice (209). Interestingly, the deletion of lpxF also leads to retention of the 3′-O-acyl chain on the core glucosamine, possibly further contributing to increased immunogenicity (209). Several studies have established the importance of LPS in the virulence of F. tularensis. “Rough” LPS phase derivatives of type A strain Schu S4 (i.e., the blue-to-gray color variants observed under obliquely transmitted light and first described by Eigelsbach in 1951) are less virulent and less immunogenic in mice than their “smooth” wild-type counterparts (52). A similar pattern of reduced virulence and immunogenicity in mice and guinea pigs is also observed with gray variants of F. tularensis LVS (53). More recent studies have revisited the blue-to-gray phase variation in F. tularensis and the importance of LPS in resistance to bactericidal factors made by host cells (7, 31, 44, 56, 88). Finally, other studies have described LPS biosynthetic mutants and shown them to be attenuated for virulence in vitro and/or in vivo (114, 124, 155, 191, 213).

(iii) Type IV pili.

Pili are complex filamentous bacterial surface structures that have been shown to play an important role in numerous physiological processes in bacteria, including association of organisms with host cell surfaces and protein secretion (153). Pili are comprised of a main structural pilus shaft that contains a terminal adhesin that promotes interactions with specific surface-exposed host cell determinants. Analysis of genomic sequences from various Francisella strains indicates that these organisms contain clusters of genes that share homology with type IV pilus determinants in other species, including Neisseria meningitidis and Pseudomonas aeruginosa (64, 77, 109, 223). Pili have been observed on the surface of several Francisella species following growth in laboratory medium (77, 223). Importantly, disruption of genes predicted to encode pilus biosynthetic factors abolishes expression of these structures on the bacterial surface of Francisella (25, 65). Interestingly, a subset of pilus-like genes does not appear to be directly involved in pilus production but rather is required for protein secretion (86, 223). Expression of pilus biosynthetic genes from Francisella has also been shown to functionally complement pilus-negative strains of Neisseria gonorrhoeae with deletions in the analogous determinant (164). Several lines of evidence indicate that production of pili or expression of pilus-like genes is important for virulence by Francisella. Pilus-like genes are regulated by MglA (223), a well-established virulence determinant of F. tularensis. Disruption of specific pilus-like genes also attenuates the ability of Francisella to adhere to various host cell types (25) or grow intracellularly (223). Finally, mutations in pilus-like genes alter virulence of Francisella in a mouse model of tularemia (25, 65, 86, 223).

The FPI.

An FPI of approximately 30 kb is present in duplicate in virulent F. tularensis subsp. tularensis and F. tularensis subsp. holarctica and in single copy in F. novicida. The FPI is comprised of roughly 17 open reading frames (137), a majority of which have been shown to be required for aspects of F. tularensis pathogenesis. It is currently thought that several FPI genes encode components of a secretion system, similar to the type VI system recently described in other organisms (46, 122). Genes present in the FPI are arranged into two major transcriptional units (138) and are induced during intracellular growth of F. tularensis (29, 111, 212). FPI genes have also been shown to be regulated by other environmental conditions, including iron (22, 23, 47, 105, 113, 177) and hydrogen peroxide (80), and by several regulatory factors, including MglAB (21, 46, 85, 111, 177), SspA (26), PmrA (134, 165), FevR (20), and MigR (22). One major transcriptional unit encodes genes of the intracellular growth locus (i.e., iglABCD). Mutation within any of these genes attenuates growth of Francisella in macrophages, in insect cells, and/or in mice (78, 83, 100, 110, 117, 161, 173, 207). IglA and IglB are interacting cytoplasmic proteins and have been predicted to be involved in effector protein secretion (46). iglC encodes a 23-kDa hypothetical protein. Although the exact function of this protein has not yet been defined, iglC mutants generated in F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. novicida are highly attenuated for growth in vitro and in vivo (12, 78, 83, 110, 111, 117, 161, 173, 191, 198, 204, 213). In particular, IglC is required for the inhibition of phagosomal maturation and/or escape from the host phagosome by F. novicida (173). Finally, iglD has been shown to be essential for intracellular replication of Francisella in human monocyte-derived macrophages as well as for intracellular replication in mice (29, 100, 171).

pdpA and pdpB represent the first two genes in the second major transcriptional unit located within the FPI. pdpA and pdpB encode hypothetical proteins that are required for virulence of F. novicida in insect cells, in chicken embryos, and/or in mice (21, 138, 161, 176, 199, 213). While little is known regarding the role of PdpB, mutants with deletions in pdpA localize with markers of lysosomes following infection into host cells (176). This phenotype is reminiscent of that observed with iglC mutants, suggesting that PdpA may be required for phagosomal maturation modification and/or escape of F. tularensis from the phagosome. Francisella PdpA contains a motif in its N-terminal region that is characteristic of proteins that interact with components of the eukaryotic ubiquitin-proteosome pathway (176). However, this protein does not contain an identifiable secretion signal, it localizes to the cytoplasm in F. novicida, and it has not been detected inside host cells following infection by the bacterium (176). Thus, the roles of PdpA and PdpB and their localization during an infection currently remain undefined.

Transcriptional regulators. (i) MglAB.

MglAB (macrophage growth locus) was first identified in a screen for suppressors of acid phosphatase expression in F. novicida (12). mglA and mglB are organized in an operon and encode proteins exhibiting homology to SspA and SspB of E. coli and Haemophilus spp. (12). SspA is an RNA polymerase-associated protein in E. coli, and it has been shown to regulate expression of numerous genes in response to stress (95, 218). Mutations in either mglA or mglB attenuate growth of F. tularensis in macrophages (12), in mosquito cells (161), in amoebas (12, 111), in fruit flies (207), and in mice (12, 111). Microarray comparisons between wild-type strains and mglA mutants indicate that MglA functions as a global transcription factor in F. tularensis, regulating expression of more than 100 genes, including those within and outside of the FPI (21). Of note, disruption of mglA attenuates the ability of Francisella to inhibit phagosome maturation and escape into the host cell cytosol, likely due to regulation of genes, including iglABCD.

(ii) SspA.

SspA is a transcription factor in the Francisella genome that exhibits high homology to MglA. In F. tularensis LVS, both SspA and MglA associate with RNA polymerase (26). Furthermore, SspA and MglA directly interact, and association of MglA with RNA polymerase requires SspA (26). Targeted gene and global gene expression studies carried out using DNA microarrays indicate that SspA and MglA coregulate the same gene set in F. tularensis, providing further evidence that these proteins likely work together in combination with RNA polymerase to regulate gene expression (26).

(iii) PmrA.

pmrA encodes an orphaned response regulator that shares homology with the Salmonella enterica serovar Typhimurium response regulator PmrA (134). Response regulators (and their cognate histidine sensor kinases) function to regulate gene expression through phosphorylation-based signal transduction cascades initiated following exposure of the histidine kinase to extracytoplasmic signals. In general, F. tularensis encodes a paucity of two-component signal transduction systems and other transcription factors relative to other organisms of similar genome size. The consequences of pmrA disruption have been examined in both F. novicida and in F. tularensis LVS (134, 165). Mutants generated in either strain background are attenuated for virulence in macrophages and in mice. In addition, pmrA mutants of F. novicida are more sensitive to antimicrobial peptides than their wild-type counterparts (134). DNA microarray studies carried out with these mutants indicate that PmrA regulates a large group of genes, including those contained within the FPI (134, 165). However, PmrA does not appear to regulate MglA or vice versa (134).

(iv) FevR.

fevR (Francisella effector of virulence regulation) encodes a hypothetical protein and was identified in a screen of genes requiring MglA/SspA for their expression (20). FevR is required for replication of F. novicida and F. tularensis LVS in murine and human macrophages (20, 22), in the human epithelial cell lines HEp-2 and A549 (22), and in mice (20). While the mechanism(s) of FevR's activity remains unclear, fevR mutants of F. tularensis LVS remain trapped within a phagosome that retains late endosomal marker LAMP-1 but not lysosomal marker cathepsin D (22). fevR mutants are also compromised in their ability to block NADPH oxidase activity following infection into neutrophils (22). Based on these and other observations not discussed here, FevR has been postulated to act in parallel with MglA/SspA in a feed-forward loop to positively regulate virulence factors essential for Francisella pathogenesis, including genes within the FPI (20). fevR expression is also positively regulated by response regulator PmrA (134). Thus, expression of this regulatory determinant is complex and is mediated by multiple input signals.

(v) MigR.

migR (macrophage intracellular growth regulator) is a newly identified regulator of virulence gene expression in F. tularensis LVS (22). Isolated in a transposon mutagenesis screen for transcriptional activators of iglB, migR encodes a hypothetical protein containing a predicted AMP binding domain (22). F. tularensis LVS migR mutants are attenuated for growth in human monocyte-derived macrophages relative to their wild-type counterparts; however, they exhibit no growth differences following infection into human epithelial cell lines, including HEp-2 and A549 pneumocytes (22). In addition to its ability to regulate the igl locus in LVS, MigR is also a positive regulator of transcription factor fevR (22). However, it remains unclear whether this regulation is direct or indirect or whether the observed regulation on igl expression occurs indirectly through FevR.

Additional virulence factors.

Apart from the genetic determinants described above, a number of other virulence factors involved in Francisella pathogenesis have also been identified (Tables 2, 3, and 4). While a comprehensive discussion of these genes is beyond the scope of this review, it is possible to make several general observations regarding genes contained within this list and phenotypes of selected mutants carrying disruptions within these genes. First, advancements in genetic tools and procedures for doing allelic replacement and transposon mutagenesis have greatly expanded the number of mutants that have been generated from type A strains of F. tularensis. While the first report of a nonspontaneous mutant of a type A strain did not occur until 2005 (204), nearly 70 Schu S4 derivatives carrying targeted or randomly generated transposon insertions have been reported to date. Second, a large percentage of the genes identified in screens for virulence factors in the various F. tularensis subspecies encode hypothetical proteins with unknown function. This suggests that many of the mechanisms utilized by this organism to infect, be maintained, and/or cause disease within its host are likely to be novel. Third, there is a high correlation between the requirement of a given gene for intracellular growth and/or survival and its requirement for F. tularensis to cause disease in vivo. While there have been exceptions to this rule, the ability of F. tularensis to infect cells, alter phagosome trafficking, egress into the host cell cytosol, and undergo extensive multiplication is an essential part of the overall life cycle of this organism. Finally, phenotypes observed following the disruption of a given gene in one Francisella species or subspecies do not necessarily correlate with the phenotype observed in another strain. This characteristic will be particularly important as more and more mutant strains are generated and target genes characterized for their potential utilization as vaccine targets.

DEVELOPING A FRANCISELLA VACCINE

Early Studies

Foshay vaccine.

As the potential for weaponization of F. tularensis has been realized for some time, efforts to develop a Francisella vaccine have been ongoing since the 1940s. Early efforts involved preparations of killed whole-cell F. tularensis. However, these materials offered limited efficacy in both human and animal studies (67). Administration of such preparations resulted in poor or incomplete protection in most cases and in adverse side effects that ultimately limited utility (205). A vaccine developed by Foshay et al. using acid extraction and preservation in phenol was less reactogenic and was used to vaccinate several thousand volunteers in Ohio between 1933 and 1941 (67). However, due to the limited sample size and inadequate reporting of infection in the general population, the effectiveness of this vaccine could not be directly determined (67). The Foshay preparation was able to protect nonhuman primates from death after challenge with 740 CFU of Schu S4, but in many cases it was unable to prevent disease symptoms (84). Further studies with mice indicated that the Foshay vaccine offered only low levels of protection against highly virulent strains (67, 99).

LVS.

The isolation of an LVS in Russia and its subsequent transfer to the United States in the 1950s constituted a significant breakthrough in vaccination against Francisella. LVS was developed by multiple passages in vitro and in vivo of a virulent F. tularensis subsp. holarctica strain (201). This strain was gifted to the United States in 1956 and was approved as an investigational new drug by the Federal Drug Administration (FDA) in the early 1960s (39a). Early evaluation of LVS as a live vaccine was performed in human volunteers by the U.S. Army in the late 1950s (174, 175). LVS was given by scarification for evaluation against subsequent subcutaneous or aerosol challenges with F. tularensis type A strain Schu S4. Vaccination with LVS protected against high-dose (1,000 CFU) subcutaneous and low-dose (10 to 100 CFU) aerosol challenge with Schu S4 but did not confer significant protection against aerosol challenge with doses approaching 1,000 CFU (174, 175). Improved efficacy of LVS was observed following vaccination of volunteers via the respiratory route; however, LVS was capable of inducing human tularemia in a subset of volunteers when given at the dose necessary to confer immunity (92). Regardless, LVS was used to vaccinate laboratory workers at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRID), after which the incidence of laboratory-acquired tularemia decreased significantly. It remains unclear whether a part of this decrease may also have resulted from improved laboratory practices (24). Despite its successes, the unknown nature of its attenuation and residual virulence following vaccination by the aerosol route has limited the overall utility of LVS and resulted in its removal from the Investigational New Drug list by the FDA. Consequently, it remains unlicensed for use in the United States. Even so, LVS remains the benchmark to which other Francisella vaccine candidates are compared.

Subunit Vaccines

Efforts toward developing subunit vaccines against F. tularensis have met with limited success (Table 5). Early attempts to identify protective antigens were based on identification of immunoreactive surface-associated proteins. While these proteins were found to stimulate in vitro responses using T cells from LVS-vaccinated animals or individuals (79, 168, 185, 193), the level of in vivo protective immunity conferred by some of these immunoreactive complexes in mice was below that observed with LVS vaccination (79). Other surface-exposed antigens, including carbohydrates and LPS, have also been investigated as to their potential to stimulate an immunoreactive and/or immunoprotective response (42, 73, 74, 169, 200). While immunization with LPS has been shown to generate a protective antibody response to challenge with less virulent Francisella species or subspecies of F. tularensis, it provides only limited protection against high-dose type A challenge (42, 73, 74, 200). A number of specific immunoreactive protein and lipoprotein antigens have also been investigated, including GroEL, KatG, and Tul4. Despite their immunogenicity, evaluations of these and other protein antigens have yet to demonstrate significant protection as subunit vaccine candidates (39a, 73, 79). These results, along with the failure of the Foshay preparation years before, indicate that generation of protective immunity against F. tularensis likely requires recognition of multiple antigenic determinants capable of stimulating both humoral and cell-mediated immune responses. Consistent with this observation, administration of an LVS boost following primary vaccination with LPS provides almost complete protection in mice against 1,000 50% lethal doses (LD₅₀s) of type A F. tularensis, a level of protection better than that seen with LVS alone (74). More recently, Huntley et al. have demonstrated that vaccination with a mixture of outer membrane preparations from LVS provides partial protection against 40 CFU of type A Schu S4 delivered via the respiratory route (94). Given these recent advances and the reduced risks associated with subunit preparations relative to other vaccination types, development of protective subunit vaccines for F. tularensis remains a plausible goal.

TABLE 5.

Francisella subunit vaccine candidates

Antigen	Adjuvant	Dose, μg (route)a	Challenge strain	Boost	Challenge route (dose, CFU)b	Host mouse strain	Protection	Reference
Ethanol-inactivated LVS	Freund's adjuvant	20 (i.p.)	Schu S4	No	i.n. (40)	C3H/HeN	Yes	94
LPS	Freund's adjuvant	20 (i.p.)	Schu S4	No	i.n. (40)	C3H/HeN	No	94
Outer membrane proteins	Freund's adjuvant	20 (i.p.)	Schu S4	No	i.n. (40)	C3H/HeN	Yes	94
LPS	Freund's adjuvant	50 (s.c.)	LVS	Yes	i.p. (2.5 × 106)	BALB/c	Yes	73
OMP	Freund's adjuvant	100	LVS	Yes	i.p. (2.5 × 106)	BALB/c	Yes	73
LPS	Bovine serum albumin	20 (s.c.)	Type B no. 108	No	i.d. (80)	BALB/c	Yes	42
					Aerosol (30)	BALB/c	No	42
			Type A no. 33	No	i.d. (60)	BALB/c	No	42
					Aerosol (50)	BALB/c	No	42

^ai.p., intraperitoneal; s.c., subcutaneous.

^bi.d., intradermal; i.n., intranasal.

Live Vaccines

The utility of live attenuated strains as vaccines has been demonstrated for a number of bacterial pathogens and has resulted in the development of commercially available vaccines for Salmonella enterica serovar Typhi (Ty21a), Vibrio cholerae (CVD 103-HgR), and Mycobacterium tuberculosis (Mycobacterium bovis BCG). A live vaccine typically contains one or more defined genetic lesions in pathways that are essential for in vivo growth of the bacterium. Vaccination with these strains is advantageous, as the organism is generally unable to survive for extended periods or at high levels within host cells but remains capable of expressing its repertoire of antigenic determinants. Conversely, an inherent caveat of working with live vaccines is that they must strike an intricate balance between attenuation and stimulation of immunity. Bacterial derivatives that are overattenuated may not prime substantive immunity. On the other hand, derivatives that are able to prime substantive immunity may retain partial virulence, undermining their use as a safe vaccination tool. Despite its limitations, the successes of LVS as a vaccine suggest that developing a rationally attenuated Francisella strain is both feasible and warranted.

F. novicida.

Many of the studies examining the initial feasibility of defined F. tularensis mutants as live attenuated vaccine candidates have been conducted using F. novicida as a surrogate organism (Table 6). Mutant derivatives examined have included those carrying gene disruptions in the FPI (iglC and pdpB), in metabolic pathways (purA, purF, carB, and fumA), in transcription factors (mglA, and pmrA), in acid phosphatases (acpABCH), in LPS biosynthesis (fmlK), in disulfide bond formation (dsbB), and in other, as-yet-uncharacterized processes. While results from these studies indicate that many of the generated mutants are able to prime robust immune responses in mice following vaccination, host responses generated to these mutants, or even to sublethal concentrations of the wild-type F. novicida parent, have led to protection only against challenge with the homologous species (Table 6). To date, no F. novicida mutants have been shown to confer adequate protective immunity against challenge with virulent type A or type B F. tularensis isolates (Table 6) (134, 158, 182). This suggests that despite the high level of genetic similarity between the Francisella species, F. novicida exhibits significant differences either in immunodominant antigens or in the profile of immunological responses that it elicits relative to the more virulent subspecies. Alternatively, the in vivo lifestyle of F. novicida may differ in some as-yet-unapparent aspect from that of its more virulent type A or type B counterparts.

TABLE 6.

Francisella live vaccine candidates

Species or subspecies	Locus tag for attenuating mutation	Annotation	Vaccination routea (dose, CFU)	Challenge strain	Boost	Challenge route (dose, CFU)	Host mouse strain(s)	Protection	Reference
F. novicida	None (wild type)		i.d. (100)	FSC033	No	Aerosol (10)	BALB/c	No	182
	FTN_1645	atpC	i.n. (149)	U112	No	i.n. (25)	C57BL/6	Yes	106
	FTN_0178	purA	i.p. (3.3 × 106)	U112	No	i.p. (170)	BALB/c	No	158
	FTN_1700	purF	i.p. (6.95 × 103)	U112	No	i.p. (170)	BALB/c	Yes	158
				Schu S4	No	i.p. (100)	BALB/c	No	158
	FTN_1465	pmrA	i.n. (106)	Schu S4	No	i.n. (100)	BALB/c	No	134
				U112	No	i.n. (106)	BALB/c	Yes	134
	FTN_1608	dsbB	i.n. (6 × 105)	U112	No	i.n. (6 × 107)	BALB/c	Yes	199
	FTN_0714	FTT0742	i.n. (6 × 107)	U112	No	i.n. (6 × 107)	BALB/c	Yes	199
	FTN_1310	pdpB	i.n. (6 × 107)	U112	No	i.n. (6 × 107)	BALB/c	Yes	199
	FTN_0337	fumA	i.n. (6 × 105)	U112	No	i.n. (6 × 107)	BALB/c	Yes	199
	FTN_0020	carB	i.n. (6 × 103)	U112	No	i.n. (8 × 105)	BALB/c	Yes	199
	FTN_1322	iglC	i.n. (106)	U112	No	i.n. (103)	BALB/c	Yes	142
						i.n. (104)	BALB/c	Yes	142
						i.n. (105)	BALB/c	No	142
	FTN_1322	iglB	i.n. (106)	U112	No	i.n. (103)	BALB/c	Yes	39
				LVS	No	i.n. (3 × 104)	BALB/c	Yes	39
			Oral (103)	Schu S4	Yes	i.n. (52)	BALB/c	No	39
	FTN_1290	mglA	Aerosol (105)	U112	No	Aerosol (35-8 × 104)	BALB/c, C57BL/6	No	214
	FTN_0050, FTN_1556, FTN_1061, FTN_0954	acpABCH	i.n. (103)	U112	No	i.n. (106)	BALB/c	Yes	135
			i.n. (106)	U112	No	i.n. (106)	BALB/c	Yes	135
	FTN_0090, FTN_1061, FTN_0954	acpCH or acpACH	i.n. (103)	U112	No	i.n. (103)	BALB/c	No	135
	FTN_0546	flmK	s.c. (400-500)	U112	No	s.c. (660)	BALB/c, C57BL/6	Yes	101
			Aerosol (100)	U112	No	i.n. (50)	BALB/c	Yes	101
F. tularensis subsp. holarctica	None (wild type)		i.n. (200)	Schu S4	No	i.n. (100)	BALB/c	Yes	146
						i.n. (2,000)	BALB/c	No	146
					Yes	i.d. (100, 2,000)	BALB/c	Yes	146
	FTL_0094	clpB	i.p. (13)	LVS	No	i.p. (1 × 104)	BALB/c	Yes	129
			i.p. (1,300)	LVS	No	i.p. (1 × 104)	BALB/c	Yes	129
	FTL0395-FTL0396	purMCD	i.n. (106)	Schu S4	No	i.n. (100, 2,000)	BALB/c	No	146
						i.d. (100, 2,000)	BALB/c	No	146
					Yes	i.n. (100)	BALB/c	Yes	146
						i.n. (2,000)	BALB/c	No	146
						i.d. (100, 2,000)	BALB/c	Yes	146
	FTL0552	pmrA	i.n. (105)	Schu S4	No	i.n. (102)	C57BL/6, BALB/c	Yes	165
	FTL0421	lpnA	i.d. (105)	LVS	No	i.d. (107)	C3H/HeN	Yes	62
	FTL_1071	guaA	(2.2 × 107)	LVS	No	2.8 × 105	BALB/c	Yes	170
	FTL_1478	guaB	(3.6 × 107)	LVS	No	2.8 × 105	BALB/c		170
	FTL_1793	sodB	i.n. (5,000)	Schu S4	No	i.n. (14 × 103)	C57BL/6	Yes	9
			i.n. (500)	LVS	Yes	i.n. (1.3 × 106)	C57BL/6	Yes	9
				Schu S4	Yes	i.n. (104)	C57BL/6	No	9
	FTL_0592	wbtA	i.d. (1.5 × 108)	FSC 108	Yes	i.d. (17)	BALB/c	Yes	180
				Schu S4	Yes	i.d. (10)	BALB/c	Yes	180
F. tularensis subsp. tularensis	FTT0107c	dsbB	i.n. (6.8 × 103)	Schu S4	No	i.n. (13-13,000)	C57BL/6	No	156
	FTT0893-FTT0894	purMCD	i.n. (104)	Schu S4	No	i.n. (100, 2,000)	BALB/c	No	146
						i.d. (100, 2,000)	BALB/c	Yes	146
					Yes	i.n. (100)	BALB/c	Yes	146
						i.n. (2,000)	BALB/c	No	146
						i.d. (100, 2,000)	BALB/c	Yes	146
	FTT0918	FTT0918	i.d. (105)	FSC033	No	i.d. (500)	BALB/c	Yes	204
						Aerosol (10)	BALB/c	Yes	204
	FTT1357c, FTT1712c	iglC, iglC1	i.d. (106-107)	FSC033	No	i.d. (500), aerosol (10)	BALB/c	No	204
	FTT1459c-FTT1461c	wbtDEF	s.c. (105)	Schu S4	No	s.c. (100)	BALB/c	No	200
	FTT1103	FTT1103	i.n. (1.6 × 108)	Schu S4	No	i.n. (1,000)	C57BL/6, BALB/c	Yes	157

^ai.d., intradermal; i.n., intranasal; i.p., intraperitoneal; s.c., subcutaneous.

Infection-vaccination studies with specific F. novicida mutants have also provided important insights into characteristics that will be necessary for successful F. tularensis vaccine development. Mutants unable to escape from the phagosome (i.e., iglC and mglA mutants) do not function well as live vaccine candidates even though they are extremely attenuated and can be administered at high doses in vivo (Table 6) (142, 214). This suggests that replication within the host cell cytosol is essential for priming a protective immune response against Francisella. The variable effectiveness of metabolic mutants as vaccine candidates also indicates that limited intracellular (or extracellular) replication is an important criterion for generating a protective immune response. For example, F. novicida mutants disrupted in purA, a gene involved in the early steps of purine biosynthesis, are unable to protect against homologous challenge (158). In contrast, protective immunity against homologous challenge is observed following vaccination of mice with mutants defective in a later step of the purine biosynthetic pathway (i.e., purF) (158). It is likely that differences in the ability of these mutants to persist in host tissues contribute to this phenotype.

F. tularensis subsp. holarctica.

While LVS remains problematic as a host for vaccine development for several reasons, its previous success as a live vaccine against type A F. tularensis challenge indicates that it may be a more suitable surrogate for evaluating the efficacies of specific genes as vaccine targets. In this regard, a variety of mutants have been generated in the LVS background and examined for their ability to confer protective immunity against challenge with type A or type B strains (Table 6) (10, 62, 129, 145, 146, 165, 170, 180). These have included LVS derivatives mutated in the clpB heat shock protein gene, purine biosynthetic genes purMCD and guaAB, response regulator gene pmrA, predicted lipoprotein gene lpnA, superoxide dismutase gene sodB, and LPS biosynthetic gene wbtA (Table 6). In general, results from these studies indicate that vaccination with sublethal doses of wild-type LVS or LVS-derived mutants confer high levels of protective immunity against challenge with wild-type LVS (10, 62, 129, 145, 170). Vaccination with these derivatives also provides variable protection against virulent type A or type B strains when administered by the parenteral route (146, 180) and against relatively low doses of Schu S4 delivered via the respiratory route (10, 146, 165). Not surprisingly, mice vaccinated with LVS mutants are not well protected against respiratory infection with higher doses of type A or type B F. tularensis (10, 146), and this level of protection is similar to that observed following sublethal vaccination with wild-type LVS (146).

F. tularensis subsp. tularensis.

It has been speculated that the limited protection offered by LVS or its derivatives against aerosol challenge with type A strains of F. tularensis might be related to differences in protective antigens and could be countered by vaccination with an attenuated type A strain (39a, 40, 84, 204, 221). Alternatively, the types of immune responses elicited or cell types encountered during type A pulmonary infection may be different than those generated with other subspecies. Consistent with this idea, Wu et al. have observed that boosting with sublethal doses of the type A strain NMFTA after LVS vaccination affords mice superior protection against NMFTA challenge compared to simply boosting with LVS (221). While only a limited number of F. tularensis subsp. tularensis mutants have been evaluated for their potential as live attenuated vaccine candidates (Table 3), results from these studies have indicated that it is possible to generate highly attenuated derivatives in this genetic background, including some that are able to provide low to modest levels of protection against virulent type A or type B isolates delivered by the respiratory route (146, 157, 204). However, given the highly infectious nature of type A strains of F. tularensis and the broad host range that these strains exhibit in nature, development of vaccine candidates that are safe, immunogenic, and highly protective will undoubtedly be a formidable task.

Heterologous hosts.

The inherent problems with the use of live attenuated Francisella vaccines, as well as the limitations of subunit/component vaccines, have led some groups to explore the use of heterologous bacteria to express Francisella antigens (73, 97, 186) (Table 7). Of the various outer membrane protein, lipoprotein, and virulence factor genes studied to date, only iglC expressed in a Listeria monocytogenes ΔactA mutant confers protection against challenge with type A or B F. tularensis (97).

TABLE 7.

Expression of Francisella antigens in heterologous hosts

F. tularensis antigen(s)	Host	Attenuating mutation(s)	Vaccination routea (dose, CFU)	Challenge strain	Boost	Challenge routeb (dose, CFU)	Host mouse strain	Protection	Reference
IglC	L. monocytogenes	ΔactA	i.d. (106-107)	LVS	Yes	i.n. (4,400)	BALB/c	Yes	97
				Schu S4	Yes	Aerosol (1 [10 LD50])	BALB/c	Yes	97
KatG	L. monocytogenes	ΔactA	i.d. (106-107)	LVS	Yes	i.n. (4,400)	BALB/c	No	97
				Schu S4	Yes	Aerosol (1 [10 LD50])	BALB/c	No	97
AcpA, Bfr, DnaK, GroEL, Pld	L. monocytogenes	ΔactA	i.d. (106-107)	LVS	Yes	i.n. (4,400)	BALB/c	No	97
Tul4	S. enterica serovar Typhimurium	Δasd, Δcya, Δcrp	i.p. (5 × 103)	LVS	No	i.v. (160-230)	BALB/cJ	No	186
FopA	S. enterica serovar Typhimurium	ΔaroA	i.v. (1 × 107)	LVS	Yes	i.p. (101-2.5 × 106)	BALB/c	No	73

^ai.d., intradermal; i.p., intraperitoneal; i.v., intravenous.

^bi.n., intranasal.

Respiratory versus parenteral vaccination.

A growing body of literature indicates that pulmonary vaccination may provide optimal protection against respiratory pathogens, rather than vaccination via the typical parenteral routes (39b, 92, 221). Aerosol delivery of a tularemia vaccine may be important for the stimulation of lung-associated lymph nodes and key immune mediators present in the lung. While inoculation of vaccines via the intradermal or intramuscular route typically induces systemic immune responses, vaccination via these routes often fails to induce substantial responses in mucosal tissues such as the lungs (130). This may in part explain the fact that while offering considerable protection against subsequent parenteral Schu S4 challenge, LVS delivered by scarification confers poor protection against respiratory challenge with Schu S4. An advantage of vaccination via the respiratory route is the generation of immune responses at local as well as at distal mucosal sites, thus generating the necessary response in lymphoid organs (130). Chen et al. demonstrated that intranasal vaccination of mice with LVS confers superior protection against subsequent aerosol challenge with 10 CFU of type A F. tularensis relative to that observed following vaccination with LVS via the intradermal route (28). Importantly, immunization via this route also maintains protective efficacy against intradermal challenge (28). These results are also consistent with findings by Wu et al. demonstrating that intranasal vaccination with LVS confers superior protection against subsequent intranasal challenge with type A strain NMFTA (221). These findings and others indicate that a live attenuated Francisella strain delivered via the respiratory route may provide optimal protection against subsequent aerosol challenge.

CONCLUDING REMARKS

F. tularensis is a highly virulent pathogen with the potential to cause severe disease in humans. Very few infectious bacteria have as broad a host range and the capacity to replicate to such high numbers in a variety of host tissues. Humans can be infected via a number of routes, resulting in a variety of disease manifestations. Inhalation of as few as 10 live organisms can result in potentially lethal disease. The biological weapons programs of several countries included F. tularensis as a bioagent due to its potential to cause debilitating disease. However, since the conclusion of these weapons programs, little work has been done to understand the genetic determinants required for Francisella disease and pathogenesis. The recent influx of investigators and funding initiatives in the Francisella field has lead to a number of advancements in the study of genetics, pathogenesis, and vaccine development for this organism. Continued support of initiatives in these areas will undoubtedly continue to uncover important new aspects of the F. tularensis lifestyle and/or the mechanisms by which this organism interacts with its host. Undoubtedly, these insights will prove crucial as studies into the development of a safe and protective Francisella vaccine continue forward.

As increasing amounts of literature examining vaccination strategies against virulent F. tularensis subspecies have become available, a picture of the characteristics necessary for protective immunity is emerging. It is likely that a successful vaccine candidate will need to induce aspects of both cell-mediated and humoral immunity. Both CD4⁺ and CD8⁺ T-cell responses are necessary for clearance of highly virulent strains, and induction of such responses is essential to generate F. tularensis immunity. Despite efforts to identify key antigenic mediators, it is evident that live attenuated vaccines may offer the best protection, as has been demonstrated in mouse infection models. Vaccination with attenuated derivatives of type A F. tularensis strains appears thus far to offer no significant advantage over vaccination with LVS, indicating that work toward developing a live attenuated vaccine candidate based on utilization of a type B background may be more suitable. Regardless of the subspecies utilized, it may be necessary to disrupt bacterial determinants involved in initial suppression of the host immune response. Particularly during respiratory infection, F. tularensis uses a number of strategies to limit the host response, allowing for significant bacterial proliferation and dissemination early after infection. It is likely no coincidence that respiratory tularemia is the most difficult disease manifestation to prevent with vaccination. Identification and elimination of F. tularensis immunosuppressive mediators in already-attenuated strains may also be a plausible strategy that leads to a safer and less reactogenic vaccine. Finally, the route of vaccination needs to be considered when developing vaccine candidates, as evidence indicates that respiratory vaccination may provide better protection against aerosol challenge. Despite difficulties in vaccinating against highly virulent F. tularensis strains, the multitude of vaccine candidates emerging as a result of recent advancements in F. tularensis genetics continues to yield important information regarding the elements necessary to elicit immunity against this highly virulent pathogen.

ADDENDUM IN PROOF

Presently, the genome for Francisella tularensis subsp. holarctica strain URFT1 (NZABAZ00000000) is undergoing shotgun sequencing.

Biography

Thomas C. Zahrt is an Associate Professor in the Department of Microbiology and Molecular Genetics and the Center for Biopreparedness and Infectious Diseases at the Medical College of Wisconsin. He received his B.A. from Kalamazoo College and his Ph.D. from the University of Illinois in Urbana-Champaign, and he conducted postdoctoral work at the University of Michigan Medical School in the laboratory of Dr. Vojo Deretic. Dr. Zahrt's interests include the study of intracellular bacterial pathogens and the regulatory factors utilized by these organisms to adapt within the host. In particular, Dr. Zahrt studies Mycobacterium tuberculosis and the importance of two-component signal transduction systems in the establishment and maintenance of this organism and in its reactivation from long-term persistent infection. Dr. Zahrt also studies Francisella tularensis pathogenesis, including the genetic programs necessary for growth and/or survival within tissues of the host, including macrophages and lung pneumocytes.

Roger Pechous obtained a B.S. in Biology from the University of Illinois at Champaign-Urbana and an M.S. in Biological Sciences from Illinois State University, analyzing the response of Staphylococcus aureus to antibiotic challenge in the laboratory of Dr. Radheshyam Jayaswal. He then obtained his Ph.D. in Microbiology and Molecular Genetics at the Medical College of Wisconsin in the laboratory of Dr. Thomas Zahrt, where he developed new genetic tools for Francisella tularensis and utilized these tools to generate live attenuated vaccine candidates and identify potential virulence determinants using in vivo expression technology. Dr. Pechous is currently a Postdoctoral Research Associate in the laboratory of Dr. Bill Goldman at the University of North Carolina at Chapel Hill, where he is studying the pathogenesis of primary pneumonic plague.

Travis McCarthy became interested in microbe/host interactions as an undergraduate student at Wartburg College. He earned his Ph.D. from the University of Iowa in the laboratory of Dr. Larry Schlesinger, where he studied the involvement of mycobacterial phosphomannomutases in surface-associated mannoglycoconjugate biosynthesis and the importance of these molecules in interactions with host macrophage receptors. Dr. McCarthy is currently a postdoctoral fellow at the Medical College of Wisconsin in the laboratory of Dr. Thomas Zahrt, where he is studying genetic programs utilized by Francisella tularensis to grow and/or survive inside macrophages and mice.