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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Environ Microbiol. 2013 Oct 21;16(2):350–358. doi: 10.1111/1462-2920.12290

Amoeba host-Legionella synchronization of amino acid auxotrophy and its role in bacterial adaptation and pathogenic evolution

Christopher T D Price 1, Ashley M Richards 1, Juanita E Von Dwingelo 1, Hala A Samara 1, Yousef Abu Kwaik 1
PMCID: PMC3946891  NIHMSID: NIHMS532757  PMID: 24112119

Summary

Legionella pneumophila, the causative agent of Legionnaires’ disease, invades and proliferates within a diverse range of free-living amoeba in the environment but upon transmission to humans the bacteria hijack alveolar macrophages. Intracellular proliferation of L. pneumophila in two evolutionarily distant hosts is facilitated by bacterial exploitation of conserved host processes that are targeted by bacterial protein effectors injected into the host cell. A key aspect of microbe-host interaction is microbial extraction of nutrients from the host but understanding of this is still limited. AnkB functions as a nutritional virulence factor and promotes host proteasomal degradation of polyubiquitinated proteins generating gratuitous levels of limiting host cellular amino acids. L. pneumophila is auxotrophic for several amino acids including cysteine, which is a metabolically preferred source of carbon and energy during intracellular proliferation, but is limiting in both amoebae and humans. We propose that synchronization of bacterial amino acids auxotrophy with the host is a driving force in pathogenic evolution and nutritional adaptation of L. pneumophila and other intracellular bacteria to life within the host cell. Understanding microbial strategies of nutrient generation and acquisition in the host will provide novel antimicrobial strategies to disrupt pathogen access to essential sources of carbon and energy.

Keywords: Legionella pneumophila, AnkB, amino acid auxotrophy

Introduction

Legionella pneumophila, the causative agent of Legionnaires’ disease and Pontiac fever, invades and proliferates intracellularly in a wide range of free-living amoebae, which normally feed on other bacteria, and is a constituent of complex biofilms in the aquatic environment (Rowbotham, 1980; Fields, 1996; Molmeret et al., 2005; Franco et al., 2009; Al-Quadan et al., 2012). Currently there are at least 14 species of amoebae, two species of ciliated protozoa and one species of slime mold that are capable of supporting intracellular replication of L. pneumophila (Rowbotham, 1980; Fields, 1996). The development of man-made water systems such as air-conditioners and cooling towers has expanded the environmental niche for L. pneumophila in association with their host amoebae, and consequently this organism is thought to become an accidental human pathogen. Inhalation of aerosolized water droplets containing L. pneumophila allows this organism to reach alveolar macrophages in the human lung where it invades and replicates rapidly similar to its life cycle within amoeba (Franco et al., 2009; Al-Quadan et al., 2012). Amoebae play a key role in the life cycle and pathogenesis of L. pneumophila and the ability of L. pneumophila to infect human macrophages is thought to be a consequence of prior adaptation to intracellular growth within various primitive eukaryotic hosts such as protozoa (Franco et al., 2009; Al-Quadan et al., 2012).

Sequestration of host nutrients from microbial access is one of the innate defense mechanisms against microbial invasion. Although microbial extraction of nutrients from the host is one of the most fundamental aspects of microbe-host interaction and clinical manifestation of diseases in mammals, our understanding of this crucial aspect is still very limited. Recent evidence indicates idiosyncrasies in novel microbial strategies to trigger generation of essential nutrients from the host to power bacterial proliferation (Alkhuder et al., 2009; Price et al., 2011; Niu et al., 2012; Winter et al., 2013). L. pneumophila, although auxotrophic for several amino acids including cysteine, preferentially metabolizes amino acids as the main sources of energy and carbon to power intracellular replication in amoeba and human cells (Pine et al., 1979; George et al., 1980; Reeves et al., 1981; Ristroph et al., 1981; Tesh and Miller, 1981; Tesh et al., 1983; Molofsky and Swanson, 2004; Wieland et al., 2005; Eylert et al., 2010). This organism has evolved an exquisite mechanism to promote host degradation of proteins to generate a plentiful supply of amino acids to power its intracellular replication in a diverse range of amoebal hosts and the evolutionarily distant human host (Al-Khodor et al., 2008; Price et al., 2009; Price et al., 2010b; Price et al., 2011). Interestingly, L. pneumophila has evolved with a remarkable synchronization of amino acid auxotrophy with many of its amoebal hosts and humans. Here we discuss how cysteine auxotrophy, high metabolic dependency of L. pneumophila on cysteine, and synchronization of L.pneumophila-amoebae amino acids auxotrophy has been a driving force in pathogenic evolution and nutritional adaptation of L. pneumophila to the intracellular life within the host cell. We discuss how diverse nutritional virulence strategies are employed by other pathogens to trigger host mechanisms to generate additional supplies of amino acids.

Establishment of the Legionella-containing vacuole

Upon invasion of amoeba or human macrophages, L. pneumophila immediately evades the default endosome-lysosome pathway, remodeling its phagosome into an endoplasmic reticulum-derived Legionella-containing vacuole (LCV) (Fig. 1) (Isberg et al., 2009; Al-Quadan et al., 2012; Luo, 2012). L. pneumophila facilitates this by translocating into the host cell ~300 effectors through its Dot/Icm Type IVB secretion apparatus (Fig. 1) (de Felipe et al., 2008; Isberg et al., 2009; Luo, 2011; Zhu et al., 2011). Although a myriad of effector proteins are injected, most do not have a detectable role in intracellular proliferation of L. pneumophila, suggesting potential functional redundancy (Isberg et al., 2009). Approximately 70 of the injected effector proteins contain eukaryotic-like domains including the ankyrin repeat, Sel1, F-box, SET, U-box and leucine rich repeats, which suggests L. pneumophila hijacks and manipulates various eukaryotic processes through molecular mimicry (Cazalet et al., 2004; Chien et al., 2004; Bruggemann et al., 2006; Cazalet et al., 2008; Isberg et al., 2009; Li et al., 2013; Rolando et al., 2013).

Figure 1.

Figure 1

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The intracellular lifecycle of L. pneumophila within its various amoebal hosts. 1) Infectious extracellular L. pneumophila expressing flagella attach to and are quickly engulfed by amoeba. 2) Upon invasion, L. pneumophila immediately translocates many effector proteins through its Dot/Icm type IV secretion system, diverting its vacuole away from the default endocytic pathway, avoiding the lysosome. The LCV matures into a rough ER-like vacuole through hijacking ER-to-golgi vesicular trafficking. 3) Once the LCV is established, L. pneumophila commences exponential growth and are non-flagellated. 4) During late stages of the infection, the bacteria escape into the cytosol and finish the last rounds of proliferation. During this stage, the bacteria respond to exhaustion of nutrients (such as amino acid and fatty acid) and trigger the proteins, RelA and SpoT, to synthesize ppGpp. This signals L. pneumophila to revert to its flagellated infectious form. Finally, L. pneumophila escape from the host amoeba into the extracellular environment whereby they can initiate another infection cycle of new amoeba cells in the aquatic environment. 5) Under nutrient starvation conditions, amoebae convert from the metabolically active trophozoite form to an inactive but highly resistant cyst form. L. pneumophila trapped within the cyst do not replicate and are protected from the harsh, nutrient poor extracellular environment. When nutrients are again abundant the cyst reverts back to the active trophozoite form and L. pneumophila can continue to proliferate.

Upon establishment of the LCV, L. pneumophila replicates rapidly within the LCV, but prior to egress from amoeba and human cells, the bacteria escape into the cytosol where they finish the last 1-2 rounds of proliferation (Fig. 1) (Molmeret et al., 2004; Al-Khodor et al., 2010a). In the host cell cytosol, L. pneumophila transits into the post-exponential (PE) phase, which is associated with dramatic phenotypic modulations that include motility and increased infectivity (Fig. 1) (Al-Khodor et al., 2010a). This dramatic phenotypic change occurs in the host cell cytosol, presumably upon exhaustion of amino acid and fatty acids (Potrykus and Cashel, 2008; Edwards et al., 2009). Limitation of these essential nutrients triggers expression of RelA and SpoT that synthesize the bacterial alarmone ppGpp, resulting in changes in gene expression leading to phenotypic modulation (Hammer and Swanson, 1999; Zusman et al., 2002; Dalebroux et al., 2009; Dalebroux et al., 2010).

Amino acid requirements of L. pneumophila

L. pneumophila relies on amino acids as the main source of energy and carbon, though carbohydrates may be a minor substrate (George et al., 1980; Ristroph et al., 1981; Tesh and Miller, 1981; Eylert et al., 2010). Even though L. pneumophila preferentially utilizes amino acids for carbon and energy, in silico analysis of the L. pneumophila genome sequence revealed that this organism is auxotrophic for the seven amino acids Arg, Cys, Ile, Leu, Met, Thr and Val (Cazalet et al., 2004; Chien et al., 2004). Furthermore, it has been shown that the amino acids Arg, Cys, Ile, Leu, Met, Ser, Thr and Val are essential for growth of L. pneumophila in vitro (Pine et al., 1979; George et al., 1980; Reeves et al., 1981; Ristroph et al., 1981; Tesh and Miller, 1981; Tesh et al., 1983; Molofsky and Swanson, 2004; Wieland et al., 2005; Eylert et al., 2010). The four amino acids, Arg, Cys, Gln and Ser are needed to support growth in vivo (Wieland et al., 2005). Cysteine is an absolute requirement for L. pneumophila to grow in vitro and rich media must be supplemented with ~3 mM cysteine (Carlsson et al., 1979; Hoffman et al., 1983). L. pneumophila metabolizes both serine and cysteine to pyruvate then to acetyl CoA to feed the TCA cycle, which is the main metabolic pathway for energy generation in L. pneumophila (Pine et al., 1979; Warren and Miller, 1979; Hoffman and Pine, 1982; Keen and Hoffman, 1984; Butler et al., 1985; Eylert et al., 2010).

The high demand for amino acids by L. pneumophila is further demonstrated by the presence of ~12 classes of ABC transporters, amino acid permeases, and several proteases (Cazalet et al., 2004; Chien et al., 2004). L. pneumophila elaborates several proteasese to host cells by using its type II secretion system and these proteases contribute to the ability of L. pneumophila to infect its amoebal hosts (DebRoy et al., 2006; Rossier et al., 2008; Tyson et al., 2013). The threonine transporter, PhtA, is essential for replication of L. pneumophila in macrophages (Sauer et al., 2005). The importance of the other transporters and proteases encoded by the L. pneumophila genome is currently unknown, but they are likely critical for successful nutrient acquisition and infection of diverse amoebal hosts. Amino acid transport across membranes in eukaryotes requires energy dependent transporters (Christensen, 1990). Therefore, for L. pneumophila to gain access to the amino acids pool in the host cell cytosol, this organism must recruit host amino acid transporters to the LCV membrane. Expression of the human amino acid transporter SLC1A5, which has broad susbtrate specificity for neutral and cationic amino acids including Cys, Gln, Iso, Leu, Met, Phe, Ser, Val, Trp and Tyr (Kekuda et al., 1996; Sloan and Mager, 1999), is induced in macrophages during infection by L. pneumophila (Wieland et al., 2005). Furthermore, replication of L. pneumophila in macrophages is dramatically reduced when SLC1A5 expression is silenced or its activity is chemically inhibited (Wieland et al., 2005). Even though SLC1A5 hasn’t yet been shown to localize to the LCV, it has been suggested by Wieland et al that SLC1A5 maybe be recruited to the LCV, enabling L. pneumophila to import amino acids from the host cytosol (Wieland et al., 2005). It is likely other host-cell amino acid transporters are localized to the LCV within both amoeba and human cells.

Triggering the host to generate excess amino acids by L. pneumophila

A constant for L. pneumophila during intracellular infection of its diverse amoebal hosts and human cells is the absolute requirement for amino acids and in particular cysteine, which is the most limiting amino acid in eukaryotes and is scarce in the environment. To overcome host limitation of cysteine and other amino acids, L. pneumophila hijacks the conserved polyubiquitination and proteasomal degradation machinery found in all eukaryotes, to elevate intracellular concentrations of free amino acids (Price et al., 2011). L. pneumophila achieves this by translocating the bona fide F-box effector, AnkB, via the Dot/Icm type IVB secretion system, into the host cell (Fig. 2). Of the ~300 effectors of L. pneumophila, AnkB is the only one indispensable for intracellular proliferation of L. pneumophila in both amoebae and human cells and for manifestation of pulmonary disease in the mouse model (Al-Khodor et al., 2008; Price et al., 2009; Lomma et al., 2010; Price et al., 2010a; Price et al., 2010b; Price et al., 2011).

Figure 2.

Figure 2

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AnkB of L. pneumophila exploits highly conserved eukaryotic pathways to generate a surplus of host amino acids in amoeba and macrophages. The AnkB effector is translocated into amoebae by the Dot/Icm type IV secretion system of L. pneumophila. AnkB is immediately farnesylated by three host enzymes FTase, RCE1, and ICMT, that are recruited to the LCV by the Dot/Icm system. Once AnkB is farnesylated, it is anchored into the cytosolic face of the LCV membrane where it interacts with the eukaryotic SCF1 ubiquitin ligase complex. The AnkB effector functions as a platform for the docking of K48-linked polyubiquitinated proteins to the LCV, which are subsequently degraded by the proteasome into 2-24 amino acid peptides that are rapidly degraded by oligo- and amino-peptidases. This generates a surplus of cellular amino acids within the cytosol of L. pneumophila-infected amoeba and macrophages. The amino acids are imported into the LCV through various host amino acid transporters present in the LCV membrane, potentially including the neutral amino acid transporter SLC1A5, and then into L. pneumophila through numerous ABC transporters and amino acid permeases such as the threonine transporter PhtA.

The eukaryotic domains of AnkB

AnkB harbors a number of eukaryotic domains that enables this protein to hijack the conserved polyubiquitination and proteasome degradation pathway. The N-terminus of AnkB harbors an F-box domain which directly interacts with the host SCF1 (Skp1, Cul1, F-box) E3 ubiquitin ligase complex (Schulman et al., 2000; Price et al., 2009; Ensminger and Isberg, 2010; Lomma et al., 2010). Directly downstream of the F-box domain are two 33-amino acid ankyrin repeat (ANK) domains, which mediate protein-protein interactions in eukaryotic cells (Mosavi et al., 2002; Al-Khodor et al., 2010b). The C-terminus of AnkB harbors a CaaX motif (C- cysteine, a- any aliphatic amino acid, X- any amino acid) that results in lipidation of AnkB by the host farnsyltransferase (FTase) in both amoebal and human cells (Ivanov et al., 2010; Price et al., 2010b; Al-Quadan and Abu Kwaik, 2011). Farnesyltransferase catalyzes the covalent linkage of a 15-carbon farnesyl lipid moiety to the conserved cysteine residue. The AnkB CaaX motif is further processed by the peptidase, RCE1, which cleaves the terminal three amino acids (aaX) downstream of the farnesylated cysteine residue and then the methyltransferase, ICMT, which methylates the now C-terminal cysteine residue (Price et al., 2010b). The combined activity of these three enzymes enables the otherwise cytosolic AnkB to embed into the outer leaflet of the LCV membrane facing the host cytosol, which is necessary for its biological function (Price et al., 2010b; Al-Quadan and Abu Kwaik, 2011).

In contrast to the 130b/AA100 strain, the C-terminus of AnkB in the Paris strain of L. pneumophila is truncated, resulting in deletion of the CaaX motif and therefore cannot be farnesylated (Lomma et al., 2010). Mutation of AnkB in either 130b/AA100 or Paris, leads to a similar intracellular growth defect in cells and disease manifestation in a mouse model of pulmonary disease, but the phenotype is more severe in the 130b/AA100 strain (Price et al., 2009; Lomma et al., 2010; Price et al., 2010b). Farnesylation of AnkB in strain AA100 is essential for its biological activity but the farnesylation motif is not present in strain Paris, which suggests functional differences between these two AnkB variants in the two strains (Price et al., 2010b). Perhaps strain Paris harbors another unknown mechanism that compensates for the loss of the AnkB CaaX motif that allows this effector to function correctly during intracellular infection, but the cellular location of the Paris AnkB protein during infection is not known.

Triggering amoeba and macrophages by L. pneumophila to generate a surplus of amino acids

Upon L. pneumophila infection of amoeba or human cells an orchestrated series of events occur leading to a rapid rise in cellular amino acid levels in the host cytosol (Fig. 2) (Price et al., 2011). L. pneumophila triggers AnkB translocation through the Dot/Icm Type IVB secretion system into host cell, where AnkB is immediately farnesylated and becomes embedded into the outer leaflet of the LCV membrane (Fig. 2). Here, AnkB likely recruits host proteins through its ANK domains which are then polyubiquitinated through interaction of the F-box domain with the SCF1 ubiquitin ligase (Fig. 2) (Price et al., 2009; Lomma et al., 2010; Price et al., 2010a). This generates an LCV that is densely decorated with polyubiquitinated proteins (Dorer et al., 2006; Price et al., 2009; Lomma et al., 2010), which can be detected immediately upon invasion of L. pneumophila into amoebae or human cells (Fig. 2).

Polyubiquitination of proteins is a conserved eukaryotic post-translational modification involved in a plethora of cellular processes including signaling, protein localization and protein turnover (Sadowski et al., 2012). Ubiquitination requires the sequential activity of three groups of enzymes, E1 the activating enzyme which transfers a 76-amino acid ubiquitin polypeptide to the conjugating enzyme, E2, and a E3 ubiquitin ligase, such as the SCF1 complex which covalently links ubiquitin moieties to the target protein (Sadowski et al., 2012). Proteins can be tagged with a polyubiquitin chain through the sequential addition of ubiquitin monomers to one of the seven lysine residues found in ubiquitin and the pattern of ubiquitin linkage dictates the fate of the tagged protein (Clague and Urbe, 2010; Sadowski et al., 2012). If a protein is tagged with a K48-linked polyubiquitin chain, the protein is targeted for proteolytic degradation by the proteasome (Clague and Urbe, 2010; Gallastegui and Groll, 2010; Sadowski et al., 2012).

AnkB embedded in the outer leaflet of the LCV membrane mediates a preferential accumulation of K48-linked polyubiquitinated proteins surrounding the LCV, which are ultimately degraded by the proteasome into small peptide fragments that are further degraded to free amino acids by cytosolic oligo- and aminopeptidases (Price et al., 2011). This leads to a rapid rise in amino acid concentrations in the host cell cytosol, which is essential for intracellular replication of L. pneumophila (Price et al., 2011). If the proteasome or the cytosolic aminopeptidases are inhibited, intracellular replication of L. pneumophila is blocked (Price et al., 2011). This indicates that L. pneumophila needs to actively elevate the basal levels of amino acids in the cytosol in both amoebae and human cells to power intracellular proliferation.

Promoting host proteasomal degradation to avert a starvation response by L. pneumophila within amoebae and macrophages

Upon invasion of amoebae or human cells, the ankB mutant of L. pneumophila immediately exhibits a starvation phenotype with elevated transcription of relA and spoT, which leads to increased production of the bacterial alarmone, ppGpp (Price et al., 2011). These changes are hallmarks of the transition of wild type L. pneumophila from the non-infectious replicative form to the motile and highly infectious non-replicative form upon exhaustion of nutrients, which normally occurs 16-18h post-infection (Molofsky and Swanson, 2004). As it is starved of nutrients from the outset of infection, the ankB mutant exhibits the infectious non-replicative phenotype (Price et al., 2011). The starvation phenotype and the intracellular and in vivo growth defect of the ankB mutant can be reversed within amoeba, human cells or in a mouse model of disease, through supplementation with an excess of an amino acids mixture, and strikingly with cysteine alone (Al-Quadan and Abu Kwaik, 2011; Price et al., 2011). These supplemented amino acids feed the L. pneumophila TCA cycle. Citrate or pyruvate, which directly feed the TCA cycle, are sufficient to restore intracellular growth to the ankB mutant, similar to cysteine supplementation (Al-Quadan and Abu Kwaik, 2011; Price et al., 2011).

AnkB is an intriguing example of a nutritional virulence factor (Abu Kwaik and Bumann, 2013) that is involved in the exploitation of multiple conserved eukaryotic host processes with the ultimate goal of overcoming host limitation of certain amino acids, providing an amino acid rich environment for L. pneumophila to proliferate within. This is essential to block a starvation response by Legionella as the basal levels of certain amino acids, such as cysteine, are not sufficient to power intracellular proliferation of the bacteria. Cysteine is the most limiting amino acid in humans and is considered semi-essential. Thus, AnkB directly links cellular microbiology and virulence of L. pneumophila to its basic metabolism through the TCA cycle in vivo to avoid a starvation response. It is fascinating that the eukaryotic farnesylation, polyubiquitination, and proteasomal degradation machineries are all highly conserved through the eukaryotic kingdom and all are exploited by AnkB to enable intracellular growth of L. pneumophila within evolutionarily distant hosts.

Synchronization of amino acid auxotrophy

L. pneumophila is an environmental organism that lives within complex biofilms or intracellularly within free living amoeba in aquatic environments where there is a dearth of simple nutrients like amino acids. Even though L. pneumophila absolutely requires amino acids to power intracellular growth, it is auxotrophic for Arg, Cys, Ile, Leu, Met, Thr and Val (Fig. 3) (Pine et al., 1979; George et al., 1980; Reeves et al., 1981; Ristroph et al., 1981; Tesh and Miller, 1981; Tesh et al., 1983; Molofsky and Swanson, 2004; Wieland et al., 2005; Eylert et al., 2010). Surprisingly, Acanthamoebae, a primary host amoeba of L. pneumophila in the aquatic environment, is auxotrophic for Arg, Ile, Leu, Met, and Val (Fig. 3) (Ingalls and Brent, 1983), which clearly overlaps with amino acid auxotrophy of L. pneumophila. Another amoebal host, Dictyostelium discoideum is auxotrophic for 11 amino acids that also overlap with L. pneumophila amino acid auxotrophy (Fig. 3) (Payne and Loomis, 2006). D. discoideum is not auxotrophic for cysteine, but if this amino acid is absent from in vitro culture media, both growth rate and yield of D. discoideum is significantly reduced (Franke and Kessin, 1977). Synchronization of amino acid auxotrophy by L. pneumophila with its natural amoebal hosts is unlikely to be coincidental.

Figure 3.

Figure 3

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Synchronization of amino acid auxotrophy between L. pneumophila and its eukaryotic hosts L. pneumophila, Acanthamoeba, Dictyostelium and humans show striking overlap in their amino acid auxotrophy. Acanthamoeba are auxotrophic for 5 of the same amino acids as L. pneumophila. Strikingly, Dictyostelium are auxotrophic for all of the essential L. pneumophila amino acids. Humans are auxotrophic for 5 of the same amino acids as L. pneumophila. Therefore, ability of L. pneumophila to acquire amino acids through proteasomal protein degradation is essential for bacterial pathogenesis. * L. pneumophila has the genetic capacity to synthesize serine, but serine is required for both in vitro and in vivo growth. † Humans and Dicytostelium are not auxotrophic for cysteine but this amino acid is the most limiting one in eukaryotic cells.

Interestingly, recent studies on other intracellular bacterial pathogens, such as Francisella tularensis and Anaplasma phagocytophilum, shows novel idiosyncratic microbial strategies to trigger generation of a surplus of certain limiting amino acids, and cysteine in particular (Alkhuder et al., 2009; Niu et al., 2012). Similar to L. pneumophila, F. tularensis and A. phagocytophilum are auxotrophic for cysteine, suggesting auxotrophy to amino acids that are very limiting within the host has driven pathogenic adaption and evolution of these organisms. It is instrumental that we understand the mechanisms that bacterial pathogens utilize to acquire essential nutrients, to develop more effective anti-microbial treatment and prevention strategies.

When nutrients are abundant, amoebae are in their metabolically active replicative trophozoite form where they grow and divide. However, when the environment is exhausted of essential nutrients such as amino acids, trophozoites differentiate into inactive highly resistant cysts (Weisman, 1976; Fouque et al., 2012). Amoebal cysts can re-differentiate back to the trophozoite form when the nutritional micro-environment is favorable for amoebal growth (Weisman, 1976; Fouque et al., 2012). Other hazardous stimuli such as osmotic stress and bacterial toxins also induce amoebal encystment (Weisman, 1976; Fouque et al., 2012). In the natural environment when nutrients become limiting, amoebae harboring L. pneumophila differentiate into cysts, forming a protective niche for intracellular bacteria. Since following encystation, amoebal protein synthesis is halted, proteasomal degradation of polyubiquitinated proteins is inhibited. The inability of L. pneumophila to obtain sufficient amino acids from the host likely halts bacterial proliferation and induces a bacterial starvation and survival program within the encysted amoebal host. We have shown that the ankB mutant of L. pneumophila, which cannot obtain sufficient amino acids to proliferate intracellularly, immediately triggers increased expression of RelA and SpoT that elevates ppGpp levels, which induces a starvation and survival program upon invasion of host cells (Price et al., 2011). Remarkably, the ankB mutant bacteria persist within host cells but do not replicate, however when a gratuitous supply of amino acids are added to cell culture media, even days post-initial infection, bacterial proliferation commences (Price et al., 2011). We postulate that this is analogous to what occurs to starved L. pneumophila found within encysted amoebae in the environment. Since encysted amoebae are resistant to invasion by L. pneumophila, it would be detrimental for L. pneumophila to continue proliferating within metabolically inactive cysts, and being released into an environment that lacks essential nutrients and susceptible amoebal hosts and other potentially hazardous conditions. Therefore, the evolution of amino acid auxotrophy to adapt to the amoebal host and relying on an active host proteasome pathway to acquire essential amino acids that are limiting in the host ensures persistence of L. pneumophila in the environment.

Conclusions

The synchronization of amino acid auxotrophy by L. pneumophila with its natural amoebal hosts and hijacking the conserved polyubiquitin and proteasomal degradation pathway represents a fascinating nutritional adaptation that facilitates persistence of L. pneumophila in the aquatic environment. Firstly, by not proliferating within starved amoebal cells and persisting in amoebal cysts, L. pneumophila prevents its own counter-selection if it were released into a nutrient poor environment where no susceptible trophozoites were present. Secondly, when environmental conditions become favorable, the ability of L. pneumophila to promote the major eukaryotic protein degradation pathway, which is highly conserved through evolution, ensures this organism can obtain essential amino acid nutrients in whatever eukaryotic host it invades. The reliance of L. pneumophila on amino acids obtained from the host cell may also represent its Achilles heel. This mechanism should be targeted to reduce L. pneumophila load in water systems such as air-conditioners and cooling towers, to reduce the risk of Legionnaires’ disease outbreaks.

Acknowledgments

YAK is supported by Public Health Service Awards R01AI43965 and R01AI069321 from NIAID and by the commonwealth of Kentucky Research Challenge Trust Fund. The authors declare no conflict of interest.

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