Subterfuge and Sabotage

Abstract

The development of a severe invasive bacterial infection in an otherwise healthy individual is one of the most striking and fascinating aspects of human medicine. A small cadre of gram-positive pathogens of the genera Streptococcus and Staphylococcus stand out for their unique invasive disease potential and sophisticated ability to counteract the multifaceted components of human innate defense. This review illustrates how these leading human disease agents evade host complement deposition and activation, impede phagocyte recruitment and activation, resist the microbicidal activities of host antimicrobial peptides and reactive oxygen species, escape neutrophil extracellular traps, and promote and accelerate phagocyte cell death through the action of pore-forming cytolysins. Understanding the molecular basis of bacterial innate immune resistance can open new avenues for therapeutic intervention geared to disabling specific virulence factors and resensitizing the pathogen to host innate immune clearance.

INTRODUCTION

Our innate immune system comprises a highly integrated and effective network of cells and effector molecules, standing ready to broadly defend us against infection in a complex environment of continual microbial exposures. Its many layers include the physical barrier functions of skin and mucosal epithelium, niche competition and antimicrobial products produced by the normal microflora, and soluble antimicrobial effectors such as host defense peptides, complement proteins, or reactive oxygen species (ROS). Sophisticated mechanisms intrinsic to the innate immune system allow the host to recognize molecular patterns of microbial origin, leading to activation and targeting of inflammatory responses. Of critical importance, specialized leukocytes such as neutrophils and macrophages undertake directed migration, phagocytosis, and production of microbicidal compounds to efficiently clear bacterial pathogens that threaten to gain access to deeper tissues.

Failures of innate immunity define the clinical field of infectious diseases. Often the cause is intrinsic to the human host, such as the developmental immaturity or senescence of immune functions at extremes of age; immunodeficiency due to genetic, acquired, or iatrogenic origin; breakdown in barrier integrity; high-risk behaviors or exposures; or general malnutrition or chronic illness. Yet in other cases, serious infection develops in previously healthy individuals, and physicians know to manage such patients by empirically targeting a short list of likely suspects. Among the most important human pathogens in this category are four coccoid-shaped, gram-positive bacterial species that consistently demonstrate the capacity to produce invasive and potentially life-threatening infections: Staphylococcus aureus, Streptococcus pneumoniae (SPN), group A Streptococcus (GAS), and group B Streptococcus (GBS). The unique pathogenic potential of these organisms is played against a background of normalcy: Each of these four bacteria colonizes the skin or mucosal surfaces of 10–30% of healthy individuals without producing symptoms. S. aureus, SPN, and GAS are also associated with a variety of mild, superficial infections.

S. aureus is currently the leading cause of serious bacterial infections in the United States and many other developed countries, with an ability to produce abscesses in every tissue and organ system. In recent decades, marked increases in disease caused by multidrug (methicillin)-resistant S. aureus (MRSA) have occurred in both health care and community settings, posing an ominous threat to public health (120). However globally, the most lethal bacterial pathogen remains SPN, estimated to cause ~10% of all deaths in children in the first 5 years of life (90). Invasive GAS infections produce a global burden estimated at more than 650,000 new cases and 160,000 deaths each year (17). Finally, GBS is the leading cause of bacterial pneumonia, sepsis, and meningitis in newborn infants in Europe, the Americas, and Australia, where universal maternal screening programs seek to guide intrapartum antibiotic prophylaxis (121). Among these invasive gram-positive pathogens, only SPN has a licensed preventive vaccine.

The propensity of S. aureus, SPN, GAS, and GBS to produce systemic infection in otherwise healthy individuals reveals a capacity of each pathogen to resist innate defense mechanisms that normally prevent microbial dissemination (Figure 1). Notably, in gaining access to the bloodstream and spreading to distant organs, the pathogen thwarts the sophisticated opsonophagocytic clearance function of neutrophils and macrophages. In this review, we examine the virulence mechanisms of these four invasive gram-positive pathogens to subvert the host phagocyte defense system, placing a special emphasis on recent discoveries validated through molecular genetic analysis, human tissue culture systems, and small animal models of infection.

Figure 1.

Inhibition of complement activation and interference with complement effectors by gram-positive pathogens. Gram-positive pathogens have multiple strategies to defend against complement activation and function, including (1) preventing the formation and activity of the C3 convertase, (2) proteasomal degradation of C3b, (3) blocking C3b deposition on bacterial surfaces, (4) preventing formation and activity of the C5 convertase, (5) proteasomal degradation of C5a, and (6) resistance to lysis by the membrane attack complex (MAC).

EVASION OF HOST COMPLEMENT DEPOSITION AND ACTIVATION

The complement system, though complex in its pathways of activation, provides two key outcomes for host innate immunity: opsonizing bacteria for phagocytosis and direct bacterial killing by membrane perturbations mediated by the membrane attack complex (MAC) (104). Activation of the complement system converges upon the formation of C3 convertases, which deposit the opsonin C3b on the bacterial surface. C3 conversion subsequently activates C5 convertases to generate C5a, a powerful chemoattractant for phagocytic cells, and C5b, which initiates the formation of the MAC (Figure 1). Although the MAC of the complement system localizes to the surface of gram-positive bacteria, it seems to have little effect on bacterial viability (12), and molecular details of the activity and function of the MAC on gram-positive bacterial surfaces are not known. Thus, the most effective functions of the complement system against gram-positive pathogens are to opsonize the bacteria via C3b and recruit phagocytes to the site of infection via C5a (Figure 1).

Complement activation is tightly controlled by endogenous regulators to limit inflammatory injury to host tissues. Nearly all of the important gram-positive pathogens express molecules that acquire the host fluid-phase inhibitory complement regulators factor H (34, 56, 93) or C4b-binding protein (C4BP) (2, 38) on the bacterial surface, allowing these regulators to interact with C3 convertases and accelerate their decay (104). Although S. aureus does not bind factor H or C4BP, it secretes proteins that themselves act as complement inhibitors in an analogous manner (104). The secreted staphylococcal complement inhibitor potently inhibits C3 convertase to prevent C3 conversion, phagocytosis, and C5a formation (103), and extracellular fibrinogen-binding protein and extracellular complement-binding protein bind to the C3b molecule directly to impair C3 convertase function (60).

Still other mechanisms prevent C3b deposition on the bacterial surface. Some GAS M proteins and S. aureus clumping factor A bind fibrinogen, which acts as a molecular shield to prevent C3b deposition (21, 45). GBS capsule and SPN serotypes 2 and 4 capsules impair bacterial opsonization with C3b by both the alternative (83) and classical complement pathways (53). Finally, S. aureus clumping factor binds the host complement inhibitory protein factor I, which cleaves C3b into iC3b (45), preventing further amplification of the complement cascade and activation via the alternative pathway.

Gram-positive pathogens additionally employ several proteolytic strategies to dispose of complement system components. Both GAS and GBS possess specific proteases capable of cleaving C5a (62, 113). Additionally, gram-positive species also elaborate broader-spectrum proteases that aid in the degradation of complement system proteins. Notable among these is the cysteine protease SpeB of GAS (50), S. aureus serine protease V8 (61), and the S. aureus metalloprotease aureolysin (73), which degrade key complement system components, including the opsonin C3b and the chemoattractant C5a. Finally, GAS streptokinase (81) and SPN PepO protein (3) can acquire and activate host plasminogen to plasmin on the bacterial surface to accelerate C3b degradation. SPN PepO protein binds plasminogen, allowing activation by urokinase-type plasminogen activator and subsequent plasmin-mediated cleavage of C3b (3). Though active plasmin can also generate fibrin, the accumulation of fibrin further prevents C3b deposition (21, 45) and additionally aids gram-positive bacteria in platelet-mediated adhesion to damaged endothelial surfaces (86a). Thus, gram-positive pathogens have evolved a repertoire of virulence factor proteins to combat the complement system. The redundancy of anti-complement factors and the multiple arms of the complement system that are targeted by these factors underscore the importance of complement defense for gram-positive bacteria to survive in blood and disseminate during invasive infection. Combating the complement defense strategies of gram-positive bacteria, while challenging, could prove to be a useful approach toward new drug therapies.

INHIBITION OF ANTIBODY-MEDIATED OPSONOPHAGOCYTOSIS

The phagocytosis of bacteria by host neutrophils and macrophages is markedly enhanced upon binding of specific antibody to allow Fc receptor-mediated uptake. Antibody binding to the bacterial surface also activates the classical pathway of complement through C1q binding to the Fc region of the immunoglobulin molecule. Pathogens can subvert antibody function through nonopsonic binding and antibody degradation.

One celebrated strategy by which gram-positive bacteria avoid recognition of antigen-bound immunoglobulins by both Fc receptors and C1q is to reorient the antibody molecule by binding to its Fc region, competitively inhibiting downstream Fc effector functions. Most notably, protein A, the most abundant protein on the surface of S. aureus, binds to the Fc gamma region of IgG to inhibit opsonophagocytic killing (67). M1 and protein H of GAS also bind immunoglobulin Fc, but the affinity of these interactions tend to be lower than antibody-antigen affinities, such that this strategy may only be viable in low Ig concentration environments (89).

Bacterial cleavage of the Ig molecule can thwart antibody binding and recognition. Certain purified bacterial proteases exhibit direct antibody cleavage activity, such as the V8 protease of S. aureus (99) and the GAS cysteine proteases SpeB (30) and Mac1/IdeS (75). However, detailed mutational and biochemical studies under physiological conditions raise a question as to whether native expression of the latter two proteases is sufficient to impede opsonophagocytosis (91, 95). Gram-positive pathogens can also coopt the activity of a host protease plasminogen to acquire antibody-degrading capacity. Staphylokinase activates plasminogen to plasmin, which can then cleave surface-bound immunoglobulins as well as C3b (105). Other gram-positive bacteria possess plasminogen-activating proteases (3, 81, 126, 128); though it has not been directly demonstrated for these proteases, one would predict the resulting activated plasmin could similarly degrade host antibodies.

Even if antibody successfully binds its antigenic target on the Gram-positive bacterial surface in the correct orientation, the pathogens have evolved additional mechanisms to thwart Fc-mediated effector functions. S. aureus microbial surface components recognizing adhesive matrix molecule (MSCRAMMs) can bind C1q in a manner that renders C1 complexes inactive (63), and SPN can use C1q to adhere to host epithelial and endothelial cells (1), concomitantly preventing its interaction with other complement proteins. The secreted GAS enzyme EndoS hydrolyzes the N-linked glycan of the constant region of the IgG heavy chain and blocks the antibody’s ability to interact with Fc receptors (111). Finally, the secreted S. aureus formyl peptide receptor-like 1 inhibitor (FLIPr) can bind Fc receptors on host phagocytes, thereby preventing their recognition of and binding to antigen-bound antibody (115).

RESISTANCE TO HOST OXIDATIVE BURST KILLING

Phagocytosis of bacterial particles induces the oxidative burst response, a key host microbicidal process in which oxygen is consumed and toxic ROS are generated. This pathway is catalyzed by two major reactions in the cell. The first reaction begins with activation and translocation of the NADPH oxidase (NOX) complex to the phagosome, producing superoxide (O₂⁻) that quickly dismutates to hydrogen peroxide (H₂O₂) (Figure 2). Both O₂⁻ and H₂O₂ can damage iron-sulfur clusters of dehydratases, releasing free iron (Fenton reaction), which when combined with H₂O₂ can generate hydroxyl radicals lethal to many bacteria (127). The second reaction involves the conversion of both O₂⁻ and H₂O₂ by the azurophilic granule enzyme myeloperoxidase (MPO), generating hypochlorite (HOCl) (127). It is thought that of all the ROS produced HOCl exerts the most bacteridal activity (127).

Figure 2.

Gram-positive pathogens interfere with ROS production and function. Engulfment of bacteria triggers NADPH oxidase (NOX)-dependent generation of superoxide. Bacterial superoxide dismutase (SOD) accelerates the generation of hydrogen peroxide from superoxide. Bacteria subsequently neutralize hydrogen peroxide by using enzymes that (1) convert peroxide to alcohol, (2) convert peroxide to molecular oxygen or (3) possess iron-containing molecules or generate nitric oxide to prevent Fenton chemistry. Depleting hydrogen peroxide prevents formation of highly bactericidal hypochlorite. Gram-positive bacteria additionally have molecules such as pigment, which acts as a molecular shield against superoxide anions. The most bactericidal ROS species are indicated in green.

To survive the oxidative burst response, gram-positive pathogens have mechanisms to either prevent the production of or dispose of harmful oxidants (129) (Figure 2). Invasive gram-positive species encode a bacterial superoxide dismutase (SOD) (65, 130) that accelerates conversion of O₂⁻ to H₂O₂. Bacteria must then further convert H₂O₂ to a less reactive molecule. Catalase is a common mechanism for gram-negative bacteria and S. aureus to oxidize H₂O₂ into molecular oxygen and water. However, all streptococci and even some strains of S. aureus are catalase-negative and must therefore deploy other systems to enzymatically remove H₂O₂ (129). Reducing pathways such as thioredoxin systems provide electrons to small molecules that can react with H₂O₂ (40, 108), whereas the tripeptide glutathione can reduce H₂O₂ directly (55). GAS additionally produces its own iron-binding protein (118), whereas S. aureus expression of bacterial nitric oxide (NO) synthase generates NO (44), both of which inhibit Fenton chemistry. The enzyme alkylhydroperoxidase can convert H₂O₂ to alcohol and water and is another means for gram-positive pathogens to eliminate H₂O₂ (129). Depletion of H₂O₂ thus reduces the opportunity for MPO to generate the more toxic HOCl species. Strategies to prevent H₂O₂ depletion may therefore improve infection outcomes. For example, the recent development of specific bacterial nitric oxide synthase (bNOS) inhibitors (48a) to counter S. aureus expression of bNOS that promotes ROS resistance (120a) opens up new antimicrobial possibilities.

In addition to the diverse enzymatic strategies to limit ROS generation, certain gram-positive pathogens have evolved antioxidant shields to enhance their intrinsic resistance to oxidant-mediated damage. The eponymous golden carotenoid pigmentation of S. aureus, the result of cell wall-associated carotenoid pigments 4′,4′-diaponeurosporene and staphyloxanthin, provides broad-based free radical scavenging properties and the ability for quenching single oxygen (80), increasing neutrophil resistance. GBS similarly produces a carotenoid pigment known as Granadaene that aids in ROS neutralization and impedes phagocyte killing (79). Pigment production therefore represents an important drug target to render the bacteria more sensitive to the killing mechanisms of the host. Recently, the indole derivative 7-benzyloxyindole was shown to inhibit staphyloxanthin synthesis, rendering S. aureus colorless and sensitive to ROS killing (74a). This chemical had an added feature of also blocking the bacterium’s expression of the toxin α-hemolysin (74a).

RESISTANCE TO PHAGOCYTIC GRANULE COMPONENTS

A critical element of bacterial killing within the phagocytic cell occurs when various intracellular granules fuse with the phagosome to deliver their cargoes of antimicrobial enzymes and peptides. Granule components include the cell wall-degrading enzyme lysozyme; serine proteases including cathepsin G, elastase, azurocidin, and proteinase 3; and small cationic antimicrobial peptides (AMPs), such as defensins and cathelicidin. Although the serine proteases have been demonstrated to have direct antimicrobial effects on gram-positive pathogens (114), the main antibacterial function of these proteases may revolve around processing of AMPs from inactive precursors to their mature form.

Gram-positive bacterial surfaces consist of a thick, dense, and complex layer of proteins and carbohydrate structures including teichoic acids, peptidoglycan, and capsule (Figure 3). Lysozyme, a muramidase present in phagocytic cells’ granules, cleaves peptidoglycan between β-1,4-linked N-acetyl glucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc). Lysosome can kill bacteria through cell wall degradation, which further generates peptidoglycan (PPG) fragments that can be recognized by pattern-recognition receptors (PRRs, see sidebar) such as NOD2 (35) and peptidoglycan recognition protein (25). Gram-positive pathogens have evolved mechanisms to PPG less recognizable to lysozyme, including PPG modification in SPN (122) and GAS (41), O-acetylation in SPN (32) and S. aureus (11), and masking of PPG with covalently linked wall teichoic acid (WTA) structures in S. aureus (8). Functionally, modification of PPG to impart lysozyme resistance results in not only evasion of detection by PRRs, but also limiting of host production of important inflammatory mediators such as interleukin (IL)-1β via the Nod-like receptor (NLR) family pyrin-domain-containing 3 (NLRP3) inflammasome (see sidebar) and interferon-β (64, 109).

Figure 3.

Gram-positive pathogens physically alter their surface to thwart antimicrobial granule contents. Gram-positive bacteria produce (1) Antimicrobial peptide (AMP) binding molecules or (2) AMP degrading enzymes, which prevent AMP access. The density and altered charge of molecules such as lipoteichoic acid (LTA), wall teichoic acid (WTA) and exopolysaccharide capsule further prevent AMPs from accessing the bacterial membrane (3). Modification of peptidoglycan (PPG) prevents cleavage and degradation by lysozyme (4).

AMPs such as cathelicidin LL-37 are small cationic molecules that form pores in or otherwise destabilize the bacterial membrane. Surface charge and access to the bacterial membrane are important factors in determining the potency of endogenous AMPs. Gram-positive bacteria achieve the first of these defenses by altering their surface charge, chiefly by biochemical modification of cell wall teichoic acids (Figure 3). Teichoic acids include both lipoteichoic acids (LTAs) and WTAs (14). All teichoic acids contain a negatively charged anionic backbone (14). Addition of d-alanyl esters to teichoic acids through the activity of enzymes encoded by the dlt operon can reduce the net negative charge of teichoic acids; accordingly, bacteria lacking the genes involved in d-alanylation are more susceptible to positively charged cationic AMPs such as human cathelicidin LL-37 and α-defensins (72, 96, 97). There is also evidence to suggest that d-alanylation prevents penetration and access of AMPs to the bacterial surface owing to increased surface density rather than charge (106). Alteration of teichoic acids with positively charged lysyl-phosphatidylglycerol also reduces AMP interaction with S. aureus (87).

Gram-positive bacteria, particularly GAS and S. aureus, also possess proteases that can cleave AMPs into nonfunctional breakdown products. S. aureus aureolysin (110) and the broad-spectrum cysteine protease SpeB of GAS (58) have been shown to cleave and inactivate human cathelicidin LL-37. GAS streptokinase can also activate plasmin on the GAS surface to degrade LL-37 (49), mirroring its protective action against C3b (81).

Another means of AMP resistance occurs through the expression of bacterial surface molecules that capture the host defense molecules, blocking them from reaching the cell membrane target of action. The hypervariable N-terminal domain of GAS M1 protein was recently shown to bind and sequester LL-37, preventing access to the bacterial membrane (74). The secreted GAS streptococcal inhibitor of complement (SIC), originally described as preventing formation of the complement MAC complex, is also capable of binding LL-37 to prevent bacterial killing (94). The binding of human neutrophil α-defensins into a complex by S. aureus staphylokinase impedes their bactericidal activity (57), and GBS surface pilus protein PilB captures cathelicidin to increase pathogen resistance (82). Finally, although the precise mechanisms remain to be elucidated, GBS penicillin-binding protein-1a and GAS hyaluronic acid capsule both increase bacterial AMP resistance in manners independent of surface charge alteration (29, 59), perhaps again acting as a molecular shield.

IMPEDING PHAGOCYTE RECRUITMENT AND ACTIVATION

A perhaps even more elegant suite of immune evasion strategies by which these pathogens limit detection and diminish the effectiveness of host innate immune clearance is emerging: virulence factors that block phagocyte recruitment and phagocyte activation to reduce bacterial exposure to antimicrobial molecules.

In addition to degrading the complement-derived chemoattractant C5a (62), GAS produces the protease SpyCEP, which specifically targets the important neutrophil chemokine IL-8 (132), whereas GBS produces a similar serine protease (CspA), which cleaves the related CXC chemokines Gro-α, Gro-β, and Gro-γ (15). Alternatively, gram-positive pathogens can block neutrophil recruitment by antagonizing the chemoattractant receptors. Formyl-methionyl-leucyl-phenylalanine (fMLP) peptides are powerful neutrophil chemoattractants that arise from bacterial protein degradation and can activate high-affinity fMLP peptide receptors on neutrophils in picomolar concentrations. Chemotaxis inhibitory protein of S. aureus (CHIPS) is secreted by the pathogen and binds specifically to the fMLP peptide (and C5a) receptors to impair their sensing function (104). Likewise, S. aureus releases FLIPr, another virulence factor capable of antagonizing the fMLP peptide receptor to impair leukocyte responses to the bacterial-derived chemoattractant agonists (98).

As a general rule, gram-positive bacteria are not sensed effectively by extracellular Toll-like receptors (TLRs), but intracellular detection is important (see sidebar). Phagocytosed gram-positive pathogens have been shown to evade detection by limiting phagosome acidification or phagosome fusion with granules or lysosomes (88, 112) or by escaping the phagosome altogether, as mediated by toxins such as GAS SLO and S. aureus leukocidins (37, 92). Other mechanisms of evading intracellular killing include using protein mimicry to blunt immune signaling. S. aureus was recently shown to express a Toll/IL-1 receptor (TIR)-domain-containing protein (TirS) that interferes with stimuli-induced TLR2-mediated NF-κB activation, c-Jun N-terminal kinase (JNK) phosphorylation, and secretion of proinflammatory cytokines (7). GAS DNase Sda1 can also cleave its own CpG-rich DNA, diminishing pathogen sensing and innate immune responses mediated by the intracellular sensor TLR-9 (119).

Molecular mimicry of host glycan structures by bacterial capsular polysaccharide capsules can promote innate immune evasion by gram-positive pathogens. The hyaluronic acid capsule of GAS is structurally identical to the nonsulfated glycosaminoglycans abundant in human skin and cartilage, thus covering the bacteria in a nonimmunogenic cloak that hides underlying opsonization targets. The terminal sialic acid residues in the capsule of GBS mimic common glycoepitopes on the surface of all mammalian cells and can engage Sia-binding immunoglobulin-like lectins (Siglecs), leukocyte receptors with inhibitory signaling properties that regulate the baseline activation (19). Recent data show that upon Siglec engagement, leukocyte responses such as oxidative burst and cytokine release are blunted, promoting GBS survival and virulence (20, 23). Furthermore, certain strains of GBS show the capacity to engage inhibitory Siglecs through their surface β-protein, once again limiting leukocyte activation (18). This demonstration of a sialic acid-independent mechanism of Siglec subversion suggests that other nonsialylated bacterial pathogens could evolve similar strategies for innate immune resistance.

ESCAPE FROM NEUTROPHIL EXTRACELLULAR TRAPS

Neutrophil extracellular traps (NETs) are a relatively recently appreciated aspect of innate immune defense in which neutrophils undergo a specialized cell death process at tissue foci of infection, whereon they release a highly decondensed chromatin structure containing histones, granule proteases, and AMPs (5, 123). Neutrophils produce NETs in response to gram-positive pathogens such as GAS and S. aureus (123) and are thought to kill microbes by exposing them to high local concentrations of antimicrobial effectors (5). The structure of NETs, held together by the DNA backbone, is critical for their antimicrobial function. A shared mechanism of bacterial escape from NET entrapment by means of extracellular DNAse production has now been described for GAS (16, 116), SPN (10), GBS (36), and S. aureus (13). For GAS, the acquisition of the potent bacteriophage-encoded DNAse Sda1 may have been a critical strep in the evolution of the hypervirulent M1T1 clone that has disseminated globally as a leading agent of severe invasive infections (124).

ACCELERATED PHAGOCYTE DEATH BY CYTOLYTIC TOXINS

A common feature of invasive gram-positive bacterial pathogens is the elaboration of potent membrane-damaging cytotoxins capable of targeting a variety of host cell membranes and important in the disruption and penetration of epithelial and endothelial barriers. Because phagocytic cell types such as neutrophils and macrophages are often vulnerable to these cytotoxic activities, another de facto mechanism for gram-positive innate immune evasion can involve triggering phagocytic cell death before bacterial killing can be fully accomplished.

S. aureus is notable for producing rapid necrosis of neutrophils following phagocytic uptake of the pathogen (69). S. aureus produces a family of two-subunit hetero-heptameric toxins that can oligomerize target leukocyte cell membranes to create pores and promote hypoosmotic cell lysis. These include γ-hemolysin, leukotoxin ED, leukotoxin GH, and the controversial bacteriophage-encoded Panton-Valentine leukocidin (PVL). Similarly the pore-forming GAS β-hemolysin streptolysin S (SLS) is cytolytic to human neutrophils and increases GAS resistance to phagocytic killing (33, 85).

Phenol-soluble modulins (PSMs) are a recently recognized group of small peptides produced by S. aureus with important roles in immune evasion and virulence (125). PSMs possess an amphipathic α-helical structure and membrane-destabilizing properties that can recruit, activate (71), and lyse neutrophils and induce a marked proinflammatory response while promoting bacterial survival. PSMs allow S. aureus to escape the phagolysosome and replicate intracellularly (43). PSMs can also act synergistically to exaggerate the cytolytic effect of PVL on human neutrophils (51).

Structurally distinct from SLS, the GAS cholesterol-binding cytolysin SLO is also toxic to human neutrophils and helps the pathogen avoid lysosomal localization (46) and is a key virulence factor in animal models of invasive GAS infection (33, 76). Upon phagocytosis, GAS mediates a program of accelerated neutrophil apoptosis relative to other common human pathogens (70). SLO was shown to represent the key GAS proapoptotic factor through mitochondrial membrane damage and activation of canonical caspase pathways (117). Similarly, the GBS β-hemolysin/cytolysin is a pore-forming toxin that induces cytolysis and apoptosis of macrophages, thereby promoting bacterial survival and virulence in animal models of pulmonary and systemic infection (79). Pneumolysin, the thiol-activated cytolysin of SPN, has a complex relationship with host defense, inducing the necrosis and apoptosis of human neutrophils (133) and macrophage cell lines (48) but also triggering IL-8 production (28) and stimulating neutrophil transendothelial migration (86).

Recent data reveal that the host innate immune system has developed the means to detect subcytolytic concentrations of pore-forming cytolysins, through a mechanism involving activation of the NLRP3 inflammasome and release of the proinflammatory cytokine IL-1β (31). Thus, pore-forming toxins can both stimulate or disable host defense depending on expression levels, site, and magnitude of infection.

FUTURE OUTLOOK AND THERAPEUTIC OPPORTUNITIES

The clinical specialty of infection disease medicine might be accurately defined as those medical conditions that arise when there has been a failure of innate immune defense. Our review has highlighted the diverse array of molecular mechanisms by which leading human gram-positive pathogens counteract or disable critical elements of host phagocyte-based innate immune defense to survive in the host and produce invasive infection. Whereas each pathogen deploys a different suite of virulence factors, encoded by unique sets of genes and possessing unique chemical structures, the cumulative effect of these features provides each pathogen significant resistance to phagocyte recruitment and activation, opsonophagocytosis, bacterial entrapment and uptake, and the microbicidal activities of key host defense factors such as AMPs and ROS.

In the present day, the cornerstone for treatment of invasive bacterial infection remains classical antibiotics, which function to kill or suppress the growth of a broad spectrum of bacteria, coupled with supportive care measures in the hospital or intensive care unit setting. However, this approach faces ever-increasing challenges owing to the continual evolution of antibiotic resistance (e.g., MRSA/vancomycin-intermediate S. aureus, penicillin-resistant SPN), epidemics of hypervirulent pathogen clones, and expanding vulnerable patient groups. Broad-spectrum antibiotic therapy also markedly perturbs the normal human microflora, which is increasingly recognized to play a critical role in maintaining healthy physiology and immune function.

The explosion of new knowledge regarding the immune evasion mechanisms of invasive gram-positive pathogens highlighted herein raises an interesting possibility: Could therapy of these leading disease agents be contemplated in a fashion that is not focused exclusively on the bacterium but rather more holistically on the host-pathogen interaction? Essentially, if a molecular mechanism of immune resistance is identified, the virulence factor itself could be explored as a therapeutic target, with the goal of resensitizing the pathogen to that particular arm of host innate defense (Figure 4). Such antivirulence therapies (22, 39) have the advantage in greater specificity and should not perturb or select for antibiotic resistance among the normal microflora. Virulence factor inhibitor treatments could also be used hand-in-hand with classical antibiotics for management of severe infections, including those complicated by antibiotic resistance or compromised host immunity. We have highlighted numerous studies employing targeted gene inactivation in leading gram-positive pathogens to produce isogenic mutants with increased susceptibility to innate immune clearance and reduced virulence in animal infection models. It is these gene-encoded pathogenicity factors that become pharmacologic targets, and literature is emerging to provide solid proofs-of-principle of therapeutic utility.

Figure 4.

Dual specificity therapeutic agents that target bacterial virulence factors to increase susceptibility to host cell clearance mechanisms and increase the host cell response could provide a multi-faceted approach to treatment. One such example is a squalene synthesis inhibitor, which blocks staphyloxanthin production (1) while simultaneously enhancing the capacity of neutrophils to produce NETs (2).

Recent approaches to limit S. aureus pathogenicity highlight this therapeutic strategy. A virulence factor discovery was exploited to design an approach to increase S. aureus sensitivity to ROS (Figure 4). The golden pigment staphyloxanthin was found to promote S. aureus virulence by neutralizing ROS-based neutrophil killing without affecting bacterial viability (80). Similarities were then recognized between the enzyme dehydrosqualene synthase (CrtM) in the biosynthetic pathway for staphyloxanthin and squalene synthase, an enzyme involved in human cholesterol biosynthesis. A cholesterol-lowering drug candidate targeting squalene synthetase (BPH-652) was found to block staphyloxanthin production, rendering the organism sensitive to ROS and neutrophil killing; treatment of mice with BPH-652 provided protection against systemic S. aureus infection (78). Additionally, given that blockade of cholesterol biosynthesis fortifies host defense against S. aureus by increasing NET production (26), dual specificity agents targeting CrtM and squalene synthesis have the potential to simultaneously boost neutrophil activity while sensitizing the pathogen to neutrophil-derived ROS (Figure 4) (77).

Targeting a specific virulence factor has also proven to be useful in limiting pathogenicity. Monoclonal antibodies to S. aureus α-hemolysin were shown to protect mice in pneumonia models (100). The lethal effects of α-hemolysin can also be blocked by administration of a cyclodextrin derivative that inhibits the assembled heptameric α-hemolysin pore in target cell membranes (101), or by treatment with novel biomimetic nanosponges composed of red blood cell membranes that absorb pore-forming toxins (52). The cellular receptor for α-hemolysin is A-disintegrin and metalloprotease 10 (ADAM10), which can be blocked with a small molecule inhibitor of ADAM10 metalloprotease activity (54). More recently, the related U.S. Food and Drug Administration-approved nonsteroidal anti-inflammatory drug diflunisol was identified as a candidate inhibitor of the AgrA response regulator, proving to block α-hemolysin production by the pathogen (66). Therapeutic targeting of the gene regulatory pathways that control the pathogen’s expression of key virulence determinants has also been investigated. One such example is the RNAII-inhibiting peptide (RIP), which has been shown to block Agr-dependent quorum sensing in S. aureus and to provide therapeutic benefit in several animal models of staphylococcal infections (9, 27).

Finally, targeting of host cell pathways to enhance bacterial clearance has also been investigated as a therapeutic strategy (Figure 4). Recently, the human immunodeficiency virus (HIV) co-receptor CCR5 was identified as a cellular determinant required for cytotoxic targeting of leukocytes by S. aureus leukotoxin ED (4). CCR5 knockout mice are more resistance to S. aureus infection, and treatment of normal mice with the HIV drug Maraviroc, a CCR5 inhibitor, conferred protection (4). Fascinatingly, in addition to increasing the capacity of neutrophils to produce NETs (26), statin treatment and lowering cholesterol levels in cell membranes reduced host cell lysis by the cholesterol-dependent SPN cytotoxin pneumolysin, helping protect against fulminant pneumococcal infection in an experimental mouse model of sickle cell anemia (105a).

In summary, a continued focus on deciphering the specific determinants responsible for innate immune evasion by invasive gram-positive pathogens so as to design specific virulence factor inhibitors can add to the future arsenal of therapeutic options available to physicians. Additionally, recent data suggest that even certain classical antibiotics may exert immune-sensitizing activities at subtherapeutic concentrations or against strains deemed resistant by classical minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) testing (6, 107). Furthermore, the development of drugs that enhance the ability of host cells to eliminate bacterial pathogens limits the development of drug-resistant bacteria and tips the balance of the host-microbe interaction in favor of the host (Figure 4). In the current era of increasing antibiotic resistance and complex hospitalized patient populations, it is hard to envision winning the battle against the leading invasive gram-positive pathogens without harnessing the power of our innate immune system.

SUMMARY POINTS.

1. Gram-positive pathogens have mechanisms to bind host complement inhibitory proteins to prevent activation of the complement system.

2. Gram-positive pathogens either prevent or reorient the binding of opsonins to the bacterial surface to prevent opsonophagocytosis.

3. Though mostly catalase-negative, gram-positive pathogens possess several antioxidant systems that provide protection against ROS and prevent the generation of HOCl.

4. All gram-positive pathogens can modify their peptidoglycan structures to prevent recognition and cleavage by lysozyme, preventing intracellular PRR detection.

5. Gram-positive bacteria modify their surface charge and prevent access to the bacterial membrane to resist the actions of AMPs or to produce proteases to directly degrade AMPs.

6. Gram-positive bacteria escape from NET-mediated killing using DNases.

7. Gram-positive bacteria are capable of either avoiding or dampening immune detection by phagocytic cells.

8. Capsules and toxins of gram-positive bacteria are multifaceted virulence factors that defend the bacteria against multiple arms of innate immunity.

Role of Pattern-Recognition Receptors in the Recognition of Gram-Positive Pathogens.

PRRs play a crucial role in the recognition of highly conserved pathogen-associated molecular patterns and subsequent activation of innate immune cells. TLRs expressed on the surface of immune cells (TLR1, 2, 4, 6) have proven to serve only minor roles in defense against gram-positive bacteria, which are classically described as extracellular pathogens. Of the extracellular TLRs, only TLR2, which generally recognizes lipoproteins, has the potential to recognize gram-positive bacteria, though there is some debate as to whether LTA specifically is recognized by TLR2 (102). However, TLR2 has been shown to be dispensable in mouse models of infection by gram-positive bacteria (42, 47, 68). Conversely, bacteria that have been phagocytosed and digested (109) reveal ligands that can be recognized by intracellular PRRs, such as TLR9, which recognizes bacterial DNA, and NOD2, which recognizes muramyldipeptide of PPG (24, 131). Activation of these intracellular PRRs leads to the formation and activation of the NLRP3 inflammasome, which activates the cytokine IL-1β, which regulates neutrophil recruitment to sites of infection (31, 84). Thus, intracellular PRRs are critical mediators of the innate immune response to gram-positive pathogens.

TERMS AND DEFINITIONS

AMPs

antimicrobial peptides, sequences of 12–50 amino acids that have direct antimicrobial activity and are generated from a precursor protein

C4BP

C4b-binding protein complement: a system of 30 proteins that aid in opsonization, chemoattraction, and direct antimicrobial activity

fMLP

formyl-methionyl-leucyl-phenylalanine peptide

GAS

group A Streptococcus

GBS

group B Streptococcus

NETs

neutrophil extracellular traps, an antimicrobial decondensed chromatin structure released from neutrophils containing histones, AMPs, and granule proteases

opsonophagocytosis

phagocytosis initiated by recognition of an opsonin by a host cell receptor

PRR

pattern-recognition receptor

PSM

phenol-soluble modulin

ROS

reactive oxygen species; highly antimicrobial reactive oxygen-containing chemical species and radicals

SPN

Streptococcus pneumonia

TLR

Toll-like receptor