Mechanisms Of Complement Evasion Research Articles

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

Plasmodium falciparum Gametes and Sporozoites Hijack Plasmin and Factor H To Evade Host Complement Killing.

Plasmodium parasites are the etiological agents of malaria, a disease responsible for over half a million deaths annually. Successful completion of the parasite's life cycle in the vertebrate host and transmission to a mosquito vector is contingent upon the ability of the parasite to evade the host's defenses. The extracellular stages of the parasite, including gametes and sporozoites, must evade complement attack in both the mammalian host and in the blood ingested by the mosquito vector. Here, we show that Plasmodium falciparum gametes and sporozoites acquire mammalian plasminogen and activate it into the serine protease plasmin to evade complement attack by degrading C3b. Complement-mediated permeabilization of gametes and sporozoites was higher in plasminogen-depleted plasma, suggesting that plasminogen is important for complement evasion. Plasmin also facilitates gamete exflagellation through complement evasion. Furthermore, supplementing serum with plasmin significantly increased parasite infectivity to mosquitoes and lowered the transmission-blocking activity of antibodies to Pfs230, a potent vaccine candidate currently in clinical trials. Finally, we show that human factor H, previously shown to facilitate complement evasion by gametes, also facilitates complement evasion by sporozoites. Plasmin and factor H simultaneously cooperate to enhance complement evasion by gametes and sporozoites. Taken together, our data show that Plasmodium falciparum gametes and sporozoites hijack the mammalian serine protease plasmin to evade complement attack by degrading C3b. Understanding of the mechanisms of complement evasion by the parasite is key to the development of novel effective therapeutics. IMPORTANCE Current approaches to control malaria are complicated by the development of antimalarial-resistant parasites and insecticide-resistant vectors. Vaccines that block transmission to mosquitoes and humans are a plausible alternative to overcome these setbacks. To inform the development of efficacious vaccines, it is imperative to understand how the parasite interacts with the host immune response. In this report, we show that the parasite can co-opt host plasmin, a mammalian fibrinolytic protein to evade host complement attack. Our results highlight a potential mechanism that may reduce efficacy of potent vaccine candidates. Taken together, our results will inform future studies in developing novel antimalarial therapeutics.

Novel Antibody-independent Method to Measure Complement Deposition on Bacteria.

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such asStaphylococcus aureusto overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such asS. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and bothLactococcus lactisandS. aureus.

Outer surface lipoproteins from the Lyme disease spirochete exploit the molecular switch mechanism of the complement protease C1s

Proteolytic cascades comprise several important physiological systems, including a primary arm of innate immunity called the complement cascade. To safeguard against complement-mediated attack, the etiologic agent of Lyme disease, Borreliella burgdorferi, produces numerous outer surface-localized lipoproteins that contribute to successful complement evasion. Recently, we discovered a pair of B.burgdorferi surface lipoproteins of the OspEF-related protein family-termed ElpB and ElpQ-that inhibit antibody-mediated complement activation. In this study, we investigate the molecular mechanism of ElpB and ElpQ complement inhibition using an array of biochemical and biophysical approaches. Invitro assays of complement activation show that an independently folded homologous C-terminal domain of each Elp protein maintains full complement inhibitory activity and selectively inhibits the classical pathway. Using binding assays and complement component C1s enzyme assays, we show that binding of Elp proteins to activated C1s blocks complement component C4 cleavage by competing with C1s-C4 binding without occluding the active site. C1s-mediated C4 cleavage is dependent on activation-induced binding sites, termed exosites. To test whether these exosites are involved in Elp-C1s binding, we performed site-directed mutagenesis, which showed that ElpB and ElpQ binding require C1s residues in the anion-binding exosite located on the serine protease domain of C1s. Based on these results, we propose a model whereby ElpB and ElpQ exploit activation-induced conformational changes that are normally important for C1s-mediated C4 cleavage. Our study expands the known complement evasion mechanisms of microbial pathogens and reveals a novel molecular mechanism for selective C1s inhibition by Lyme disease spirochetes.

Lipopolysaccharides and outer membrane proteins as main structures involved in complement evasion strategies of non-typhoidal Salmonella strains

Non-typhoidal Salmonella (NTS) infections pose a serious public health problem. In addition to the typical course of salmonellosis, an infection with Salmonella bacteria can often lead to parenteral infections and sepsis, which are particularly dangerous for children, the elderly and immunocompromised. Bacterial resistance to serum is a key virulence factor for the development of systemic infections. Salmonella, as an enterobacterial pathogen, has developed several mechanisms to escape and block the antibacterial effects of the complement system. In this review, we discuss the relevance of outer membrane polysaccharides to the complement evasion mechanisms of NTS strains. These include the influence of the overall length and density of the lipopolysaccharide molecules, modifications of the O-antigen lipopolysaccharide composition and the role of capsular polysaccharides in opsonization and protection of the outer membrane from the lytic action of complement. Additionally, we discuss specific outer membrane protein complement evasion mechanisms, such as recruitment of complement regulatory proteins, blocking assembly of late complement components to form the membrane attack complex and the proteolytic cleavage of complement proteins.

Streptococcus pneumoniae interactions with the complement system.

Host innate and adaptive immunity to infection with Streptococcus pneumoniae is critically dependent on the complement system, demonstrated by the high incidence of invasive S. pneumoniae infection in people with inherited deficiency of complement components. The complement system is activated by S. pneumoniae through multiple mechanisms. The classical complement pathway is activated by recognition of S. pneumoniae by C-reactive protein, serum amyloid P, C1q, SIGN-R1, or natural or acquired antibody. Some S. pneumoniae strains are also recognised by ficolins to activate the mannose binding lectin (MBL) activation pathway. Complement activation is then amplified by the alternative complement pathway, which can also be activated by S. pneumoniae directly. Complement activation results in covalent linkage of the opsonic complement factors C3b and iC3b to the S. pneumoniae surface which promote phagocytic clearance, along with complement-mediated immune adherence to erythrocytes, thereby protecting against septicaemia. The role of complement for mucosal immunity to S. pneumoniae is less clear. Given the major role of complement in controlling infection with S. pneumoniae, it is perhaps unsurprising that S. pneumoniae has evolved multiple mechanisms of complement evasion, including the capsule, multiple surface proteins, and the toxin pneumolysin. There is considerable variation between S. pneumoniae capsular serotypes and genotypes with regards to sensitivity to complement which correlates with ability to cause invasive infections. However, at present we only have a limited understanding of the main mechanisms causing variations in complement sensitivity between S. pneumoniae strains and to non-pathogenic streptococci.

Staphylococcal Complement Evasion Protein Sbi Stabilises C3d Dimers by Inducing an N-Terminal Helix Swap.

Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.

Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing

Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vector–vertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host–pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

C5b-9 Membrane Attack Complex Formation andExtracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett’s Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Hijacking Factor H for Complement Immune Evasion.

The complement system is an essential player in innate and adaptive immunity. It consists of three pathways (alternative, classical, and lectin) that initiate either spontaneously (alternative) or in response to danger (all pathways). Complement leads to numerous outcomes detrimental to invaders, including direct killing by formation of the pore-forming membrane attack complex, recruitment of immune cells to sites of invasion, facilitation of phagocytosis, and enhancement of cellular immune responses. Pathogens must overcome the complement system to survive in the host. A common strategy used by pathogens to evade complement is hijacking host complement regulators. Complement regulators prevent attack of host cells and include a collection of membrane-bound and fluid phase proteins. Factor H (FH), a fluid phase complement regulatory protein, controls the alternative pathway (AP) both in the fluid phase of the human body and on cell surfaces. In order to prevent complement activation and amplification on host cells and tissues, FH recognizes host cell-specific polyanionic markers in combination with complement C3 fragments. FH suppresses AP complement-mediated attack by accelerating decay of convertases and by helping to inactivate C3 fragments on host cells. Pathogens, most of which do not have polyanionic markers, are not recognized by FH. Numerous pathogens, including certain bacteria, viruses, protozoa, helminths, and fungi, can recruit FH to protect themselves against host-mediated complement attack, using either specific receptors and/or molecular mimicry to appear more like a host cell. This review will explore pathogen complement evasion mechanisms involving FH recruitment with an emphasis on: (a) characterizing the structural properties and expression patterns of pathogen FH binding proteins, as well as other strategies used by pathogens to capture FH; (b) classifying domains of FH important in pathogen interaction; and (c) discussing existing and potential treatment strategies that target FH interactions with pathogens. Overall, many pathogens use FH to avoid complement attack and appreciating the commonalities across these diverse microorganisms deepens the understanding of complement in microbiology.

Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing.

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

Complement in malaria: immune evasion strategies and role in protective immunity.

The malaria parasite has for long been thought to escape host complement attack as a survival strategy. However, it was only recently that complement evasion mechanisms of the parasite were described. Simultaneously, the role of complement in antibody‐mediated naturally acquired and vaccine‐induced protection against malaria has also been reported. Such findings should be considered in future vaccine design, given the current need to develop more efficacious vaccines against malaria. Parasite antigens derived from molecules mediating functions crucial for parasite survival, such as complement evasion, or parasite antigens against which antibody responses lead to an efficient complement attack could present new candidates for vaccines. In this review, we discuss recent findings on complement evasion by the malaria parasites and the emerging role of complement in antibody‐mediated protection against malaria. We emphasize that immune responses to vaccines based on complement inhibitors should not only induce complement‐activating antibodies but also neutralize the escape mechanisms of the parasite.

Complement evasion mechanisms of the systemic pathogens Yersiniae and Salmonellae.

Pathogens that colonize deep tissues and spread systemically encounter the innate host resistance mechanism of complement-mediated lysis and complement opsonization leading to engulfment and degradation by phagocytic cells. Yersinia and Salmonella species have developed numerous strategies to block the antimicrobial effects of complement. These include recruitment of complement regulatory proteins factor H, C4BP, and vitronectin (Vn) as well as interference in late maturation events such as assembly of C9 into the membrane attack complex that leads to bacterial lysis. This review will discuss the contributions of various surface structures (proteins, lipopolysaccharide, and capsules) to evasion of complement-mediated immune clearance of the systemic pathogens Yersiniae and Salmonellae. Bacterial proteins required for recruitment of complement regulatory proteins will be described, including the details of their interaction with host regulatory proteins, where known. The potential role of the surface proteases Pla (Yersiniapestis) and PgtE (Salmonella species) on the activity of complement regulatory proteins will also be addressed. Finally, the implications of complement inactivation on host cell interactions and host cell targeting for type 3 secretion will be discussed.

Structural determination of the complement inhibitory domain of Borrelia burgdorferi BBK32 provides insight into classical pathway complement evasion by Lyme disease spirochetes.

The carboxy-terminal domain of the BBK32 protein from Borrelia burgdorferi sensu stricto, termed BBK32-C, binds and inhibits the initiating serine protease of the human classical complement pathway, C1r. In this study we investigated the function of BBK32 orthologues of the Lyme-associated Borrelia burgdorferi sensu lato complex, designated BAD16 from B. afzelii strain PGau and BGD19 from B. garinii strain IP90. Our data show that B. afzelii BAD16-C exhibits BBK32-C-like activities in all assays tested, including high-affinity binding to purified C1r protease and C1 complex, and potent inhibition of the classical complement pathway. Recombinant B. garinii BGD19-C also bound C1 and C1r with high-affinity yet exhibited significantly reduced in vitro complement inhibitory activities relative to BBK32-C or BAD16-C. Interestingly, natively produced BGD19 weakly recognized C1r relative to BBK32 and BAD16 and, unlike these proteins, BGD19 did not confer significant protection from serum killing. Site-directed mutagenesis was performed to convert BBK32-C to resemble BGD19-C at three residue positions that are identical between BBK32 and BAD16 but different in BGD19. The resulting chimeric protein was designated BXK32-C and this BBK32-C variant mimicked the properties observed for BGD19-C. To query the disparate complement inhibitory activities of BBK32 orthologues, the crystal structure of BBK32-C was solved to 1.7Å limiting resolution. BBK32-C adopts an anti-parallel four-helix bundle fold with a fifth alpha-helix protruding from the helical core. The structure revealed that the three residues targeted in the BXK32-C chimera are surface-exposed, further supporting their potential relevance in C1r binding and inhibition. Additional binding assays showed that BBK32-C only recognized C1r fragments containing the serine protease domain. The structure-function studies reported here improve our understanding of how BBK32 recognizes and inhibits C1r and provide new insight into complement evasion mechanisms of Lyme-associated spirochetes of the B. burgdorferi sensu lato complex.

Complement Activation as a Helping Hand for Inflammophilic Pathogens and Cancer.

The complement system, an evolutionarily ancient component of innate immunity, is capable of protecting hosts from invading pathogens, either directly, by lysis of target cells, or indirectly, by mobilization of host immune mechanisms. However, this potentially cytotoxic cascade must be tightly regulated, since improperly controlled complement can damage healthy cells and tissues. The practical importance of this axis is highlighted when impairment of complement regulators or bacterial mechanisms of complement evasion result in pathogenic conditions. Recognition of complement as a “double-edged sword” is widely acknowledged, but another, currently underappreciated aspect of complement function has emerged as an important player in homeostatic balance—the dual outcome of complement-mediated inflammation. In most cases, the proinflammatory properties of complement are beneficial to the host. However, certain pathogens have developed the ability to utilize local inflammation as a source of nutrients and as a way to establish a niche for further colonization. Such a strategy can be illustrated in the example of periodontitis. Interestingly, certain tumors also seem to benefit from complement activation products, which promote a proangiogenic and immunosuppressive microenvironment.

Ail provides multiple mechanisms of serum resistance to Yersinia pestis.

Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery and serum resistance activities. Resistance to complement proteins in serum is critical for the survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b-binding protein (C4BP) and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co-sedimentation assays, we demonstrate that two surface-exposed amino acids, F80 and F130, are required for the interaction of Y. pestis Ail with Vn, factor H and C4BP. However, although Ail-F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000-fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site-directed mutagenesis, we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early-stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.

Molecular mechanisms of classical pathway complement evasion by Borrelia spp.

Molecular mechanisms of classical pathway complement evasion by Borrelia spp.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism

ABSTRACT Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (Ide Ssuis ) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of Ide Ssuis abrogated IgM cleavage completely. In contrast to wt rIde Ssuis , point mutated rIde Ssuis _C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIde Ssuis depends on the IgM proteolytic activity. A S. suis mutant expressing Ide Ssuis _C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab’)2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIde Ssuis and rIde Ssuis _C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies Ide Ssuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

The two-component system VicRK regulates functions associated with Streptococcus mutans resistance to complement immunity.

Streptococcus mutans, a dental caries pathogen, can promote systemic infections upon reaching the bloodstream. The two-component system (TCS) VicRKSm of S.mutans regulates the synthesis of and interaction with sucrose-derived exopolysaccharides (EPS), processes associated with oral and systemic virulence. In this study, we investigated the mechanisms by which VicRKSm affects S.mutans susceptibility to blood-mediated immunity. Compared with parent strain UA159, the vicKSm isogenic mutant (UAvic) showed reduced susceptibility to deposition of C3b of complement, low binding to serum immunoglobulin G (IgG), and low frequency of C3b/IgG-mediated opsonophagocytosis by polymorphonuclear cells in a sucrose-independent way (P<.05). Reverse transcriptase quantitative polymerase chain reaction analysis comparing gene expression in UA159 and UAvic revealed that genes encoding putative peptidases of the complement (pepO and smu.399) were upregulated in UAvic in the presence of serum, although genes encoding murein hydrolases (SmaA and Smu.2146c) or metabolic/surface proteins involved in bacterial interactions with host components (enolase, GAPDH) were mostly affected in a serum-independent way. Among vicKSm -downstream genes (smaA, smu.2146c, lysM, atlA, pepO, smu.399), only pepO and smu.399 were associated with UAvic phenotypes; deletion of both genes in UA159 significantly enhanced levels of C3b deposition and opsonophagocytosis (P<.05). Moreover, consistent with the fibronectin-binding function of PepO orthologues, UAvic showed increased binding to fibronectin. Reduced susceptibility to opsonophagocytosis was insufficient to enhance ex vivo persistence of UAvic in blood, which was associated with growth defects of this mutant under limited nutrient conditions. Our findings revealed that S.mutans employs mechanisms of complement evasion through peptidases, which are controlled by VicRKSm.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.