Sarika Agarwal

Human Factor H Interacts Selectively with Neisseria gonorrhoeae and Results in Species-Specific Complement Evasion

Complement forms a key arm of innate immune defenses against gonococcal infection. Sialylation of gonococcal lipo-oligosaccharide, or expression of porin 1A (Por1A) protein, enables Neisseria gonorrhoeae to bind the alternative pathway complement inhibitor, factor H (fH), and evade killing by human complement. Using recombinant fH fragment-murine Fc fusion proteins, we localized two N. gonorrhoeae Por1A-binding regions in fH: one in complement control protein domain 6, the other in complement control proteins 18–20. The latter is similar to that reported previously for sialylated Por1B gonococci. Upon incubation with human serum, Por1A and sialylated Por1B strains bound full-length human fH (HufH) and fH-related protein 1. In addition, Por1A strains bound fH-like protein 1 weakly. Only HufH, but not fH from other primates, bound directly to gonococci. Consistent with direct HufH binding, unsialylated Por1A gonococci resisted killing only by human complement, but not complement from other primates, rodents or lagomorphs; adding HufH to these heterologous sera restored serum resistance. Lipo-oligosaccharide sialylation of N. gonorrhoeae resulted in classical pathway regulation as evidenced by decreased C4 binding in human, chimpanzee, and rhesus serum but was accompanied by serum resistance only in human and chimpanzee serum. Direct-binding specificity of HufH only to gonococci that prevents serum killing is restricted to humans and may in part explain species-specific restriction of natural gonococcal infection. Our findings may help to improve animal models for gonorrhea while also having implications in the choice of complement sources to evaluate neisserial vaccine candidates.

Relative Contributions of Dectin-1 and Complement to Immune Responses to Particulate β-Glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-d-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4+ T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3−/−) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1−/− mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3−/− mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1−/−, and C3−/− mice with GP-OVA resulted in similar Ag-specific IgG1 and IgG2c type Ab and CD4+ T cell lymphoproliferative responses. Moreover, while CD4+ Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3−/− mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3−/− mice.

The Gonococcal Fur Regulon: Identification of Additional Genes Involved in Major Catabolic, Recombination, and Secretory Pathways

In this study, we have characterized the in vitro binding of Neisseria gonorrhoeae Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in Escherichia coli HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog), and the opa family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, the Fe-S homolog gene, and the opa gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11 opa genes (opaA through -K), we did not detect binding of purified E. coli Fur with 8 of the 11 opa members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the opa family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.

An evaluation of the role of properdin in alternative pathway activation on Neisseria meningitidis and Neisseria gonorrhoeae.

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive Neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological “native” (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the “conventional” mechanism of stabilizing AP C3 convertases.

Characterization of a novel Neisseria meningitidis Fur and iron‐regulated operon required for protection from oxidative stress: utility of DNA microarray in the assignment of the biological role of hypothetical genes

We have previously shown that in the human pathogen Neisseria meningitidis group B (MenB) more than 200 genes are regulated in response to growth with iron. Among the Fur‐dependent, upregulated genes identified by microarray analysis was a putative operon constituted by three genes, annotated as NMB1436, NMB1437 and NMB1438 and encoding proteins with so far unknown function. The operon was remarkably upregulated in the presence of iron and, on the basis of gel retardation analysis, its regulation was Fur dependent. In this study, we have further characterized the role of iron and Fur in the regulation of the NMB1436–38 operon and we have mapped the promoter and the Fur binding site. We also demonstrate by mutant analysis that the NMB1436–38 operon is required for protection of MenB to hydrogen peroxide‐mediated killing. By using both microarray analysis and S1 mapping, we demonstrate that the operon is not regulated by oxidative stress signals. We also show that the deletion of the NMB1436–38 operon results in an impaired capacity of MenB to survive in the blood of mice using an adult mouse model of MenB infection. Finally, we show that the NMB1436–38 deletion mutant exhibits increased susceptibility to the killing activity of polymorphonuclears (PMNs), suggesting that the ‘attenuated’ phenotype is mediated in part by the increased sensitivity to reactive oxygen species‐producing cells. This study represents one of the first examples of the use of DNA microarray to assign a biological role to hypothetical genes in bacteria.

Linkage Specificity and Role of Properdin in Activation of the Alternative Complement Pathway by Fungal Glycans

ABSTRACT Fungal cell walls are predominantly composed of glucans, mannans, and chitin. Recognition of these glycans by the innate immune system is a critical component of host defenses against the mycoses. Complement, an important arm of innate immunity, plays a significant role in fungal pathogenesis, especially the alternative pathway (AP). Here we determine that the glycan monosaccharide composition and glycosidic linkages affect AP activation and C3 deposition. Furthermore, properdin, a positive regulator of the AP, contributes to these functions. AP activation by glycan particles that varied in composition and linkage was measured by C3a generation in serum treated with 10 mM EGTA and 10 mM Mg2+ (Mg-EGTA-treated serum) (AP specific; properdin functional) or Mg-EGTA-treated serum that lacked functional properdin. Particles that contained either β1→3 or β1→6 glucans or both generated large and similar amounts of C3a when the AP was intact. Blocking properdin function resulted in 5- to 10-fold-less C3a production by particulate β1→3 glucans. However, particulate β1→6 glucans generated C3a via the AP only in the presence of intact properdin. Interestingly, zymosan and glucan-mannan particles (GMP), which contain both β-glucans and mannans, also required properdin to generate C3a. The β1→4 glycans chitin and chitosan minimally activated C3 even when properdin was functional. Finally, properdin binding to glucan particles (GP) and zymosan in serum required active C3. Properdin colocalized with bound C3, suggesting that in the presence of serum, properdin bound indirectly to glycans through C3 convertases. These findings provide a better understanding of how properdin facilitates AP activation by fungi through interaction with the cell wall components. IMPORTANCE Invasive fungal infections have increased in incidence with the widespread use of immunosuppressive therapy and invasive procedures. Activation of the complement system contributes to innate immunity against fungi by generating chemoattractants that recruit white blood cells and by coating the pathogen with complement fragments that “mark” them for phagocytosis. The fungal cell wall activates complement in an antibody-independent manner through the alternative pathway (AP). Properdin is a positive regulator of the AP. This study elucidates how the specificity of cell wall glycan linkages affects AP activation and the role properdin plays in this process. Particulate β1→3 glucans activated the AP even in the absence of properdin, while β1→6 glucans required properdin for AP activation. In contrast, the β1→4 glycans chitin and chitosan failed to activate the AP. These findings enhance our mechanistic understanding of how fungi activate complement and have implications for the use of glycans in biomedical applications. Invasive fungal infections have increased in incidence with the widespread use of immunosuppressive therapy and invasive procedures. Activation of the complement system contributes to innate immunity against fungi by generating chemoattractants that recruit white blood cells and by coating the pathogen with complement fragments that “mark” them for phagocytosis. The fungal cell wall activates complement in an antibody-independent manner through the alternative pathway (AP). Properdin is a positive regulator of the AP. This study elucidates how the specificity of cell wall glycan linkages affects AP activation and the role properdin plays in this process. Particulate β1→3 glucans activated the AP even in the absence of properdin, while β1→6 glucans required properdin for AP activation. In contrast, the β1→4 glycans chitin and chitosan failed to activate the AP. These findings enhance our mechanistic understanding of how fungi activate complement and have implications for the use of glycans in biomedical applications.

Flavobacterium johnsoniae SprA Is a Cell Surface Protein Involved in Gliding Motility

Flavobacterium johnsoniae cells glide rapidly over surfaces by an unknown mechanism. Transposon-induced sprA mutants formed nonspreading colonies on agar, and the cells examined in wet mounts were deficient in attachment to surfaces and were almost completely nonmotile. Exposure of intact cells to proteinase K cleaved the 270-kDa SprA into several large peptides, suggesting that it is partially exposed on the cell surface.

The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Properdin Is Critical for Antibody-Dependent Bactericidal Activity against Neisseria gonorrhoeae That Recruit C4b-Binding Protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.

Meningococcal Group W-135 and Y Capsular Polysaccharides Paradoxically Enhance Activation of the Alternative Pathway of Complement

Although capsular polysaccharide (CPS) is critical for meningococcal virulence, the molecular basis of alternative complement pathway (AP) regulation by meningococcal CPSs remains unclear. Using serum with only the AP active, the ability of strains to generate C3a (a measure of C3 activation) and subsequently deposit C3 fragments on bacteria was studied in encapsulated group A, B, C, W-135, and Y strains and their isogenic unencapsulated mutants. To eliminate confounding AP regulation by membrane-bound factor H (fH; AP inhibitor) and lipooligosaccharide sialic acid, the meningococcal fH ligands (fHbp and NspA) and lipooligosaccharide sialylation were deleted in all strains. Group A CPS expression did not affect C3a generation or C3 deposition. C3a generated by encapsulated and unencapsulated group B and C strains was similar, but CPS expression was associated with reduced C3 deposition, suggesting that these CPSs blocked C3 deposition on membrane targets. Paradoxically, encapsulated W-135 and Y strains (including the wild-type parent strains) enhanced C3 activation and showed marked C3 deposition as early as 10 min; at this time point C3 was barely activated by the unencapsulated mutants. W-135 and Y CPSs themselves served as a site for C3 deposition; this observation was confirmed using immobilized purified CPSs. Purified CPSs bound to unencapsulated meningococci, simulated findings with naturally encapsulated strains. These data highlight the heterogeneity of AP activation on the various meningococcal serogroups that may contribute to differences in their pathogenic mechanisms.