Biomedical subjects

Sanjay Ram

Publications and source records attributed to Sanjay Ram.

12 recordsLinked to original sources

PubMed2007

lgtC expression modulates resistance to C4b deposition on an invasive nontypeable Haemophilus influenzae.

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.

Antibodies, Viral↗

PubMed2006

Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants.

Nontypeable (NT) Haemophilus influenzae is an important cause of otitis media in children. We have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding ( approximately 5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an approximately 1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.

Animals↗

PubMed2006

The meningococcal vaccine candidate GNA1870 binds the complement regulatory protein factor H and enhances serum resistance.

Neisseria meningitidis binds factor H (fH), a key regulator of the alternative complement pathway. A approximately 29 kD fH-binding protein expressed in the meningococcal outer membrane was identified by mass spectrometry as GNA1870, a lipoprotein currently under evaluation as a broad-spectrum meningococcal vaccine candidate. GNA1870 was confirmed as the fH ligand on intact bacteria by 1) abrogation of fH binding upon deleting GNA1870, and 2) blocking fH binding by anti-GNA1870 mAbs. fH bound to whole bacteria and purified rGNA1870 representing each of the three variant GNA1870 families. We showed that the amount of fH binding correlated with the level of bacterial GNA1870 expression. High levels of variant 1 GNA1870 expression (either by allelic replacement of gna1870 or by plasmid-driven high-level expression) in strains that otherwise were low-level GNA1870 expressers (and bound low amounts of fH by flow cytometry) restored high levels of fH binding. Diminished fH binding to the GNA1870 deletion mutants was accompanied by enhanced C3 binding and increased killing of the mutants. Conversely, high levels of GNA1870 expression and fH binding enhanced serum resistance. Our findings support the hypothesis that inhibiting the binding of a complement down-regulator protein to the neisserial surface by specific Ab may enhance intrinsic bactericidal activity of the Ab, resulting in two distinct mechanisms of Ab-mediated vaccine efficacy. These data provide further support for inclusion of this molecule in a meningococcal vaccine. To reflect the critical function of this molecule, we suggest calling it fH-binding protein.

Adjuvants, Immunologic↗

PubMed2006

Binding of vitronectin by the Moraxella catarrhalis UspA2 protein interferes with late stages of the complement cascade.

Many Moraxella catarrhalis strains are resistant to the bactericidal activity of normal human serum (NHS). The UspA2 protein of the serum-resistant strain O35E has previously been shown to be directly involved in conferring serum resistance on this strain. Testing of 11 additional serum-resistant M. catarrhalis wild-type isolates and their uspA1 and uspA2 mutants showed that the uspA1 mutants of all 11 strains were consistently serum resistant and that the uspA2 mutants of these same 11 strains were always serum sensitive. Analysis of complement deposition on four different serum-resistant M. catarrhalis strains and their serum-sensitive uspA2 mutants showed that, for three of these four strain sets, the wild-type and mutant strains bound similar amounts of early complement components. In contrast, there was a significant reduction in the amount of the polymerized C9 on the wild-type strains relative to that on the uspA2 mutants. These same three wild-type strains bound more vitronectin than did their uspA2 mutants. UspA2 proteins from these three strains, when expressed in Haemophilus influenzae, bound vitronectin and conferred serum resistance on this organism. Furthermore, vitronectin-depleted NHS exhibited bactericidal activity against these same three serum-resistant wild-type strains; addition of purified vitronectin to this serum restored serum resistance. In contrast, binding of the complement regulator C4b-binding protein by the M. catarrhalis strains used in this study was found to be highly variable and did not appear to correlate with the serum-resistant phenotype. These results indicate that binding of vitronectin by UspA2 is involved in the serum resistance of M. catarrhalis; this represents the first example of vitronectin-mediated serum resistance on a microbe.

Bacterial Outer Membrane Proteins↗

PubMed2005

Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection.

Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease that is restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind the classical pathway of complement down-regulatory protein C4b-binding protein (C4bp) to evade killing by human complement. Strains of N. gonorrhoeae that resisted killing by human serum complement were killed by serum from rodent, lagomorph, and primate species, which cannot be readily infected experimentally with this organism and whose C4bp molecules did not bind to N. gonorrhoeae. In contrast, we found that Yersinia pestis, an organism that can infect virtually all mammals, bound species-specific C4bp and uniformly resisted serum complement-mediated killing by these species. Serum resistance of gonococci was restored in these sera by human C4bp. An exception was serotype Por1B-bearing gonococcal strains that previously had been used successfully in a chimpanzee model of gonorrhea that simulates human disease. Por1B gonococci bound chimpanzee C4bp and resisted killing by chimpanzee serum, providing insight into the host restriction of gonorrhea and addressing why Por1B strains, but not Por1A strains, have been successful in experimental chimpanzee infection. Our findings may lead to the development of better animal models for gonorrhea and may also have implications in the choice of complement sources to evaluate neisserial vaccine candidates.

Amino Acid Sequence↗

PubMed2005

Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis.

Neisseria meningitidis (meningococcus) is an important cause of meningitis and sepsis. Currently, there is no effective vaccine against serogroup B meningococcal infection. Host defense against neisseriae requires the complement system (C) as indicated by the fact that individuals deficient in properdin or late C components (C6-9) have an increased susceptibility to recurrent neisserial infections. Because the classical pathway (CP) is required to initiate efficient complement activation on neisseriae, meningococci should be able to evade it to cause disease. To test this hypothesis, we studied the interactions of meningococci with the major CP inhibitor C4b-binding protein (C4bp). We tested C4bp binding to wild-type group B meningococcus strain (H44/76) and to 11 isogenic mutants thereof that differed in capsule expression, lipo-oligosaccharide sialylation, and/or expression of either porin (Por) A or PorB3. All strains expressing PorA bound radiolabeled C4bp, whereas the strains lacking PorA bound significantly less C4bp. Increased binding was observed under hypotonic conditions. Deleting PorB3 did not influence C4bp binding, but the presence of polysialic acid capsule reduced C4bp binding by 50%. Bound C4bp remained functionally active in that it promoted the inactivation of C4b by factor I. PorA-expressing strains were also more resistant to C lysis than PorA-negative strains in a serum bactericidal assay. Binding of C4bp thus helps Neisseria meningitidis to escape CP complement activation.

Amino Acid Sequence↗

PubMed2005

Enhanced factor H binding to sialylated Gonococci is restricted to the sialylated lacto-N-neotetraose lipooligosaccharide species: implications for serum resistance and evidence for a bifunctional lipooligosaccharide sialyltransferase in Gonococci.

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galalpha1 --> 4 [corrected] Galbeta1 --> 4Glcbeta1 --> 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galbeta1 --> 4GlcNAcbeta1 --> 3Galbeta1 --> 4Glcbeta1 --> 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an alpha2 --> 6 linkage and, as expected, to the terminal Gal of LNT LOS via an alpha2--> 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.

Amino Acid Sequence↗

PubMed2005

Killing of dsrA mutants of Haemophilus ducreyi by normal human serum occurs via the classical complement pathway and is initiated by immunoglobulin M binding.

Previously, we showed that serum resistance in Haemophilus ducreyi type strain 35000HP required expression of the outer membrane protein DsrA because the isogenic dsrA mutant FX517 is highly serum susceptible. In this study, we confirmed this finding by construction of additional serum-susceptible dsrA mutants in more recently isolated serum-resistant strains. We also demonstrated that killing of dsrA mutants required an intact classical complement cascade but not the alternative or mannan-binding lectin pathways. Between 5- and 10-fold more purified human immunoglobulin M (IgM) but not IgG was deposited onto dsrA mutant FX517 than onto parent strain 35000HP, consistent with IgM initiation of the classical cascade. Depletion of IgM, but not IgG, from complement-intact serum inhibited killing of FX517. As predicted from the amounts of IgM bound, more of the individual complement components were bound by FX517 than by parent strain 35000HP. Examination of the binding of negative regulators of complement as an explanation for serum resistance indicated that parent strain 35000HP bound more C4 binding protein and vitronectin than FX517 but not factor H. However, the degree and pattern of complement component binding observed suggested that IgM binding to the serum-susceptible mutant FX517 was responsible for the activation of the classical pathway and the observed killing of FX517 as opposed to binding of negative regulators of complement by the serum-resistant parent. We speculate that an undefined neo-epitope, possibly carbohydrate, is exposed in the dsrA mutant that is recognized by naturally occurring bactericidal IgM antibodies present in human sera.

Bacterial Outer Membrane Proteins↗

PubMed2003

Neisserial lipooligosaccharide is a target for complement component C4b. Inner core phosphoethanolamine residues define C4b linkage specificity.

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement.

Amides↗

PubMed2003

The Lip lipoprotein from Neisseria gonorrhoeae stimulates cytokine release and NF-kappaB activation in epithelial cells in a Toll-like receptor 2-dependent manner.

The human pathogen Neisseria gonorrhoeae produces an array of diseases ranging from urethritis to disseminated gonococcal infections. Early events in the establishment of infection involve interactions between N. gonorrhoeae and the mucosal epithelium, which leads to the local release of inflammatory mediators. Because of this, it is important to identify the bacterial virulence factors and host cell components that contribute to inflammation. Using a series of column chromatography steps, we purified a lipoprotein from N. gonorrhoeae strain F62 called Lip. This outer membrane antigen expresses a conserved epitope known as H.8, which is common to all pathogenic Neisseria species. We found the purified preparation of Lip to be a potent inflammatory mediator capable of inducing the release of the chemokine interleukin (IL)-8 and the cytokine IL-6 by immortalized human endocervical epithelial cells and the production of IL-8 and the activation of the transcription factor NF-kappaB by human embryonic kidney 293 (HEK) cells transfected with toll-like receptor (TLR) 2. Upon removal of Lip by immunoprecipitation, the ability of the H.8/Lip preparation to stimulate NF-kappaB activation was abolished. In addition to TLR2, the activation of NF-kappaB by H.8/Lip in HEK cells was enhanced upon coexpression of TLR1 but not TLR6. These observations provide evidence that Lip is capable of inducing the release of inflammatory mediators from epithelial cells in a TLR2-dependent manner.

Bacterial Outer Membrane Proteins↗

PubMed2003

The role of porins in neisserial pathogenesis and immunity.

Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.

Adjuvants, Immunologic↗

PubMed2002

Regulation of the mannan-binding lectin pathway of complement on Neisseria gonorrhoeae by C1-inhibitor and alpha 2-macroglobulin.

We examined complement activation by Neisseria gonorrhoeae via the mannan-binding lectin (MBL) pathway in normal human serum. Maximal binding of MBL complexed with MBL-associated serine proteases (MASPs) to N. gonorrhoeae was achieved at a concentration of 0.3 microg/ml. Preopsonization with MBL-MASP at concentrations as low as 0.03 microg/ml resulted in approximately 60% killing of otherwise fully serum-resistant gonococci. However, MBL-depleted serum (MBLdS) reconstituted with MBL-MASP before incubation with organisms (postopsonization) failed to kill at a 100-fold higher concentration. Preopsonized organisms showed a 1.5-fold increase in C4, a 2.5-fold increase in C3b, and an approximately 25-fold increase in factor Bb binding; enhanced C3b and factor Bb binding was classical pathway dependent. Preopsonization of bacteria with a mixture of pure C1-inhibitor and/or alpha(2)-macroglobulin added together with MBL-MASP, all at physiologic concentrations before adding MBLdS, totally reversed killing in 10% reconstituted serum. Reconstitution of MBLdS with supraphysiologic (24 microg/ml) concentrations of MBL-MASP partially overcame the effects of inhibitors (57% killing in 10% reconstituted serum). We also examined the effect of sialylation of gonococcal lipooligosaccharide (LOS) on MBL function. Partial sialylation of LOS did not decrease MBL or C4 binding but did decrease C3b binding by 50% and resulted in 80% survival in 10% serum (lacking bacteria-specific Abs) even when sialylated organisms were preopsonized with MBL. Full sialylation of LOS abolished MBL, C4, and C3b binding, resulting in 100% survival. Our studies indicate that MBL does not participate in complement activation on N. gonorrhoeae in the presence of "complete" serum that contains C1-inhibitor and alpha(2)-macroglobulin.

Adult↗