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C Elkins

Publications and source records attributed to C Elkins.

32 records · Page 2Linked to original sources
PubMed1996

Transferrin increases adherence of iron-deprived Neisseria gonorrhoeae to human endometrial cells.

OBJECTIVE: Our purpose was to study the effects of iron deprivation with and without human transferrin supplementation on the adherence and invasion of Neisseria gonorrhoeae to human endometrial cells. STUDY DESIGN: N. gonorrhoeae grown with our without iron was placed in media alone or media containing 2.5 mg/ml saturated human transferrin or unsaturated transferrin. N. gonorrhoeae was inoculated onto polarized human endometrial carcinoma cell (HEC 1-B) monolayers, and at various intervals monolayers were washed and incubated with media containing gentamicin or media alone. Colony-forming units per milliliter of N. gonorrhoeae associated with HEC 1-B cells were then determined. N. gonorrhoeae strains tested included both a transferrin receptor-positive (wild-type) and a transferrin receptor-negative mutant. Differences in percent of original inoculum remaining at varying time points were analyzed by the Mann-Whitney U test. Transmission electron microscopy using a primary endometrial cell line was used to verify findings. RESULTS: Iron-negative N. gonorrhoeae exhibited less adherence than did iron-positive N. gonorrhoeae. No difference in HEC 1-B adherence was seen when either saturated transferrin or unsaturated transferrin was added to the iron-positive N. gonorrhoeae. With iron-negative N. gonorrhoeae addition of either saturated transferrin or unsaturated transferrin significantly increased N. gonorrhoeae adherence although unsaturated transferrin did not permit growth of iron-negative N. gonorrhoeae in tissue culture media alone. Transmission electron microscopy confirmed increased adherence of iron-negative N. gonorrhoeae supplemented with unsaturated transferrin. An iron-negative N. gonorrhoeae mutant lacking the transferrin receptor exhibited no adherence regardless of addition of saturated transferrin or unsaturated transferrin. Invasion could not be quantitated reliably because of persistence of gentamicin effect. CONCLUSION: Iron and transferrin increased attachment of N. gonorrhoeae to human endometrial cells.

Bacterial Adhesion↗
PubMed1996

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.

Bacterial Outer Membrane Proteins↗
PubMed1996

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Amino Acid Sequence↗
PubMed1995

Identification and purification of a conserved heme-regulated hemoglobin-binding outer membrane protein from Haemophilus ducreyi.

A hemoglobin-binding protein (HgbA) from Haemophilus ducreyi was identified and purified. The 100-kDa HgbA was detected in all strains of H. ducreyi tested, and a somewhat larger hemoglobin-binding protein was found in one strain of Haemophilus influenzae. HgbA was purified and the amino acid sequence of the N terminus of HgbA revealed no significant homologies with known proteins. Two different antisera to HgbA from H. ducreyi 35000 recognized HgbA proteins from all tested H. ducreyi strains; they did not recognize proteins from the H. influenzae strain. Expression of HgbA was regulated by the level of heme but not by iron present in the medium. Animal species of hemoglobin competed with iodinated human hemoglobin for binding to whole cells of H. ducreyi and supported the growth of H. ducreyi. The lack of immunological cross-reactivity and the differences in hemoglobin specificities between the H. ducreyi and the H. influenzae hemoglobin-binding proteins suggest that they are unrelated.

Amino Acid Sequence↗
PubMed1995

Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.

Amino Acid Sequence↗
PubMed1994

Vaccines for bacterial sexually transmitted infections: a realistic goal?

Bacterial infections of the genital tract (gonorrhea, chlamydia, chancroid, syphilis) are common and cause significant morbidity. Their importance is heightened by recent appreciation of their roles in facilitation of transmission of the human immunodeficiency virus (HIV). Each is capable of causing repeated infections, suggesting lack of permanent broadly effective immunity. An effective vaccine has yet to be developed for any of these diseases. Rapid progress in understanding the molecular basis for pathogenesis of infection, including mechanisms for escape from otherwise effective immune surveillance and mechanisms for causing injury to host cells, has stimulated renewed efforts to make vaccines for some of these infections. Progress has been greatest for Neisseria gonorrhoeae and Chlamydia trachomatis. Present emphasis is on the major or principal outer membrane proteins of N. gonorrhoeae and C. trachomatis, based on evidence for neutralizing antibodies directed against surface-exposed variable domains of each of these proteins. Other surface-exposed proteins, including the iron-repressible transferrin receptor in gonococci and certain heat-shock proteins in chlamydia, also may be targets for vaccines. Although much remains to be learned, cautious optimism is warranted.

Humans↗
PubMed1994

Cloning and constitutive expression of structural genes encoding gonococcal porin protein in Escherichia coli and attenuated Salmonella typhimurium vaccine strains.

Previous reports [Gotschlich et al., Proc. Natl. Acad. Sci. USA 84 (1987) 8135-8139; Carbonetti and Sparling, Proc. Natl. Acad. Sci. USA 84 (1987) 9084-9088; Carbonetti et al., Proc. Natl. Acad. Sci. USA 85 (1988) 6841-6845] concluded that synthesis of the porin protein (Por) from Neisseria gonorrhoeae in Escherichia coli was toxic to that organism, which limited studies of the biology of Por in foreign hosts. We assembled intact por genes from the gonococcal strains, FA19 (serogroup PIA) and FA6434 (a hybrid Por containing epitopes from serogroups PIA and PIB), and observed stable expression in E. coli without evident toxicity. Expression of por from strain MS11 (serogroup PIB) in E. coli was difficult, but por from MS11 was expressed without toxicity when the -35 region of the por promoter was removed. Encouraged by this, we moved por from E. coli into attenuated Salmonella typhimurium strains and expressed por either in single copy from the chromosome or in multiple copy from plasmids. Expression levels of por in S. typhimurium were higher from plasmids than from the chromosome, probably due to a gene dosage effect. This work will enable study of the immune response to Por in mice vaccinated orally with live S. typhimurium.

Bacterial Vaccines↗
PubMed1994

Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae.

Gonococcal porins (Por) from strains FA19 (Por-1, serogroup A), MS11 (Por-2, serogroup B) and FA6434 (Por-5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non-denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole-cell radioimmunoprecipitation and were broadly cross-reactive within serogroups. Post-immune, but not pre-immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outer-membrane protein Rmp.

Amino Acid Sequence↗
PubMed1992

Antibodies to N-terminal peptides of gonococcal porin are bactericidal when gonococcal lipopolysaccharide is not sialylated.

Six synthetic 25-mer peptides corresponding to certain presumed surface-exposed regions of gonococcal porin protein I (PI) were made from strains FA19 (PIA) and MS11 (PIB). Four peptides were immunogenic in rabbits. Affinity-purified antisera against both PIA and PIB N-terminal peptides were bactericidal for homologous gonococci and many heterologous PI serovars. However, sialylation of gonococcal lipopolysaccharide (LPS) by growth of gonococci in the presence of cytidine monophosphate-neuraminic acid (CMP-NANA) abrogated the bactericidal activity of these antisera. Binding of anti-PI monoclonal antibodies to whole gonococci was reduced two- to fourfold by sialylation of LPS, suggesting that sialylation may inhibit bactericidal activity by masking porin epitopes. However, binding of anti-PII (Opa) monoclonal antibodies was not inhibited, yet complement-mediated killing was inhibited by sialylated LPS. Binding of complement components C3 and C9 was inhibited in the presence of either anti-PI or anti-PII monoclonals when gonococci were grown in the presence of CMP-NANA. Thus sialylation inhibited both anti-PI antibody binding and complement deposition, with a resultant decrease in bactericidal activity.

Amino Acid Sequence↗
PubMed1991

Species-specific uptake of DNA by gonococci is mediated by a 10-base-pair sequence.

Piliated Neisseria gonorrhoeae are known to be transformed less readily if transforming DNA competes with DNA containing the 10-bp sequence GCCGTCTGAA. It has been postulated that the 10-bp sequence is a recognition sequence which is required for efficient DNA uptake. We show that the presence of various forms of this 10-bp sequence results in increased uptake of double-stranded DNA into a DNase-resistant state and allows genetic transformation by an otherwise nontransformable plasmid.

Base Sequence↗
PubMed1990

Construction of isogenic gonococci with variable porin structure: effects on susceptibility to human serum and antibiotics.

Protein I (PI) is the most abundant protein on the gonococcal cell surface and besides its porin function it may have important properties contributing to pathogenicity. By allelic exchange using cloned PI genes from FA19 (PIA) and MS11 (PIB) and a selectable marker introduced closely downstream of these genes, we constructed sets of isogenic gonococcal strains that differ only in their PI gene. Analysis revealed that PI has a major effect on stable resistance to normal human serum, and a slight effect on low-level resistance to antibiotics. All PIA/B hybrids were hypersusceptible to serum, suggesting a possible explanation for why such hybrids do not occur in nature.

Alleles↗
PubMed1990

Monoclonal antibodies to outer membrane protein PII block interactions of Neisseria gonorrhoeae with human neutrophils.

Nonopsonic binding of gonococci to human neutrophils appears to be mediated by a family of heat-modifiable outer membrane proteins termed protein IIs (PIIs). We studied the ability of a wide variety of antigonococcal monoclonal antibodies (MAbs) to inhibit the interactions of nonpiliated PII+ gonococci with human neutrophils by measuring gonococcal adherence to neutrophils and subsequent luminol-enhanced neutrophil chemiluminescence. From one set of 95 MAbs reacting with whole gonococci, only two, 7VA2 and 7B9, inhibited the ability of gonococci to induce neutrophil chemiluminescence. 7VA2 and 7B9 both reacted only with PII. MAb 53C4, from a smaller set of anti-PII MAbs, inhibited adherence to neutrophils of PII variants that bound 53C4, but not of PII variants that did not. It also inhibited gonococcus-induced neutrophil chemiluminescence. Using a whole-cell binding assay and Western blotting (immunoblotting), we showed that MAb 53C4 bound to one PII (PII4) of strain F62 and to two PIIs (PIIb and PIId) of strain FA1090. The present studies confirm and extend the role of PII in gonococcal adherence to and stimulation of human neutrophils and show intrastrain conservation of PII epitopes. The results indicate that PII is the only outer membrane component involved in adherence of nonpiliated gonococci to human neutrophils.

Antibodies, Monoclonal↗
PubMedDate not supplied

The contrasting mechanisms of serum resistance of Neisseria gonorrhoeae and group B Neisseria meningitidis.

Neisseria gonorrhoeae and Neisseria meningitidis have evolved intricate mechanisms to evade complement-mediated killing. Sialylation of gonococcal lipooligosaccharide (LOS) results in conversion of previously serum sensitive strains to unstable serum resistance, which is mediated by factor H binding. Porin (Por) is also instrumental in mediating stable serum resistance in gonococci. The 5th loop of certain gonococcal PorlAs binds factor H, which efficiently inactivates C3b to iC3b. Factor H glycan residues may be essential for factor H binding to certain Por1A strains. Por1A strains can also regulate the classical pathway by binding to C4b-binding protein (C4bp) probably via the 1st loop of the Por molecule. Certain serum resistant Por1 B strains can also regulate complement by binding C4bp through a loop other than loop 1. Purified C4b can inhibit binding of C4bp to Por 1B, but not Por1A, suggesting different binding sites on C4bp for the two Por types. Unlike serum resistant gonococci, resistant meningococci have abundant C3b on their surface, which is only partially processed to iC3b. The main mechanism of complement evasion by group B meningococci is inhibition of membrane attack complex (MAC) insertion by their polysaccharide capsule. LOS structure may act in concert with capsule to prevent MAC insertion. Meningococcal strains with Class 3 Por preferentially bind factor H, suggesting Class 3 Por acts as a receptor for factor H.

Blood Bactericidal Activity↗