Biomedical subjects

C Elkins

Publications and source records attributed to C Elkins.

At least 19 recordsLinked to original sources

PubMed2001

Invasion of human fallopian tube epithelium by Escherichia coli expressing combinations of a gonococcal porin, opacity-associated protein, and chimeric lipo-oligosaccharide.

The transepithelial migration of Escherichia coli that expressed all possible combinations of a plasmid-encoded gonococcal porin (Por), opacity-associated protein (Opa), and 3F11 lipo-oligosaccharide (LOS) epitope was investigated. Surface expression of Por mediated selective changes in E. coli antibiotic susceptibility, and coexpression of Opa and the 3F11 LOS epitope mediated bacterial clumping (P<.01). In the human fallopian tube organ-culture model, Opa-producing variants attached up to 44-fold better than control bacteria (P<.01), and Por-producing variants exceeded submucosal invasion of control bacteria by 500-fold (P<.01). Opa and Por each facilitated intracellular invasion 20-40-fold (P<.01). In dual expresser variants, the 3F11 LOS epitope markedly reduced attachment and invasion mediated by Opa or Por. The LOS inhibitory effect was curbed when all 3 factors were expressed, which suggests an additional interaction of the 3 factors at the bacterial surface. Por, Opa, and LOS play important roles in Neisseria gonorrhoeae trafficking across human fallopian tube epithelium.

Antigens, Bacterial↗

PubMed2001

Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae.

We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

Amino Acid Sequence↗

PubMed2001

C4bp binding to porin mediates stable serum resistance of Neisseria gonorrhoeae.

Screening of 29 strains of Neisseria gonorrhoeae revealed that 16/21 serum resistant strains and 0/8 serum sensitive strains bound C4bp, suggesting that C4bp binding to gonococci could contribute to serum resistance. C4bp bound to gonococci retained cofactor (C4b-degrading) function. Using allelic exchange to construct strains with hybrid Por1A/B molecules, we demonstrate that the N-terminal loop (loop 1) of Por1A is required for C4bp binding. Serum resistant Por1B gonococcal strains also bind C4bp via their Por molecule. Using allelic exchange and site-directed mutagenesis, we have shown that loops 5 and 7 together form a negatively charged C4bp binding domain. C4bp-Por1B interactions are ionic in nature (inhibited by high salt as well as by heparin), while the C4bp-Por1A bond is hydrophobic. mAbs directed against SCR1 of the alpha-chain of C4bp inhibit C4bp binding to both Por1A and Por1B. Furthermore, only recombinant C4bp mutant molecules that contain alpha-chain SCR1 bind both Por1A and Por1B gonococci, confirming that SCR1 contains Por binding sites. C4bp alpha-chain monomers do not bind strains with either Por molecule, suggesting that the polymeric form of C4bp is required for binding to gonococci. Inhibition of C4bp binding to serum resistant Por1A and Por1B strains in a serum bactericidal assay using fAb fragments against C4bp SCR1 results in complete killing at 30 min of otherwise fully serum resistant strains in only 10% normal serum, underscoring the role of C4bp in mediating gonococcal serum resistance.

Amino Acid Sequence↗

PubMed2001

DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers.

Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P = 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

Adult↗

PubMed2001

Cloning, overexpression, purification, and immunobiology of an 85-kilodalton outer membrane protein from Haemophilus ducreyi.

We have identified an 85-kDa outer membrane protein that is expressed by all tested strains of Haemophilus ducreyi. Studies of related proteins from other pathogenic bacteria, including Haemophilus influenzae, Pasteurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, and Shigella dysenteriae, suggested a role for these proteins in pathogenesis and immunity. In keeping with the first such described protein from Haemophilus influenzae type B, we termed the H. ducreyi protein D15. The gene encoding the H. ducreyi D15 protein was cloned and sequenced, and the deduced amino acid sequence was found to be most similar to sequences of the D15-related proteins from other Pasteurella spp. The arrangement of the flanking genes was similar to that of H. influenzae Rd and suggested that D15 was part of a multigene operon. Attempts to make a null mutation of the D15 gene were unsuccessful, paralleling results in other D15 gene studies. Overexpression of H. ducreyi D15 in Escherichia coli resulted in a source of recombinant D15 (rD15) from which it was readily purified. rD15 was immunogenic, and it was found that immunization of rabbits with an rD15 vaccine preparation conferred partial protection against a virulent challenge infection. Antisera to an N-terminal peptide recognized all tested strains of H. ducreyi.

Amino Acid Sequence↗

PubMed2000

An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection.

Haemophilus ducreyi expresses a conserved hemoglobin-binding outer-membrane protein (HgbA). To test the role of HgbA in pathogenesis, we infected 9 adults with isolate 35000 and its isogenic hgbA-inactivated mutant (FX504) on their upper arms in a double-blinded, escalating dose-response study. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule-formation rate was 55% (95% confidence interval [CI], 30. 8%-78.5%) at parent sites and 0 (95% CI, 0-10.5%) at mutant sites (P<.0001). The recovery rate of H. ducreyi from surface cultures was 16% (n=142) from parent sites and 0 (n=213) from mutant sites (P<. 0001). H. ducreyi was recovered at biopsy from 6 of 7 parent sites and from 0 of 3 mutant sites. The results indicate that hemoglobin may be a critical source of heme or iron for the establishment of H. ducreyi infection in humans.

Adult↗

PubMed2000

Serum resistance in Haemophilus ducreyi requires outer membrane protein DsrA.

Haemophilus ducreyi is resistant to killing by normal serum antibody and complement. We discovered an H. ducreyi outer membrane protein required for expression of serum resistance and termed it DsrA (for "ducreyi serum resistance A"). The dsrA locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express dsrA. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of H. ducreyi tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring dsrA-nonexpressing strains were complemented in trans with a plasmid expressing dsrA. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in H. ducreyi does not require expression of full-length LOS but does require expression of dsrA. The dsrA locus from eight additional H. ducreyi strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.

Amino Acid Sequence↗

PubMed2000

Development of a serological test for Haemophilus ducreyi for seroprevalence studies.

We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from Haemophilus ducreyi strain 35000 were cloned, overexpressed, and purified from Escherichia coli for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors.

Antibodies, Bacterial↗

PubMed2000

True-lumen collapse in aortic dissection: part I. Evaluation of causative factors in phantoms with pulsatile flow.

PURPOSE: To investigate the causative factors in true-lumen collapse in a model of aortic dissection. MATERIALS AND METHODS: Phantoms with an aortic arch, true and false lumina with abdominal branch vessels, and a distal bifurcation were used to model a Stanford type B aortic dissection. The effects of anatomic factors (entry-tear size, branch-vessel flow distribution, fenestrations, distal reentry communication) and physiologic factors (peripheral resistance in the branch vessels, pump output and rate, vascular compliance) on true-lumen collapse were investigated. The morphology of the true lumen was observed. Branch pressures and flow rates were measured. RESULTS: True-lumen collapse was induced and was exacerbated by an increase in the size of the entry tear, a decrease in the false-lumen outflow caused by occluding the false-lumen branch vessels, and an increase in the true-lumen outflow caused by lowering the peripheral resistance in true-lumen branch vessels. Two kinds of true-lumen collapse depended on pump output. With low pump output and low outflow resistance from the true lumen, the true lumen collapsed. With high pump output and low inflow resistance in the false lumen, the true lumen was compressed. Distal reentry communication between the true and false limbs was more effective than aortic fenestrations in preventing true-lumen collapse. CONCLUSION: True-lumen collapse in this dissection model strongly depends on the difference in the ratios of inflow capacity to outflow capacity in the true and false lumina. Both anatomic and physiologic factors can affect true-lumen collapse.

Aortic Dissection↗

PubMed2000

True-lumen collapse in aortic dissection: part II. Evaluation of treatment methods in phantoms with pulsatile flow.

PURPOSE: To discover and evaluate the effective treatment methods to prevent or relieve true-lumen collapse in models of aortic dissection. MATERIALS AND METHODS: Two phantoms were built to simulate type B aortic dissection. After true-lumen collapse was induced, experiments were conducted to evaluate the effectiveness of clinically relevant variables in relieving the collapse. Variables included entry-tear size, branch-vessel flow distribution, distal reentry communication between the true and false limbs, aortic fenestrations, and pump output. To test the effect of closing the entry tear, a stent-graft was deployed over the entry tear under physiologic conditions in a mock-flow loop. The difference in the effect of each variable on the prevention and relief of true-lumen collapse was also investigated. RESULTS: It was more difficult to relieve true-lumen collapse than it was to prevent it. Placement of a stent-graft over the entry tear was the most effective method of relieving true-lumen collapse. Less-effective procedures included opening a false-lumen outflow branch and opening the distal reentry branch. Opening the fenestration-branch loops, meant to simulate the creation of artificial fenestrations in the intimal flap, did not relieve true-lumen collapse. CONCLUSION: The definitive treatment for true-lumen collapse in aortic dissection is direct repair of the entry tear to decrease false-lumen inflow. Otherwise, increasing the false-lumen outflow and/or creating distal fenestrations between the true and false lumina distal to the level of the compromised aortic branch are less-effective alternatives.

Aortic Dissection↗

PubMed1999

Cardiac Whipple's disease without digestive symptoms.

Whipple's disease is a systemic illness that can affect the heart, causing pericarditis, myocarditis, and valvular endocarditis. We describe a 43-year-old man without gastrointestinal symptoms who underwent mitral and aortic valve replacement for endocarditis, in whom a diagnosis of Whipple's disease was made at operation.

Adult↗

PubMed1998

Binding of complement factor H to loop 5 of porin protein 1A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae.

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.

Amino Acid Sequence↗

PubMed1998

Role of the Haemophilus ducreyi Ton system in internalization of heme from hemoglobin.

By cloning into Escherichia coli and construction of isogenic mutants of Haemophilus ducreyi, we showed that the hemoglobin receptor (HgbA) is TonB dependent. An E. coli hemA tonB mutant expressing H. ducreyi hgbA grew on low levels of hemoglobin as a source of heme only when an intact H. ducreyi Ton system plasmid was present. In contrast, growth on heme by the E. coli hemA tonB mutant expressing hgbA was observed only at high concentrations of heme, was TonB independent, and demonstrated that H. ducreyi HgbA was not sufficient to function as a typical TonB-dependent heme receptor in E. coli. Allelic replacement of the wild-type H. ducreyi exbB, exbD, and tonB loci with the exbB, exbD, and tonB deletion resulted in an H. ducreyi isogenic mutant unable to utilize hemoglobin but able to utilize hemin at the same levels as the parent strain to fulfill its heme requirement. This finding confirms the TonB dependence of HgbA-mediated hemoglobin utilization and suggests that uptake of hemin in H. ducreyi is TonB independent. Additionally, the H. ducreyi Ton system mutant synthesized increased amounts of HgbA and other heme-regulated outer membrane proteins, consistent with derepression of these proteins due to lower intracellular heme and/or iron concentrations in the mutant. Sequencing of the Ton system genes revealed that the arrangement of the genes was exbB exbD tonB. The proximity and structure of these genes suggested that they are transcribed as an operon. This arrangement, as well as the DNA and deduced amino acid sequences of these H. ducreyi genes, was most similar to those from other pasteurellae.

Aldehyde Oxidoreductases↗

PubMed1998

Neisseria gonorrhoeae heme biosynthetic mutants utilize heme and hemoglobin as a heme source but fail to grow within epithelial cells.

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding gamma-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.

Aldehyde Oxidoreductases↗

PubMed1998

Phase variation of hemoglobin utilization in Neisseria gonorrhoeae.

Most Neisseria gonorrhoeae isolates are unable to use human hemoglobin as the sole source of iron for growth (Hgb-), but a minor population is able to do so (Hgb+). This minor population grows luxuriously on hemoglobin, expresses two outer membrane proteins of 42 kDa (HpuA) and 89 kDa (HpuB), and binds hemoglobin under iron-stressed conditions. In addition to the previously reported HpuB, we identified and characterized HpuA, which is encoded by the gene hpuA, located immediately upstream of hpuB. Expression of both proteins was found to be controlled at the translational level by frameshift mutations in a run of guanine residues within the hpuA sequence encoding the mature HpuA protein. The "on-phase" hemoglobin-utilizing variants contained 10 G's, while the "off-phase" variants contained 9 G's. Insertional hpuB mutants of FA19 Hgb+ and FA1090 Hgb+ no longer expressed HpuB but still produced HpuA. A polar insertional mutation of the upstream hpuA gene in FA1090 Hgb+ eliminated production of both HpuA and HpuB, whereas a nonpolar insertional mutant expressed HpuB only. Insertional mutagenesis of either hpuA or hpuB or both substantially decreased the hemoglobin binding ability of the FA1090 Hgb+ variant and prevented growth on hemoglobin plates. Therefore, both HpuA and HpuB were required for the utilization of hemoglobin for growth.

Amino Acid Sequence↗

PubMed1998

Cloning and characterization of tdhA, a locus encoding a TonB-dependent heme receptor from Haemophilus ducreyi.

Haemophilus ducreyi is unable to synthesize heme and must acquire it from its only known host, humans. We cloned and sequenced a gene encoding an outer membrane receptor for heme. It was designated tdhA (for TonB-dependent heme receptor A) since it was related by sequence homology to the family of TonB-dependent receptors. TdhA was strikingly similar to open reading frame HI0113 from the genome of Haemophilus influenzae Rd and also shared homology with five other heme receptors, including HxuC, HemR, HmuR, ChuA, and ShuA, from gram-negative bacteria. An Escherichia coli hemA tonB mutant strongly expressing H. ducreyi tdhA grew on low levels of heme as a source of heme only when an intact H. ducreyi Ton system plasmid was present, formally demonstrating functional TonB dependence. tdhA was expressed poorly in vitro by H. ducreyi and only under conditions of heme limitation. A survey of H. ducreyi revealed that all tested strains but one synthesized small amounts of TdhA in vitro under heme-limiting conditions. Surprisingly, an isogenic mutant of tdhA as well as its parent, 35000, both required the same high levels of heme for growth (50 microgram/ml [77 microM] on agar medium). This result, together with previous findings, suggests that in vitro, the uptake of heme by H. ducreyi is mediated by a TonB- and TdhA-independent mechanism, possibly diffusion.

Amino Acid Sequence↗

PubMed1997

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.

Azirines↗

PubMed1996

Cloning and sequencing of a Haemophilus ducreyi fur homolog.

Iron regulation in a growing number of bacterial species is being attributed to the presence of a fur (ferric uptake regulation) regulatory system. In the presence of iron, Fur acts as a classical negative regulator, binding conserved sequences within the promoter of iron-repressible genes and blocking transcription. Western blot analysis utilizing Escherichia coli Fur antisera detected a band of approximately 17 kDa in soluble extracts of Haemophilus ducreyi. Additionally, Southern blot hybridization of the H. ducreyi chromosome with a meningococcal fur probe indicated that H. ducreyi might contain a fur homolog. This putative fur homolog was cloned into the E. coli vector pACYC184. This clone was capable of repressing expression of a normally Furregulated lacZ fusion in the fur-background of E. coli strain H1780. The deduced amino acid sequence shows H. ducreyi fur to be 54% identical and 73% similar to E. coli fur, containing putative DNA-binding and metal-binding domains. These data demonstrate that H. ducreyi has a functional fur system.

Amino Acid Sequence↗