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

ABSTRACT To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.

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The conserved 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi has homology to PAL.

Infection and Immunity ◽

10.1128/iai.64.6.1950-1955.1996 ◽

1996 ◽

Vol 64 (6) ◽

pp. 1950-1955 ◽

Cited By ~ 24

Author(s):

S M Spinola ◽

T J Hiltke ◽

K Fortney ◽

K L Shanks

Keyword(s):

Molecular Weight ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Haemophilus Ducreyi

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HmbR, a Hemoglobin-Binding Outer Membrane Protein of Neisseria meningitidis, Undergoes Phase Variation

Journal of Bacteriology ◽

10.1128/jb.181.7.2067-2074.1999 ◽

1999 ◽

Vol 181 (7) ◽

pp. 2067-2074 ◽

Cited By ~ 70

Author(s):

Anthony R. Richardson ◽

Igor Stojiljkovic

Keyword(s):

Membrane Protein ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Phase Variation ◽

Membrane Receptors ◽

Phenotypic Switch ◽

Reading Frame ◽

Single Colony ◽

Hemoglobin Binding

ABSTRACT Neisseria meningitidis uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the hmbR expression pattern in strains in which the hpuB gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some hpuB mutants is suggestive of phase variation of hmbR. The length of the poly(G) tract starting at position +1164 of hmbR absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring thehmbR reading frame. Inactivation of hmbR in these strains resulted in a loss of Hb utilization, demonstrating that the change in the hmbR gene was responsible for the phenotypic switch. The switching rates from hmbR phase off to phase on were ∼5 × 10−4 in four serogroup C strains, 2 × 10−2 in the serogroup A isolate, and 7 × 10−6 in the serogroup B isolate.

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Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E)

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1997.tb12725.x ◽

2006 ◽

Vol 156 (2) ◽

pp. 187-191 ◽

Cited By ~ 8

Author(s):

B. Fouz ◽

R. Mazoy ◽

F. Vázquez ◽

M.L. Lemos ◽

C. Amaro

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Vibrio Vulnificus ◽

Hemoglobin Binding

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The major outer membrane protein of Haemophilus ducreyi consists of two OmpA homologs.

Journal of Bacteriology ◽

10.1128/jb.179.5.1764-1773.1997 ◽

1997 ◽

Vol 179 (5) ◽

pp. 1764-1773 ◽

Cited By ~ 36

Author(s):

J Klesney-Tait ◽

T J Hiltke ◽

I Maciver ◽

S M Spinola ◽

J D Radolf ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Major Outer Membrane Protein ◽

Haemophilus Ducreyi

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The LspB Protein Is Involved in the Secretion of the LspA1 and LspA2 Proteins by Haemophilus ducreyi

Infection and Immunity ◽

10.1128/iai.72.4.1874-1884.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1874-1884 ◽

Cited By ~ 25

Author(s):

Christine K. Ward ◽

Jason R. Mock ◽

Eric J. Hansen

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Culture Supernatant ◽

Cell Envelope ◽

Rabbit Model ◽

Haemophilus Ducreyi ◽

Pcr Analysis ◽

Supernatant Fluid ◽

Reading Frame

ABSTRACT The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

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