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Berlin Brandenburg

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  • Male  (18)
  • Chancroid  (18)
  • Virulence
  • 1
    Language: English
    In: The Journal of infectious diseases, November 2012, Vol.206(9), pp.1407-14
    Description: Haemophilus ducreyi encounters several classes of antimicrobial peptides (APs) in vivo and utilizes the sensitive-to-antimicrobial-peptides (Sap) transporter as one mechanism of AP resistance. A mutant lacking the periplasmic solute-binding component, SapA, was somewhat more sensitive to the cathelicidin LL-37 than the parent strain and was partially attenuated for virulence. The partial attenuation led us to question whether the transporter is fully abrogated in the sapA mutant. We generated a nonpolar sapBC mutant, which lacks both inner membrane permeases of the Sap transporter, and tested the mutant for virulence in human volunteers. In vitro, we compared LL-37 resistance phenotypes of the sapBC and sapA mutants. Unlike the sapA mutant, the sapBC mutant was fully attenuated for virulence in human volunteers. In vitro, the sapBC mutant exhibited significantly greater sensitivity than the sapA mutant to killing by LL-37. Similar to the sapA mutant, the sapBC mutant did not affect H. ducreyi's resistance to human defensins. Compared with the sapA mutant, the sapBC mutant exhibited greater attenuation in vivo, which directly correlated with increased sensitivity to LL-37 in vitro. These results strongly suggest that the SapBC channel retains activity when SapA is removed.
    Keywords: Drug Resistance, Bacterial ; Antimicrobial Cationic Peptides -- Pharmacology ; Haemophilus Ducreyi -- Enzymology ; Membrane Transport Proteins -- Metabolism ; Virulence Factors -- Metabolism
    ISSN: 00221899
    E-ISSN: 1537-6613
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  • 2
    Language: English
    In: Infection and Immunity, 2012, Vol. 80(2), p.679
    Description: Sialylated glycoconjugates on the surfaces of mammalian cells play important roles in intercellular communication and self-recognition. The sialic acid preferentially expressed in human tissues is N-acetylneuraminic acid (Neu5Ac). In a process called molecular mimicry, many bacterial pathogens decorate their cell surface glycolipids with Neu5Ac. Incorporation of Neu5Ac into bacterial glycolipids promotes bacterial interactions with host cell receptors called Siglecs. These interactions affect bacterial adherence, resistance to serum killing and phagocytosis, and innate immune responses. Haemophilus ducreyi, the etiologic agent of chancroid, expresses lipooligosaccharides (LOS) that are highly sialylated. However, an H. ducreyi sialyltransferase (lst) mutant, whose LOS contain reduced levels of Neu5Ac, is fully virulent in human volunteers. Recently, a second sialyltransferase gene (Hd0053) was discovered in H. ducreyi, raising the possibility that Hd0053 compensated for the loss of lst during human infection. CMP-Neu5Ac is the obligate nucleotide sugar donor for all bacterial sialyltransferases; LOS derived from an H. ducreyi CMP-Neu5Ac synthetase (neuA) mutant has no detectable Neu5Ac. Here, we compared an H. ducreyi neuA mutant to its wild-type parent in several models of pathogenesis. In human inoculation experiments, the neuA mutant formed papules and pustules at rates that were no different than those of its parent. When grown in media with and without Neu5Ac supplementation, the neuA mutant and its parent had similar phenotypes in bactericidal, macrophage uptake, and dendritic cell activation assays. Although we cannot preclude a contribution of LOS sialylation to ulcerative disease, these data strongly suggest that sialylation of LOS is dispensable for H. ducreyi pathogenesis in humans.
    Keywords: Bacterial Proteins -- Metabolism ; Chancroid -- Microbiology ; Haemophilus Ducreyi -- Metabolism ; Lipopolysaccharides -- Metabolism ; N-Acetylneuraminic Acid -- Metabolism;
    ISSN: 1098-5522
    ISSN: 10985522
    ISSN: 00199567
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  • 3
    Language: English
    In: Infection and immunity, August 2014, Vol.82(8), pp.3492-502
    Description: (p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi, the causative agent of chancroid, has homologs of both relA and spoT, which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli. We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli, overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.
    Keywords: Guanosine Pentaphosphate -- Deficiency ; Haemophilus Ducreyi -- Pathogenicity ; Ligases -- Metabolism ; Pyrophosphatases -- Metabolism
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 4
    Language: English
    In: Infection and immunity, August 2015, Vol.83(8), pp.3281-92
    Description: The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence of Haemophilus ducreyi in humans. Here, we constructed an H. ducreyi dksA mutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation in H. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% of H. ducreyi open reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to the dksA mutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion of dksA downregulated lspB and rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in the dksA mutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators of H. ducreyi gene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.
    Keywords: Bacterial Proteins -- Metabolism ; Chancroid -- Microbiology ; Guanosine Tetraphosphate -- Metabolism ; Haemophilus Ducreyi -- Metabolism
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 5
    Language: English
    In: Infection and immunity, May 2016, Vol.84(5), pp.1514-1525
    Description: Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. MunsonJr, E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.
    Keywords: Adaptation, Physiological ; Gene Expression Profiling ; Stress, Physiological ; Carbon -- Metabolism ; Chancroid -- Microbiology ; Haemophilus Ducreyi -- Physiology
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 6
    In: Infection and Immunity, 2001, Vol. 69(3), p.1483
    Description: Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyi were subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, and lspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.
    Keywords: Chancroid -- Microbiology ; Haemophilus Ducreyi -- Genetics ; RNA, Bacterial -- Biosynthesis ; RNA, Messenger -- Biosynthesis;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 7
    Language: English
    In: The Journal of infectious diseases, 01 March 2009, Vol.199(5), pp.684-92
    Description: A gene expression study of Haemophilus ducreyi identified the hypothetical lipoprotein HD0192, renamed here "fibrinogen binder A" (FgbA), as being preferentially expressed in vivo. To test the role played by fgbA in virulence, an isogenic fgbA mutant (35000HPfgbA) was constructed using H. ducreyi 35000HP, and 6 volunteers were experimentally infected with 35000HP or 35000HPfgbA. The overall pustule-formation rate was 61.1% at parent sites and 22.2% at mutant sites (P = .019). Papules were significantly smaller at mutant sites than at parent sites (13.3 vs. 37.9 mm(2); P = .002) 24 h after inoculation. Thus, fgbA contributed significantly to the virulence of H. ducreyi in humans. In vitro experiments demonstrated that fgbA encodes a fibrinogen-binding protein; no other fibrinogen-binding proteins were identified in 35000HP. fgbA was conserved among clinical isolates of both class I and II H. ducreyi strains, supporting the finding that fgbA is important for H. ducreyi infection.
    Keywords: Bacterial Proteins -- Metabolism ; Chancroid -- Microbiology ; Fibrinogen -- Metabolism ; Haemophilus Ducreyi -- Genetics ; Lipoproteins -- Metabolism
    ISSN: 0022-1899
    E-ISSN: 15376613
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  • 8
    Language: English
    In: The Journal of infectious diseases, 01 August 2009, Vol.200(3), pp.409-16
    Description: Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P 〈 .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.
    Keywords: Bacterial Proteins -- Metabolism ; Carbon-Sulfur Lyases -- Metabolism ; Chancroid -- Microbiology ; Haemophilus Ducreyi -- Genetics
    ISSN: 0022-1899
    E-ISSN: 15376613
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  • 9
    Language: English
    In: The Journal of infectious diseases, 15 April 2008, Vol.197(8), pp.1103-9
    Description: Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%-75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%-73.9%) at 18 mutant-strain sites (P = .51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine.
    Keywords: Bacterial Outer Membrane Proteins -- Biosynthesis ; Bacterial Proteins -- Biosynthesis ; Chancroid -- Microbiology ; Haemophilus Ducreyi -- Pathogenicity ; Membrane Proteins -- Biosynthesis
    ISSN: 0022-1899
    E-ISSN: 15376613
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  • 10
    Language: English
    In: The Journal of infectious diseases, 01 June 2008, Vol.197(11), pp.1531-6
    Description: Haemophilus ducreyi 35000HP contains a cluster of homologues of genes required for the synthesis of enterobacterial common antigen (ECA), suggesting that H. ducreyi may express a putative ECA-like glycoconjugate. WecA initiates the synthesis of ECA by transferring N-acetylglucosamine to undecaprenyl-P, to form lipid I. A wecA mutant (35000HPwecA) was constructed, and 5 volunteers were inoculated at 3 sites with fixed doses of 35000HP on one arm and at 3 sites with varying doses of 35000HPwecA on the other arm. 35000HPwecA caused pustules to form at 3 sites inoculated with a dose 2.5-fold higher than that of 35000HP. However, at sites inoculated with similar doses of 35000HP and 35000HPwecA, pustules developed at 46.7% (95% confidence interval [CI], 23.3%-70.0%) of 15 parent-strain sites and at 8.3% (95% CI, 0.01%-23.6%) of 12 mutant-strain sites (P = .013). Thus, the expression of wecA contributes to the ability of H. ducreyi to cause pustules in humans.
    Keywords: Multigene Family ; Antigens, Bacterial -- Genetics ; Chancroid -- Microbiology ; Haemophilus Ducreyi -- Genetics
    ISSN: 0022-1899
    E-ISSN: 15376613
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