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  • 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: Journal of bacteriology, August 2013, Vol.195(15), pp.3486-502
    Description: Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates the transmission of human immunodeficiency virus type 1. In humans, H. ducreyi is surrounded by phagocytes and must adapt to a hostile environment to survive. To sense and respond to environmental cues, bacteria frequently use two-component signal transduction (2CST) systems. The only obvious 2CST system in H. ducreyi is CpxRA; CpxR is a response regulator, and CpxA is a sensor kinase. Previous studies by Hansen and coworkers showed that CpxR directly represses the expression of dsrA, the lspB-lspA2 operon, and the flp operon, which are required for virulence in humans. They further showed that CpxA functions predominantly as a phosphatase in vitro to maintain the expression of virulence determinants. Since a cpxA mutant is avirulent while a cpxR mutant is fully virulent in humans, CpxA also likely functions predominantly as a phosphatase in vivo. To better understand the role of H. ducreyi CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. Activation of CpxR by deletion of cpxA repressed nearly 70% of its targets, including seven established virulence determinants. Inactivation of CpxR by deletion of cpxR differentially regulated few genes and increased the expression of one virulence determinant. We identified a CpxR binding motif that was enriched in downregulated but not upregulated targets. These data reinforce the hypothesis that CpxA phosphatase activity plays a critical role in controlling H. ducreyi virulence in vivo. Characterization of the downregulated genes may offer new insights into pathogenesis.
    Keywords: Gene Expression Regulation, Bacterial ; Bacterial Proteins -- Metabolism ; Haemophilus Ducreyi -- Genetics ; Phosphoprotein Phosphatases -- Metabolism ; Protein Kinases -- Metabolism ; Repressor Proteins -- Metabolism ; Virulence Factors -- Biosynthesis
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 3
    Language: English
    In: Infection and immunity, September 2010, Vol.78(9), pp.3898-904
    Description: Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HPDeltacpxA. In human inoculation experiments, 35000HPDeltacpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HPDeltacpxA, confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HPDeltacpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.
    Keywords: Bacterial Proteins -- Physiology ; Haemophilus Ducreyi -- Pathogenicity ; Protein Kinases -- Physiology
    ISSN: 00199567
    E-ISSN: 1098-5522
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  • 4
    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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  • 5
    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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  • 6
    Language: English
    In: Microbial Pathogenesis, 2006, Vol.40(3), pp.110-115
    Description: express two porin proteins, termed OmpP2A and OmpP2B. To test whether expression of OmpP2A and OmpP2B was necessary for virulence in humans, eight volunteers were experimentally infected with the parent (35000HP) in one arm and a double OmpP2A OmpP2B mutant (35000HP::P2AB) in the other arm. The pustule formation rates were 58.3% (95% CI, 33.2–83.5%) for the parent and 41.7% (95% CI, 19.3–64.0%) for the mutant ( =0.25). Biopsy of 35000HP and 35000HP::P2AB-infected sites yielded similar amounts of bacteria in quantitative culture. These results indicate that expression of OmpP2A and OmpP2B is not necessary to initiate disease or to progress to pustule formation in humans.
    Keywords: Pustule Formation ; Haemophilus Ducreyi ; Porin Proteins ; Uirulence ; Human Challenge Model ; Biology ; Chemistry
    ISSN: 0882-4010
    E-ISSN: 1096-1208
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