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  • Baker, Beth  (6)
  • Spinola, Stanley M.  (6)
  • 1
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    Online Resource
    Activation of the CpxRA System by Deletion of cpxA Impairs the Ability of Haemophilus ducreyi To Infect Humans
    American Society for Microbiology ; 2010
    In:  Infection and Immunity Vol. 78, No. 9 ( 2010-09), p. 3898-3904
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 78, No. 9 ( 2010-09), p. 3898-3904
    Abstract: 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, 35000HPΔ cpxA . In human inoculation experiments, 35000HPΔ cpxA 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 35000HPΔ cpxA , 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. 35000HPΔ cpxA 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.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00432-10
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
    detail.hit.zdb_id: 1483247-1
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  • 2
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    Online Resource
    DksA and (p)ppGpp Have Unique and Overlapping Contributions to Haemophilus ducreyi Pathogenesis in Humans
    American Society for Microbiology ; 2015
    In:  Infection and Immunity Vol. 83, No. 8 ( 2015-08), p. 3281-3292
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 83, No. 8 ( 2015-08), p. 3281-3292
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00692-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 1483247-1
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  • 3
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    Haemophilus ducreyi Hfq Contributes to Virulence Gene Regulation as Cells Enter Stationary Phase
    American Society for Microbiology ; 2014
    In:  mBio Vol. 5, No. 1 ( 2014-02-28)
    Details
    In: mBio, American Society for Microbiology, Vol. 5, No. 1 ( 2014-02-28)
    Abstract: Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi , which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.01081-13
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 2557172-2
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  • 4
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    Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers
    American Society for Microbiology ; 2016
    In:  Infection and Immunity Vol. 84, No. 5 ( 2016-05), p. 1514-1525
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 84, No. 5 ( 2016-05), p. 1514-1525
    Abstract: 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. Munson, Jr., 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.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00048-16
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 1483247-1
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  • 5
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    Activation of CpxRA in Haemophilus ducreyi Primarily Inhibits the Expression of Its Targets, Including Major Virulence Determinants
    American Society for Microbiology ; 2013
    In:  Journal of Bacteriology Vol. 195, No. 15 ( 2013-08), p. 3486-3502
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 195, No. 15 ( 2013-08), p. 3486-3502
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00372-13
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 6
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    Haemophilus ducreyi RpoE and CpxRA Appear To Play Distinct yet Complementary Roles in Regulation of Envelope-Related Functions
    American Society for Microbiology ; 2014
    In:  Journal of Bacteriology Vol. 196, No. 23 ( 2014-12), p. 4012-4025
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 196, No. 23 ( 2014-12), p. 4012-4025
    Abstract: Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi , does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi .
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.02034-14
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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