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  • 1
    Language: English
    In: Infection and immunity, February 2013, Vol.81(2), pp.608-17
    Description: The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.
    Keywords: Bacterial Proteins -- Metabolism ; Carbon -- Metabolism ; Chancroid -- Metabolism ; Haemophilus Ducreyi -- Metabolism
    ISSN: 00199567
    E-ISSN: 1098-5522
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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, 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: 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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  • 6
    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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  • 7
    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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  • 8
    In: Infection and Immunity, 2008, Vol. 76(4), p.1608
    Description: The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 9
    In: Infection and Immunity, 2006, Vol. 74(2), p.1394
    Description: Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.
    Keywords: Mutation ; Bacterial Outer Membrane Proteins -- Genetics ; Chancroid -- Pathology ; Haemophilus Ducreyi -- Pathogenicity;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 10
    In: Infection and Immunity, 2004, Vol. 72(8), p.4528
    Description: Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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