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Berlin Brandenburg

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  • Chancroid  (25)
  • Virulence
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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, December 2014, Vol.196(23), pp.4012-25
    Description: 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.
    Keywords: Gene Expression Regulation, Bacterial ; Stress, Physiological ; Bacterial Proteins -- Metabolism ; Cell Membrane -- Physiology ; Haemophilus Ducreyi -- Physiology ; Protein Kinases -- Metabolism ; Sigma Factor -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 3
    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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  • 4
    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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  • 5
    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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  • 6
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(4), p.e0124373
    Description: Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 7
    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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  • 8
    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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  • 9
    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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  • 10
    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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