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  • 1
    Language: English
    In: Journal of Bacteriology, 2015, Vol.197(1-2), p.277(9)
    Description: The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H2O2), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H2O2. Conversely, the pgdX mutant was more resistant to H2O2 due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H2O2-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.
    Keywords: Overlapping Genes – Research ; Haemophilus Influenzae – Risk Factors ; Haemophilus Influenzae – Care and Treatment ; Haemophilus Influenzae – Genetic Aspects ; Oxidative Stress – Research ; Gene Mutation – Research
    ISSN: 0021-9193
    E-ISSN: 10985530
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  • 2
    Language: English
    In: Journal of Bacteriology, 2013, Vol.195(15-16), p.3486(17)
    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: Chancroid – Causes of ; Gene Expression – Research ; Hemophilus Infections – Research ; Virulence (Microbiology) – Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: Journal of Infectious Diseases, Sept 1, 2013, Vol.208(5), p.720(8)
    Keywords: Haemophilus Influenzae -- Genetic Aspects ; Haemophilus Influenzae -- Research ; Genetic Variation -- Research
    ISSN: 0022-1899
    Source: Cengage Learning, Inc.
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  • 4
    Language: English
    In: Journal of Bacteriology, 2014, Vol.196(23-24), p.4012(14)
    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: Hemophilus Infections – Causes of ; Sexually Transmitted Diseases – Risk Factors ; Transcription (Genetics) – Analysis
    ISSN: 0021-9193
    E-ISSN: 10985530
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  • 5
    In: The Journal of Bacteriology, 2001, Vol. 183(19), p.5756
    Description: DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3' sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.
    Keywords: Mutation ; Haemophilus Ducreyi -- Drug Effects ; Lipopolysaccharides -- Metabolism ; Pyocins -- Pharmacology;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 6
    In: The Journal of Bacteriology, 2007, Vol. 189(3), p.1004
    Description: Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.
    Keywords: Biology;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 7
    In: Journal of Bacteriology, July, 1993, Vol.175(13-14), p.4569(3)
    Description: Protein D binds an I-labeled IgD myeloma protein, produced by a cloned hpd gene from Haemophilus influenzae, exhibits glycerophosphodiester phosphodiesterase activity. This is determined by a spectrophotometric analysis involving sn-glycerol 3 phosphate dehydrogenase and NAD. Protein D of H. influenzae is homologous to the periplasmic glycerophosphodiester phsophodiesterase produced by glpQ gene of Escherichia coli. An increased activity in E. coli extracts produces recombinant protein D and the subcellular localization of the recombinant activity.
    Keywords: Haemophilus Influenzae -- Influence ; Spectrophotometry -- Usage
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 8
    Language: English
    In: Journal of Infectious Diseases, June 1, 2008, Vol.197(11), p.1531(6)
    Description: 〈p〉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 wee A contributes to the ability of H. ducreyi to cause pustules in humans.〈/p〉
    Keywords: Hemophilus Infections -- Development And Progression ; Virulence (Microbiology) -- Research ; Chancroid -- Development And Progression ; Chancroid -- Research
    ISSN: 0022-1899
    E-ISSN: 15376613
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  • 9
    In: Proceedings of the National Academy of Sciences of the United States, April 15, 1997, Vol.94(8), p.4056(6)
    Description: Little is known about the virulence mechanisms employed by Haemophilus ducreyi in the production of genital ulcers. This Gram-negative bacterium previously has been shown to produce a soluble cytotoxic activity that kills HeLa and HEp-2 cells. We have now identified a cluster of three H. ducreyi genes that encode this cytotoxic activity. The predicted proteins encoded by these genes are most similar to the products of the Escherichia coli cdtABC genes that comprise the cytolethal distending toxin (CDT) of this enteric pathogen. Eleven of 12 H. ducreyi strains were shown to possess this gene cluster and culture supernatants from these strains readily killed HeLa cells. The culture supernatant from a single strain of H. ducreyi that lacked these genes was unable to kill HeLa cells. When the H. ducreyi cdtABC gene cluster was cloned into E. coli, culture supernatant from the recombinant E. coli clone killed HeLa cells. A monoclonal antibody that neutralized this soluble cytotoxic activity of H. ducreyi was shown to bind to the H. ducreyi cdtC gene product. This soluble H. ducreyi cytotoxin may play a role in the development or persistence of the ulcerative lesions characteristic of chancroid.
    Keywords: Hemophilus Infections -- Research ; Blood Serum -- Research ; Ulcers
    ISSN: 0027-8424
    Source: Cengage Learning, Inc.
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  • 10
    In: Journal of Bacteriology, April, 2000, Vol.182(7-8), p.2292(7)
    Description: Results indicate that the lipooligosaccharide galactosyltransferase II gene in Haemophilus ducreyi codes for a 33,400 dalton protein which bears homology to glycosyltransferases of other bacteria. Data show that the biosynthetic defect lgtB in this gene does not influence serum-sensitive phenotype.
    Keywords: Chancroid -- Genetic Aspects ; Endotoxins -- Genetic Aspects ; Pathogenic Bacteria -- Physiological Aspects ; Hemophilus Infections -- Physiological Aspects
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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