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Berlin Brandenburg

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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 bacteriology, 2012, Vol.194(8), pp.1927-1933
    Description: We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic. ; p. 1927-1933.
    Keywords: Vaccines ; Haemophilus Influenzae ; Biogenesis ; Pathogenesis ; Humans ; Pathogens ; Multigene Family ; Mutants ; Fimbriae ; Biofilm ; Mass Spectrometry ; Operon ; Bacteriology ; Proteins
    ISSN: 0021-9193
    E-ISSN: 10985530
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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
    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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  • 9
    In: The Journal of Bacteriology, 2005, Vol. 187(13), p.4627
    Description: In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.
    Keywords: Bacterial Genetics ; Genetics and Evolution;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 10
    In: The Journal of Bacteriology, 2000, Vol. 182(8), p.2292
    Description: Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the los of strain A77 lacks the galactose residue found in the N- acetyllactosamine portion of the strain 35000HP los as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex los glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the los of strain A77 and lacks the 3F11- binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the los mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum- resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.
    Keywords: Haemophilus Ducreyi ; Chancroid ; Genetic Analysis ; Etiology ; Virulence ; N-Acetyllactosaminide a-1,3-Galactosaminyltransferase ; Genetics and Evolution;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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