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  • Bacterial Proteins
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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, 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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  • 3
    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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  • 4
    Language: English
    In: Infection and immunity IAI, 2010, Vol.78(9), pp.3898-3904
    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, 35000HPΔcpxA. In human inoculation experiments, 35000HPΔcpxA 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 35000HPΔcpxA, 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. 35000HPΔcpxA 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. ; Includes references ; p. 3898-3904.
    Keywords: Bacterial Proteins -- Physiology ; Haemophilus Ducreyi -- Pathogenicity ; Protein Kinases -- Physiology;
    ISSN: 0019-9567
    E-ISSN: 10985522
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  • 5
    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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  • 6
    In: Infection and Immunity, 2005, Vol. 73(1), p.599
    Description: Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of SALMONELLA: We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low- passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla beta- defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.
    Keywords: Haemophilus Influenzae ; Salmonella ; Haemophilus Influenzae ; Salmonella ; Operons ; Otitis Media ; Antimicrobial Peptides ; Middle Ear ; Sapa Gene ; Immunity ; Mutation ; Respiratory Tract ; Promoters ; Nasopharynx ; Plasmids ; Defensins ; Operons ; Otitis Media ; Antimicrobial Peptides ; Middle Ear ; Sapa Gene ; Immunity ; Mutation ; Respiratory Tract ; Promoters ; Nasopharynx ; Plasmids ; Defensins ; Ear, Nose and Respiratory Tract ; Bacterial Genetics ; Eap Operon ; Eap Operon;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 7
    In: Infection and Immunity, 2007, Vol. 75(1), p.113
    Description: Haemophilus ducreyi is a gram-negative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of H. ducreyi strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains D-glycero-D-manno-heptose in the main oligosaccharide chain extension; the lbgB gene has been shown to encode the DD-heptosyltransferase. The lbgB gene is found in a gene cluster together with the lbgA gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes, rpmE and xthA, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of H. ducreyi strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks DD-heptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the DD-heptose-deficient 33921 LOS, we cloned and sequenced the corresponding lbgAB genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains xthA and rpmE. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP lbgAB genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the lbgAB genes can serve as one indicator of the classification of H. ducreyi strains.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 8
    In: Infection and Immunity, 2005, Vol. 73(10), p.6727
    Description: Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha 2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.
    Keywords: Transposons ; Galactose ; N-Acetyllactosamine ; Monoclonal Antibodies ; ABC Transporter ; Chancroid ; Agglutinins ; Lectins ; Infection ; Gel Electrophoresis ; Sialic Acids ; Lipooligosaccharides ; Haemophilus Ducreyi ; Genetics and Evolution ; Bacteria ; Microorganisms & Parasites;
    ISSN: 0019-9567
    ISSN: 00199567
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  • 9
    In: Infection and Immunity, 2002, Vol. 70(10), p.5887
    Description: Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. In the present study, the Neisseria gonorrhoeae lgtA lipooligosaccharide glycosyltransferase gene was used to identify a homologue in the genome of H. ducreyi. The putative H. ducreyi glycosyltransferase gene (designated lgtA) was cloned and insertionally inactivated, and an isogenic mutant was constructed. Structural studies demonstrated that the lipooligosaccharide isolated from the mutant strain lacked N-acetylglucosamine and distal sugars found in the lipooligosaccharide produced by the parental strain. The isogenic mutant was transformed with a recombinant plasmid containing the putative glycosyltransferase gene. This strain produced the lipooligosaccharide glycoforms produced by the parental strain, confirming that the lgtA gene encodes the N-acetylglucosamine glycosyltransferase.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 10
    In: Infection and Immunity, 2000, Vol. 68(6), p.3352
    Description: To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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