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1.
AJPM Focus ; 3(4): 100242, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38993712

ABSTRACT

Introduction: Veterans commonly experience both poor health and employment difficulty. However, the research examining potential relationships between chronic physical and mental health conditions and employment in veterans has important limitations. This study examines those potential relationships using large-scale, nationally representative data. The authors' hypothesis was that veterans experiencing these conditions would be less likely to be employed than veterans without the conditions and, further, that there may be differences in these relationships when comparing male veterans with female veterans. Methods: The study team conducted a pooled cross-sectional analysis of nationally representative data from the 2004-2019 administrations of the Medical Expenditures Panel Survey, which had items addressing health conditions, employment, and military experience. The authors assessed the relationship between health conditions and employment using multivariate logistic regression. Control variables included demographics, SES, family size, and survey year. Results: Veterans experiencing diabetes, high blood pressure, stroke, emphysema, arthritis, serious hearing loss, poor self-reported mental health, poor self-reported health, depression, or psychological distress were less likely to be employed than veterans without those conditions, even after adjusting for potential confounding factors. Veterans with diabetes had 25% lesser odds of being employed than veterans without the condition (95% CI=0.65, 0.85). Veterans with increased likelihood of depression had 35% lesser odds of being employed than veterans without depression (95% CI=0.52, 0.81). Conclusions: This study adds evidence to the understanding of the role of chronic health conditions in employment status of veterans. The results support arguments for programs that aid veterans with both their health and their employment.

2.
Infect Immun ; 83(1): 146-60, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25312959

ABSTRACT

There are a paucity of data concerning gene products that could contribute to the ability of Moraxella catarrhalis to colonize the human nasopharynx. Inactivation of a gene (mesR) encoding a predicted response regulator of a two-component signal transduction system in M. catarrhalis yielded a mutant unable to grow in liquid media. This mesR mutant also exhibited increased sensitivity to certain stressors, including polymyxin B, SDS, and hydrogen peroxide. Inactivation of the gene (mesS) encoding the predicted cognate sensor (histidine) kinase yielded a mutant with the same inability to grow in liquid media as the mesR mutant. DNA microarray and real-time reverse transcriptase PCR analyses indicated that several genes previously shown to be involved in the ability of M. catarrhalis to persist in the chinchilla nasopharynx were upregulated in the mesR mutant. Two other open reading frames upregulated in the mesR mutant were shown to encode small proteins (LipA and LipB) that had amino acid sequence homology to bacterial adhesins and structural homology to bacterial lysozyme inhibitors. Inactivation of both lipA and lipB did not affect the ability of M. catarrhalis O35E to attach to a human bronchial epithelial cell line in vitro. Purified recombinant LipA and LipB fusion proteins were each shown to inhibit human lysozyme activity in vitro and in saliva. A lipA lipB deletion mutant was more sensitive than the wild-type parent strain to killing by human lysozyme in the presence of human apolactoferrin. This is the first report of the production of lysozyme inhibitors by M. catarrhalis.


Subject(s)
Moraxella catarrhalis/growth & development , Moraxella catarrhalis/metabolism , Muramidase/antagonists & inhibitors , Protein Kinases/metabolism , Signal Transduction , Transcription Factors/metabolism , Cell Adhesion , Cell Line , Culture Media/chemistry , Epithelial Cells/microbiology , Gene Deletion , Gene Expression Profiling , Genetic Complementation Test , Histidine Kinase , Microarray Analysis , Protein Kinases/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Saliva/immunology , Saliva/microbiology , Transcription Factors/genetics
3.
mBio ; 5(3): e01178-14, 2014 May 20.
Article in English | MEDLINE | ID: mdl-24902122

ABSTRACT

UNLABELLED: Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. IMPORTANCE: Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcγR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcγR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response.


Subject(s)
Bacterial Proteins/immunology , Chancroid/enzymology , Chancroid/immunology , Haemophilus ducreyi/immunology , Phagocytosis , src-Family Kinases/immunology , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , CSK Tyrosine-Protein Kinase , Chancroid/microbiology , Enzyme Activation , Haemophilus ducreyi/chemistry , Haemophilus ducreyi/genetics , Host-Pathogen Interactions , Humans , Lectins/chemistry , Lectins/genetics , Lectins/immunology , Protein Structure, Tertiary , src-Family Kinases/chemistry , src-Family Kinases/genetics
4.
Infect Immun ; 82(6): 2287-99, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24643539

ABSTRACT

Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being upregulated and 291 being downregulated. Among the upregulated genes was one (ORF113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but it did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.


Subject(s)
Bacterial Outer Membrane Proteins/physiology , Moraxella catarrhalis/pathogenicity , Moraxellaceae Infections/microbiology , Nasopharyngeal Diseases/microbiology , Animals , Bacterial Adhesion/physiology , Cell Line , Chinchilla , Disease Models, Animal , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Humans , Membrane Proteins/metabolism , Microbial Sensitivity Tests , Moraxella catarrhalis/drug effects , Moraxella catarrhalis/genetics , Oligonucleotide Array Sequence Analysis , Peptides/pharmacology , Protease Inhibitors/pharmacology
5.
mBio ; 5(1): e01081-13, 2014 Feb 11.
Article in English | MEDLINE | ID: mdl-24520065

ABSTRACT

UNLABELLED: To adapt to stresses encountered in stationary phase, Gram-negative bacteria utilize the alternative sigma factor RpoS. However, some species lack RpoS; thus, it is unclear how stationary-phase adaptation is regulated in these organisms. Here we defined the growth-phase-dependent transcriptomes of Haemophilus ducreyi, which lacks an RpoS homolog. Compared to mid-log-phase organisms, cells harvested from the stationary phase upregulated genes encoding several virulence determinants and a homolog of hfq. Insertional inactivation of hfq altered the expression of ~16% of the H. ducreyi genes. Importantly, there were a significant overlap and an inverse correlation in the transcript levels of genes differentially expressed in the hfq inactivation mutant relative to its parent and the genes differentially expressed in stationary phase relative to mid-log phase in the parent. Inactivation of hfq downregulated genes in the flp-tad and lspB-lspA2 operons, which encode several virulence determinants. To comply with FDA guidelines for human inoculation experiments, an unmarked hfq deletion mutant was constructed and was fully attenuated for virulence in humans. Inactivation or deletion of hfq downregulated Flp1 and impaired the ability of H. ducreyi to form microcolonies, downregulated DsrA and rendered H. ducreyi serum susceptible, and downregulated LspB and LspA2, which allow H. ducreyi to resist phagocytosis. We propose that, in the absence of an RpoS homolog, Hfq serves as a major contributor of H. ducreyi stationary-phase and virulence gene regulation. The contribution of Hfq to stationary-phase gene regulation may have broad implications for other organisms that lack an RpoS homolog. IMPORTANCE: Pathogenic bacteria encounter a wide range of stresses in their hosts, including nutrient limitation; the ability to sense and respond to such stresses is crucial for bacterial pathogens to successfully establish an infection. Gram-negative bacteria frequently utilize the alternative sigma factor RpoS to adapt to stresses and stationary phase. However, homologs of RpoS are absent in some bacterial pathogens, including Haemophilus ducreyi, which causes chancroid and facilitates the acquisition and transmission of HIV-1. Here, we provide evidence that, in the absence of an RpoS homolog, Hfq serves as a major contributor of stationary-phase gene regulation and that Hfq is required for H. ducreyi to infect humans. To our knowledge, this is the first study describing Hfq as a major contributor of stationary-phase gene regulation in bacteria and the requirement of Hfq for the virulence of a bacterial pathogen in humans.


Subject(s)
Gene Expression Regulation, Bacterial , Haemophilus ducreyi/growth & development , Haemophilus ducreyi/genetics , Host Factor 1 Protein/metabolism , Virulence Factors/biosynthesis , Adult , Chancroid/microbiology , Chancroid/pathology , Female , Gene Expression Profiling , Gene Knockout Techniques , Haemophilus ducreyi/pathogenicity , Healthy Volunteers , Host Factor 1 Protein/genetics , Humans , Male , Middle Aged
6.
Infect Immun ; 81(11): 4160-70, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23980107

ABSTRACT

Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression levels of genes encoding other important H. ducreyi virulence factors, including DsrA and Flp1, suggesting a possible global role for Fis in the control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.


Subject(s)
Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Proteins/biosynthesis , Factor For Inversion Stimulation Protein/metabolism , Gene Expression Regulation, Bacterial , Haemophilus ducreyi/genetics , Operon , Artificial Gene Fusion , Factor For Inversion Stimulation Protein/genetics , Gene Deletion , Gene Expression Profiling , Genes, Reporter , Lectins/biosynthesis , Microarray Analysis , Transcription, Genetic , Up-Regulation , Virulence Factors/metabolism , beta-Galactosidase/analysis , beta-Galactosidase/genetics
7.
Plasmid ; 69(2): 180-5, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23219721

ABSTRACT

The lack of a transcriptional reporter system for use in Moraxella catarrhalis has hindered studies of gene regulation in this pathogen. PCR and recombinant DNA methods were used to insert a multicloning site (MCS) and promoterless full-length Escherichia coli lacZ gene, flanked by transcriptional terminators both immediately upstream and downstream, into the M. catarrhalis recombinant plasmid pWW115. Insertion into the MCS in the newly constructed plasmid pASE222 of M. catarrhalis promoter regions controlled by either a repressor (i.e., NsrR) or activator (i.e., PhoB) yielded transcriptional fusion constructs that were appropriately responsive to signal inputs dependent on the host strain genotype, as measured quantitatively by means of a Miller ß-galactosidase assay. The transcriptional reporter plasmid pASE222 should prove to be a useful tool for rapid screening of factors affecting gene expression in M. catarrhalis.


Subject(s)
Genes, Reporter , Lac Operon/genetics , Moraxella catarrhalis/genetics , Transcription, Genetic , Base Sequence , Escherichia coli/enzymology , Escherichia coli/genetics , Molecular Sequence Data , Moraxella catarrhalis/drug effects , Phosphates/pharmacology , Plasmids/genetics , Promoter Regions, Genetic/genetics , Transcription, Genetic/drug effects
8.
Appl Environ Microbiol ; 78(19): 6829-37, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22820329

ABSTRACT

Mycobacterial shuttle vectors contain dual origins of replication for growth in both Escherichia coli and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-kan-MCS2) enabled green fluorescent protein expression in E. coli, Mycobacterium smegmatis, and M. avium at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a lacZα promoter. This high-level fluorescent protein expression allowed easy visualization of M. smegmatis and M. avium in infected macrophages. The M. tuberculosis gene esat-6 was cloned in place of the green fluorescence protein gene (gfp) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-kan-MCS2) yielded high levels of ESAT-6 expression in M. smegmatis. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide. M. smegmatis clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in E. coli, was insufficient to suppress a strong inflammatory response elicited by M. smegmatis or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.


Subject(s)
Gene Expression , Genetic Vectors , Genetics, Microbial/methods , Macrophages/microbiology , Molecular Biology/methods , Mycobacterium/genetics , Antigens, Bacterial/biosynthesis , Antigens, Bacterial/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli/genetics , Fluorescence , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Immune Evasion , Immune Tolerance , Mycobacterium/pathogenicity , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Virulence Factors/biosynthesis , Virulence Factors/genetics
9.
Infect Immun ; 80(3): 982-95, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22184412

ABSTRACT

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames (ORFs) encoding proteins that are involved in a truncated denitrification pathway or in the oxidative stress response, as well as several putative transcriptional regulators. Additionally, 200 M. catarrhalis genes were found to be downregulated when this bacterium was introduced into the nasopharynx. These downregulated genes included ORFs encoding several well-characterized M. catarrhalis surface proteins including Hag, McaP, and MchA1. Real-time reverse transcriptase PCR (RT-PCR) was utilized as a stringent control to validate the results of in vivo gene expression patterns as measured by DNA microarray analysis. Inactivation of one of the genes (MC ORF 1550) that was upregulated in vivo resulted in a decrease in the ability of M. catarrhalis to survive in the chinchilla nasopharynx over a 3-day period. This is the first evaluation of global transcriptome expression by M. catarrhalis cells in vivo.


Subject(s)
Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , Moraxella catarrhalis/pathogenicity , Moraxellaceae Infections/microbiology , Nasopharynx/microbiology , Animals , Chinchilla , Disease Models, Animal , Gene Expression Profiling , Histocytochemistry , Male , Microarray Analysis , Nasopharynx/pathology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
10.
J Infect Dis ; 203(12): 1859-65, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21606544

ABSTRACT

Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.


Subject(s)
Bacterial Proteins/genetics , Chancroid/microbiology , Haemophilus ducreyi/genetics , Haemophilus ducreyi/pathogenicity , Blotting, Western , Female , Humans , Male , Middle Aged , Phagocytosis , Sequence Deletion , Virulence Factors/genetics
11.
J Bacteriol ; 193(11): 2804-13, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21441505

ABSTRACT

Moraxella catarrhalis is a Gram-negative obligate aerobe that is an important cause of human respiratory tract infections. The M. catarrhalis genome encodes a predicted truncated denitrification pathway that reduces nitrate to nitrous oxide. We have previously shown that expression of both the M. catarrhalis aniA (encoding a nitrite reductase) and norB (encoding a putative nitric oxide reductase) genes is repressed by the transcriptional regulator NsrR under aerobic conditions and that M. catarrhalis O35E nsrR mutants are unable to grow in the presence of low concentrations of nitrite (W. Wang, et al., J. Bacteriol. 190:7762-7772, 2008). In this study, we constructed an M. catarrhalis norB mutant and showed that planktonic growth of this mutant is inhibited by low levels of nitrite, whether or not an nsrR mutation is present. To determine the importance of NorB in this truncated denitrification pathway, we analyzed the metabolism of nitrogen oxides by norB, aniA norB, and nsrR norB mutants. We found that norB mutants are unable to reduce nitric oxide and produce little or no nitrous oxide from nitrite. Furthermore, nitric oxide produced from nitrite by the AniA protein is bactericidal for a Moraxella catarrhalis O35E norB mutant but not for wild-type O35E bacteria under aerobic growth conditions in vitro, suggesting that nitric oxide catabolism in M. catarrhalis is accomplished primarily by the norB gene product. Measurement of bacterial protein S-nitrosylation directly implicates nitrosative stress resulting from AniA-dependent nitric oxide formation as a cause of the growth inhibition of norB and nsrR mutants by nitrite.


Subject(s)
Moraxella catarrhalis/enzymology , Nitric Oxide/metabolism , Oxidoreductases/metabolism , Gene Knockout Techniques , Moraxella catarrhalis/drug effects , Moraxella catarrhalis/genetics , Moraxella catarrhalis/growth & development , Nitrites/metabolism , Nitrites/toxicity , Nitrous Oxide/metabolism , Nitrous Oxide/toxicity , Oxidation-Reduction , Oxidoreductases/genetics
12.
Infect Immun ; 79(2): 745-55, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21098105

ABSTRACT

Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990-3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H2O2). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H2O2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H2O2. DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrhalis O35E katA and ahpC mutants for their abilities to scavenge exogenous H2O2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H2O2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Moraxella catarrhalis/drug effects , Moraxella catarrhalis/metabolism , Oxidative Stress , Amino Acid Sequence , Bacterial Proteins/genetics , Catalase/genetics , Catalase/metabolism , Gene Deletion , Gene Expression Regulation, Enzymologic/physiology , Hydrogen Peroxide/pharmacology , Molecular Sequence Data , Oxidative Stress/physiology
13.
Infect Immun ; 78(11): 4779-91, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20805330

ABSTRACT

The Haemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in Escherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Haemophilus ducreyi/metabolism , Protein Kinases/metabolism , Signal Transduction , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Electrophoretic Mobility Shift Assay , Haemophilus ducreyi/genetics , Haemophilus ducreyi/pathogenicity , Humans , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Kinases/chemistry , Protein Kinases/genetics , Regulon , Reverse Transcriptase Polymerase Chain Reaction , Virulence , Virulence Factors/genetics , Virulence Factors/metabolism
14.
BMC Microbiol ; 10: 64, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20184753

ABSTRACT

BACKGROUND: Intracellular bacterial pathogens depend on acquisition of iron for their success as pathogens. The host cell requires iron as an essential component for cellular functions that include innate immune defense mechanisms. The transferrin receptor TfR1 plays an important part for delivering iron to the host cell during infection. Its expression can be modulated by infection, but its essentiality for bacterial intracellular survival has not been directly investigated. RESULTS: We identified two distinct iron-handling scenarios for two different bacterial pathogens. Francisella tularensis drives an active iron acquisition program via the TfR1 pathway program with induction of ferrireductase (Steap3), iron membrane transporter Dmt1, and iron regulatory proteins IRP1 and IRP2, which is associated with a sustained increase of the labile iron pool inside the macrophage. Expression of TfR1 is critical for Francisella's intracellular proliferation. This contrasts with infection of macrophages by wild-type Salmonella typhimurium, which does not require expression of TfR1 for successful intracellular survival. Macrophages infected with Salmonella lack significant induction of Dmt1, Steap3, and IRP1, and maintain their labile iron pool at normal levels. CONCLUSION: The distinction between two different phenotypes of iron utilization by intracellular pathogens will allow further characterization and understanding of host-cell iron metabolism and its modulation by intracellular bacteria.


Subject(s)
Francisella tularensis/physiology , Iron/metabolism , Macrophages/metabolism , Receptors, Transferrin/metabolism , Salmonella typhimurium/physiology , Animals , Cell Growth Processes/physiology , Cell Line , Cytoplasmic Vesicles/metabolism , Francisella tularensis/pathogenicity , Gene Expression Regulation , Homeostasis , Intracellular Space/microbiology , Macrophages/microbiology , Metabolic Networks and Pathways , Mice , Receptors, Transferrin/genetics , Salmonella typhimurium/pathogenicity , Up-Regulation
15.
BMC Microbiol ; 9: 207, 2009 Sep 25.
Article in English | MEDLINE | ID: mdl-19781080

ABSTRACT

BACKGROUND: Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing. RESULTS: Moraxella catarrhalis strain E22 containing plasmid pLQ510 was shown to inhibit the growth of M. catarrhalis strain O35E. Two genes (mcbA and mcbB) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (mcbC) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (mcbI) immediately downstream from mcbC encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the mcbI gene in M. catarrhalis O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the mcbA, mcbB, mcbC, and mcbI ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several M. catarrhalis strains including O12E. Inactivation of the mcbA, mcbB, or mcbC ORFs in M. catarrhalis O12E eliminated the ability of this strain to inhibit the growth of M. catarrhalis O35E. In co-culture experiments involving a M. catarrhalis strain containing the mcbABCI locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth. CONCLUSION: This is the first description of a bacteriocin and its cognate immunity factor produced by M. catarrhalis. The killing activity of the McbC protein raises the possibility that it might serve to lyse other M. catarrhalis strains that lack the mcbABCI locus, thereby making their DNA available for lateral gene transfer.


Subject(s)
Antibiosis , Bacteriocins/genetics , Moraxella catarrhalis/genetics , Amino Acid Sequence , Bacteriocins/immunology , Cloning, Molecular , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Moraxella catarrhalis/immunology , Multigene Family , Open Reading Frames , Plasmids , Sequence Deletion
16.
J Infect Dis ; 200(3): 409-16, 2009 Aug 01.
Article in English | MEDLINE | ID: mdl-19552526

ABSTRACT

Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.


Subject(s)
Bacterial Proteins/metabolism , Carbon-Sulfur Lyases/metabolism , Chancroid/microbiology , Haemophilus ducreyi/genetics , Haemophilus ducreyi/pathogenicity , Adult , Bacterial Proteins/genetics , Biological Assay , Carbon-Sulfur Lyases/genetics , Chancroid/pathology , Female , Humans , Male , Middle Aged , Mutation , Skin/microbiology , Skin/pathology , Virulence
17.
Infect Immun ; 77(8): 3402-11, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19451237

ABSTRACT

The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.


Subject(s)
Bacterial Outer Membrane Proteins/biosynthesis , Bacterial Proteins/biosynthesis , Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial , Haemophilus ducreyi/physiology , Bacterial Proteins/genetics , DNA, Bacterial/metabolism , Electrophoretic Mobility Shift Assay , Gene Expression Profiling , Gene Knockout Techniques , Humans , Lectins/biosynthesis , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Binding , Signal Transduction
18.
J Bacteriol ; 190(23): 7762-72, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18820017

ABSTRACT

Growth of Moraxella catarrhalis in a biofilm resulted in marked upregulation of two open reading frames (ORFs), aniA and norB, predicted to encode a nitrite reductase and a nitric oxide reductase, respectively (W. Wang, L. Reitzer, D. A. Rasko, M. M. Pearson, R. J. Blick, C. Laurence, and E. J. Hansen, Infect. Immun. 75:4959-4971, 2007). An ORF designated nsrR, which was located between aniA and norB, was shown to encode a predicted transcriptional regulator. Inactivation of nsrR resulted in increased expression of aniA and norB in three different M. catarrhalis strains, as measured by both DNA microarray analysis and quantitative reverse transcriptase PCR. Provision of a wild-type nsrR gene in trans in an nsrR mutant resulted in decreased expression of the AniA protein. DNA microarray analysis revealed that two other ORFs (MC ORF 683 and MC ORF 1550) were also consistently upregulated in an nsrR mutant. Consumption of both nitrite and nitric oxide occurred more rapidly with cells of an nsrR mutant than with wild-type cells. However, growth of nsrR mutants was completely inhibited by a low level of sodium nitrite. This inhibition of growth by nitrite was significantly reversed by introduction of an aniA mutation into the nsrR mutant and was completely reversed by the presence of a wild-type nsrR gene in trans. NsrR regulation of the expression of aniA was sensitive to nitrite, whereas NsrR regulation of norB was sensitive to nitric oxide.


Subject(s)
Bacterial Proteins/metabolism , Moraxella catarrhalis/metabolism , Bacterial Proteins/genetics , Base Sequence , DNA, Bacterial/genetics , Gene Expression Profiling , Gene Expression Regulation, Bacterial/physiology , Nitric Oxide/metabolism , Nitrites/metabolism , Open Reading Frames , Transcription Factors/genetics , Transcription Factors/metabolism
19.
Infect Immun ; 76(11): 5322-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18678656

ABSTRACT

The Moraxella catarrhalis ubiquitous surface proteins (UspAs) are autotransporter molecules reported to interact with a variety of different host proteins and to affect processes ranging from serum resistance to cellular adhesion. The role of UspA1 as an adhesin has been confirmed with a number of different human cell types and is mediated by binding to eukaryotic proteins including carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), fibronectin, and laminin. A distinct difference in the ability of prototypical M. catarrhalis strains to adhere to CEACAM-expressing cell lines prompted us to perform strain-specific structure-function analyses of UspA1 proteins. In this study, we characterized CEACAM binding by a diverse set of UspA1 proteins and showed that 3 out of 10 UspA1 proteins were incapable of binding CEACAM. This difference resulted from the absence of a distinct CEACAM binding motif in nonadhering strains. Our sequence analysis also revealed a single M. catarrhalis isolate that lacked the fibronectin-binding motif and was defective in adherence to Chang conjunctival epithelial cells. These results clearly demonstrate that UspA1-associated adhesive functions are not universally conserved. Instead, UspA1 proteins must be considered as variants with the potential to confer both different cell tropisms and host cell responses.


Subject(s)
Bacterial Adhesion/genetics , Bacterial Outer Membrane Proteins/genetics , Moraxella catarrhalis/genetics , Moraxella catarrhalis/pathogenicity , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Blotting, Western , Cell Adhesion Molecules/metabolism , Cells, Cultured , Humans , Molecular Sequence Data , Moraxella catarrhalis/metabolism , Sequence Homology, Amino Acid
20.
Infect Immun ; 76(11): 5330-40, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18678659

ABSTRACT

Ubiquitous surface protein A molecules (UspAs) of Moraxella catarrhalis are large, nonfimbrial, autotransporter proteins that can be visualized as a "fuzzy" layer on the bacterial surface by transmission electron microscopy. Previous studies attributed a wide array of functions and binding activities to the closely related UspA1, UspA2, and/or UspA2H protein, yet the molecular and phylogenetic relationships among these activities remain largely unexplored. To address this issue, we determined the nucleotide sequence of the uspA1 genes from a variety of independent M. catarrhalis isolates and compared the deduced amino acid sequences to those of the previously characterized UspA1, UspA2, and UspA2H proteins. Rather than being conserved proteins, we observed a striking divergence of individual UspA1, UspA2, and UspA2H proteins resulting from the modular assortment of unrelated "cassettes" of peptide sequence. The exchange of certain variant cassettes correlates with strain-specific differences in UspA protein function and confers differing phenotypes upon these mucosal surface pathogens.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , DNA, Bacterial/genetics , Moraxella catarrhalis/chemistry , Moraxella catarrhalis/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Base Sequence , Consensus Sequence/genetics , Molecular Sequence Data , Moraxella catarrhalis/metabolism , Phenotype , Polymerase Chain Reaction , Protein Structure, Tertiary/genetics , Sequence Homology, Amino Acid
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