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1.
Virulence ; 4(8): 707-15, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24104465

RESUMO

Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Interações Microbianas , Infecções por Orthomyxoviridae/complicações , Pneumonia Estafilocócica/complicações , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Coinfecção/patologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Macaca fascicularis , Masculino , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/patologia
2.
Infect Immun ; 81(7): 2488-98, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23630963

RESUMO

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.


Assuntos
Antígenos de Bactérias/metabolismo , Borrelia burgdorferi/patogenicidade , Inativação Gênica , Ixodes/microbiologia , Doença de Lyme/transmissão , Animais , Antígenos de Bactérias/genética , Vetores Aracnídeos/microbiologia , Vetores Aracnídeos/fisiologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Comportamento Alimentar/fisiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Ixodes/fisiologia , Larva/microbiologia , Larva/fisiologia , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Mutagênese Insercional , Transcrição Gênica
3.
Proc Natl Acad Sci U S A ; 107(16): 7515-20, 2010 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-20368453

RESUMO

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.


Assuntos
Borrelia burgdorferi/metabolismo , Regulação da Expressão Gênica , Genes Bacterianos/genética , Doença de Lyme/microbiologia , Alelos , Animais , Feminino , Genes Bacterianos/fisiologia , Teste de Complementação Genética , Ixodes , Larva/metabolismo , Larva/microbiologia , Camundongos , Modelos Genéticos , Mutação , Fenótipo , Spirochaetales/genética
4.
Infect Immun ; 76(6): 2498-511, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18390998

RESUMO

Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the sigma(N)-sigma(S) regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia burgdorferi/metabolismo , Doença de Lyme/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Imunocompetência , Camundongos , Família Multigênica , Filogenia
5.
Infect Immun ; 75(6): 2753-64, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17371862

RESUMO

Members of the Borrelia burgdorferi paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from re-isolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in Borrelia residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for B. burgdorferi dissemination, tissue tropism, or evasion of the adaptive immune response.


Assuntos
Antígenos de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Expressão Gênica/fisiologia , Doença de Lyme/microbiologia , Transcrição Gênica , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Ensaio de Imunoadsorção Enzimática , Doença de Lyme/genética , Doença de Lyme/metabolismo , Camundongos
6.
Mol Microbiol ; 61(1): 243-58, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16824109

RESUMO

When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT-PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian-like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb-infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out-compete for binding. bba66 promoter mapping revealed putative sigma70 and sigmaS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the -35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either sigmaN or sigmaS resulted in loss of BBA66. Promoter-GFP fusion analysis indicated that the sigma70 and/or sigmaS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , Animais , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Borrelia burgdorferi/crescimento & desenvolvimento , Borrelia burgdorferi/imunologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipoproteínas/metabolismo , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Família Multigênica/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/genética , Fator sigma/metabolismo , Temperatura , Transcrição Gênica/genética
7.
Infect Immun ; 74(6): 3547-53, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16714587

RESUMO

We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.


Assuntos
Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa/fisiologia , Borrelia burgdorferi/patogenicidade , Doença de Lyme/imunologia , Fatores de Virulência/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Antígenos de Bactérias/análise , Borrelia burgdorferi/crescimento & desenvolvimento , Borrelia burgdorferi/imunologia , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Peritônio/microbiologia , Ratos , Ratos Sprague-Dawley
8.
Proteomics ; 6(7): 2121-34, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16485259

RESUMO

Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.


Assuntos
Proteínas de Bactérias/análise , Borrelia burgdorferi/química , Eletroforese em Gel Bidimensional , Proteômica , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Focalização Isoelétrica/métodos , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Frações Subcelulares/química
9.
Int J Med Microbiol ; 295(4): 267-78, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16128401

RESUMO

Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkappaBalpha and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kappaB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkappaBalpha (to render NF-kappaB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1alpha or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kappaB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kappaB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kappaB, subtle autocrine effects of newly synthesized IL-1alpha may contribute, in part, to the control of NF-kappaB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia infection and provide important insights to further our understanding of the pathogenesis of spotted fever group rickettsioses.


Assuntos
Quimiocina CCL2/metabolismo , Células Endoteliais/microbiologia , Interleucina-8/metabolismo , NF-kappa B/fisiologia , Rickettsia rickettsii/fisiologia , Células Cultivadas , Quimiocina CCL2/genética , Células Endoteliais/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Transcrição Gênica
10.
Infect Immun ; 73(7): 3860-8, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15972471

RESUMO

Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.


Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydia/química , Tirosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Dados de Sequência Molecular , Fosforilação , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , Especificidade da Espécie
11.
Infect Immun ; 73(1): 155-65, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15618150

RESUMO

Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-kappaB (NF-kappaB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-kappaB subunits, p65 and p50, and IkappaB proteins, IkappaBalpha and IkappaBbeta. The transcript and protein levels of p50, p65, and IkappaBbeta remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IkappaBalpha at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IkappaBalpha mRNA. The level of IkappaBalpha mRNA gradually returned toward baseline, whereas that of total IkappaBalpha protein remained lower than the corresponding controls. The activities of IKKalpha and IKKbeta, the catalytic subunits of IkappaB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-kappaB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKalpha and IKKbeta, leading to attenuation of nuclear translocation of NF-kappaB. Also, increased activity of IKKalpha was evident later during the infection, coinciding with the late phase of NF-kappaB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-kappaB activation. Since NF-kappaB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.


Assuntos
Células Endoteliais/microbiologia , Proteínas I-kappa B/metabolismo , NF-kappa B/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Rickettsia rickettsii/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Ativação Enzimática , Humanos , Quinase I-kappa B , Lipopolissacarídeos/farmacologia , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Fosforilação , RNA Mensageiro/análise , Transdução de Sinais
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