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1.
Mol Microbiol ; 97(6): 1158-67, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26075961

RESUMO

Bacteria encode heat shock proteins that aid in survival during stressful growth conditions. In addition, the major heat shock proteins of the intracellular bacterium Chlamydia trachomatis have been associated with immune pathology and disease. We developed a ChIP-qPCR method to study the regulation of chlamydial heat shock gene regulation during an intracellular infection. This approach allowed us to show that chlamydial heat shock genes are regulated by the transcription factor HrcA within an infected cell, providing validation for previous in vitro findings. Induction of chlamydial heat shock gene expression by elevated temperature was due to loss of HrcA binding to heat shock promoters, supporting a mechanism of derepression. This heat shock response was rapid, whereas recovery of HrcA binding and return to non-stress transcript levels occurred more slowly. We also found that control of heat shock gene expression was differentially regulated over the course of the intracellular Chlamydia infection. There was evidence of HrcA-mediated regulation of heat shock genes throughout the chlamydial developmental cycle, but the level of repression was lower at early times. This is the first study of Chlamydia-infected cells showing the effect of an environmental signal on transcription factor-DNA binding and target gene expression in the bacterium.


Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/fisiologia , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Animais , Proteínas de Bactérias/metabolismo , Fibroblastos/metabolismo , Camundongos , Fatores de Transcrição/metabolismo
2.
Curr Genet ; 62(3): 547-51, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26886234

RESUMO

Intracellular bacteria that reside within a host cell use a variety of strategies to exploit this unique niche. While these organisms are technically challenging to study in the context of an infected host cell, recent advances have led to an improved understanding of how the intracellular environment impacts bacterial gene expression. We recently demonstrated that chromatin immunoprecipitation (ChIP) can be used to quantify transcription factor binding in the obligate intracellular pathogen Chlamydia trachomatis within infected cells. Furthermore, we showed it was possible to experimentally modulate transcription factor binding while simultaneously measuring changes in transcription. Here we discuss these findings as well as other recent work that has used ChIP to study intracellular pathogens within infected cells. We also discuss technical considerations associated with this approach and its possible future applications.


Assuntos
Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Espaço Intracelular/genética , Espaço Intracelular/microbiologia , Animais , Chlamydia/fisiologia , Imunoprecipitação da Cromatina/métodos , Interações Hospedeiro-Parasita/genética , Humanos , Espaço Intracelular/parasitologia
3.
J Bacteriol ; 197(20): 3238-44, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26216849

RESUMO

UNLABELLED: The Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia. IMPORTANCE: This study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.


Assuntos
Chlamydia trachomatis/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Transcrição Gênica/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Fator sigma/genética , Fator sigma/metabolismo , Sistemas de Secreção Tipo III/genética
4.
Infect Immun ; 83(4): 1497-506, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25644003

RESUMO

The bacterial cell envelope is a crucial first line of defense for a systemic pathogen, with production of capsular polysaccharides and maintenance of the peptidoglycan cell wall serving essential roles in survival in the host environment. The LytR-CpsA-Psr proteins are important for cell envelope maintenance in many Gram-positive species. In this study, we examined the role of the extracellular domain of the CpsA protein of the zoonotic pathogen group B Streptococcus in capsule production and cell wall integrity. CpsA has multiple functional domains, including a DNA-binding/transcriptional activation domain and a large extracellular domain. We demonstrated that episomal expression of extracellularly truncated CpsA causes a dominant-negative effect on capsule production when expressed in the wild-type strain. Regions of the extracellular domain essential to this phenotype were identified. The dominant-negative effect could be recapitulated by addition of purified CpsA protein or a short CpsA peptide to cultures of wild-type bacteria. Changes in cell wall morphology were also observed when the dominant-negative peptide was added to wild-type cultures. Fluorescently labeled CpsA peptide could be visualized bound at the mid-cell region near the division septae, suggesting a novel role for CpsA in cell division. Finally, expression of truncated CpsA also led to attenuation of virulence in zebrafish models of infection, to levels below that of a cpsA deletion strain, demonstrating the key role of the extracellular domain in virulence of GBS.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Streptococcus agalactiae/patogenicidade , Peixe-Zebra/microbiologia , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Divisão Celular/genética , Membrana Celular/metabolismo , Parede Celular , Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Estrutura Terciária de Proteína , Infecções Estreptocócicas , Streptococcus agalactiae/citologia , Streptococcus agalactiae/genética
5.
Mol Microbiol ; 94(4): 888-97, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25250726

RESUMO

The pathogenic bacterium Chlamydia replicates in a eukaryotic host cell via a developmental cycle marked by temporal waves of gene expression. We have previously shown that late genes transcribed by the major chlamydial RNA polymerase, σ(66) RNA polymerase, are regulated by a transcriptional repressor EUO. We now report that EUO also represses promoters for a second subset of late genes that are transcribed by an alternative polymerase called σ(28) RNA polymerase. EUO bound in the vicinity of six σ(28) -dependent promoters and inhibited transcription of each promoter. We used a mutational analysis to demonstrate that the EUO binding site functions as an operator that is necessary and sufficient for EUO-mediated repression of σ(28) -dependent transcription. We also verified specific binding of EUO to σ(66) -dependent and σ(28) -dependent promoters with a DNA immunoprecipitation assay. These findings support a model in which EUO represses expression of both σ(66) -dependent and σ(28) -dependent late genes. We thus propose that EUO is the master regulator of late gene expression in the chlamydial developmental cycle.


Assuntos
Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/metabolismo , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Imunoprecipitação , Regiões Promotoras Genéticas , Ligação Proteica , Fator sigma/metabolismo
6.
J Bacteriol ; 194(7): 1668-78, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22287515

RESUMO

Streptococcal pathogens, such as the group B streptococcus (GBS) Streptococcus agalactiae, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the ΔcpsA strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the ΔcpsA strain, was attenuated in human whole blood. However, the ΔcpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Animais , Cápsulas Bacterianas/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Eritrócitos/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Streptococcus agalactiae/química , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Virulência , Peixe-Zebra
7.
J Bacteriol ; 193(2): 411-20, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21097630

RESUMO

Many streptococcal pathogens require a polysaccharide capsule for survival in the host during systemic infection. The highly conserved CpsA protein is proposed to be a transcriptional regulator of capsule production in streptococci, although the regulatory mechanism is unknown. Hydropathy plots of CpsA predict an integral membrane protein with 3 transmembrane domains and only 27 cytoplasmic residues, whereas other members of the LytR_cpsA_psr protein family are predicted to have a single transmembrane domain. This unique topology, with the short cytoplasmic domain, membrane localization, and large extracellular domain, suggests a novel mechanism of transcriptional regulation. Therefore, to determine the actual membrane topology of CpsA, specific protein domains were fused to beta-galactosidase or alkaline phosphatase. Enzymatic assays confirmed that the predicted membrane topology for CpsA is correct. To investigate how this integral membrane protein may be functioning in regulation of capsule transcription, purified full-length and truncated forms of CpsA were used in electrophoretic mobility shift assays to characterize the ability to bind the capsule operon promoter. Assays revealed that full-length, purified CpsA protein binds specifically to DNA containing the capsule promoter region. Furthermore, the large extracellular domain is not required for DNA binding, but all cytoplasmic regions of CpsA are necessary and sufficient for specific binding to the capsule operon promoter. This is the first demonstration of a member of this protein family interacting with its target DNA. Taken together, CpsA, as well as other members of the LytR_cpsA_psr protein family, appears to utilize a unique mechanism of transcriptional regulation.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus/enzimologia , Transativadores/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cápsulas Bacterianas/biossíntese , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Proteínas de Membrana/isolamento & purificação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Streptococcus/química , Streptococcus/metabolismo , Transativadores/isolamento & purificação , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
8.
Methods Mol Biol ; 1689: 147-155, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29027172

RESUMO

Chromatin immunoprecipitation is used to measure the binding of transcription factors to target DNA sequences in order to better understand transcriptional regulation. Here, we describe a process to analyze bacterial transcription factor binding in the context of an infected eukaryotic host cell. Using this approach, we measured the binding kinetics of three Chlamydia trachomatis transcription factors within infected cells, and demonstrated temporal changes in binding.


Assuntos
Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Imunoprecipitação da Cromatina/métodos , DNA Bacteriano , Fibroblastos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Ligação Proteica
9.
Curr Opin Microbiol ; 15(2): 204-10, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22236805

RESUMO

The cell surface of Gram-positive pathogens represents a complex association of glycopolymers that control cell division, homeostasis, immune evasion, tissue invasion, and resistance to antimicrobials. These glycopolymers include the peptidoglycan cell wall, wall-teichoic acids, lipoteichoic acids, and capsular polysaccharide. Disruption of individual factors often results in pleiotropic effects, making it difficult to discern regulation and function. In this review we collate recent work describing these pleiotropic phenotypes, and propose that this is due to coordinated regulation of biosynthesis or modification of these cell surface components. A better understanding of the regulatory networks that control the relative prevalence of each factor on the cell surface or their modulated functions may help facilitate the identification of new targets for antimicrobial therapy.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Membrana Celular/química , Parede Celular/química , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Peptidoglicano/metabolismo , Polissacarídeos Bacterianos/metabolismo , Ácidos Teicoicos/metabolismo
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