RESUMO
The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.
Assuntos
Infecções por Chlamydia , Chlamydia muridarum , Humanos , Camundongos , Animais , Chlamydia muridarum/genética , Técnicas de Inativação de Genes , Deleção de Genes , Chlamydia trachomatis/genética , Replicon , Modelos Animais , Infecções por Chlamydia/microbiologiaRESUMO
The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia's ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.
Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidade , Mutagênese/genética , Actinas/genética , Alelos , Animais , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Fluorescência , Deleção de Genes , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C3H , Fosfoproteínas/genética , Virulência/genéticaRESUMO
Enterotoxigenic Escherichia coli is a major cause of acute diarrheal illness worldwide and is responsible for high infant and child mortality rates in developing nations. Two types of enterotoxins, one heat labile and the other heat stable, are known to cause diarrhea. The expression of soluble heat-labile toxin is subject to catabolite (glucose) activation, and three binding sites for cAMP receptor protein (CRP or CAP) were identified upstream and within the toxin promoter by DNase I footprinting. One CRP operator is centered at -31.5, thus encompassing the promoter's -35 hexamer. Potassium permanganate footprinting revealed that the occupancy of this operator prevents RNA polymerase from forming an open complex in vitro. However, the operator centered at -31.5 is not sufficient for full repression in vivo because the deletion of the other two CRP binding sites partially relieved the CRP-dependent repression of the heat-labile toxin promoter. In contrast to heat-labile toxin, CRP positively regulates the expression of heat-stable toxin. Thus, the conditions for the optimal expression of one enterotoxin limit the expression of the other. Since glucose inhibits the activity of CRP by suppressing the pathogen's synthesis of cyclic AMP (cAMP), the concentration of glucose in the lumen of the small intestine may determine which enterotoxin is maximally expressed. In addition, our results suggest that the host may also modulate enterotoxin expression because cells intoxicated with heat-labile toxin overproduce and release cAMP.
Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Receptores de AMP Cíclico/metabolismo , Adulto , Toxinas Bacterianas/genética , Sequência de Bases , Escherichia coli Enterotoxigênica/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Humanos , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of infant and child mortality in developing countries. This enteric pathogen causes profuse watery diarrhea by elaborating one or more enterotoxins that intoxicate eukaryotic cells and ultimately leads to a loss of water to the intestinal lumen. Virulence is also dependent upon fimbrial adhesins that facilitate colonization of the small intestine. RESULTS: The expression of CS1 fimbriae is positively regulated by Rns, a member of the AraC/XylS superfamily of transcriptional regulators. Based on fimbrial protein homology, CS1 fimbriae have been categorized as subclass 5b along with CS17, CS19, and PCFO71 fimbriae. In this study we show that Rns positively regulates the expression of these other subclass 5b members. DNase I footprinting revealed a Rns binding site adjacent to the -35 hexamer of each fimbrial promoter. The CS17 and PCFO71 fimbrial promoters carry a second Rns binding site centered at -109.5, relative to the Rns-dependent transcription start site. This second binding site is centered at -108.5 for the CS19 promoter. Mutagenesis of either site reduced Rns-dependent transcription from each promoter indicating that the molecules bound to these sites apparently function independently of one another, with each having an additive effect upon fimbrial promoter activation. CONCLUSION: This study demonstrates that the ETEC virulence regulator Rns is required for the expression of all known 5b fimbriae. Since Rns is also known to control the expression of additional ETEC fimbriae, including those within subclasses 5a and 5c, the inactivation or inhibition of Rns could be an effective strategy to prevent ETEC infections.
Assuntos
Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Transativadores/genética , Sítios de Ligação , Pegada de DNA , Escherichia coli Enterotoxigênica/patogenicidade , Mutagênese , Plasmídeos , Mutação Puntual , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição , Transcrição Gênica , VirulênciaRESUMO
Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.
Assuntos
DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transativadores/genética , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Dimerização , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glutaral/química , Mutação , Plasmídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transativadores/química , Transativadores/metabolismoRESUMO
H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions -23 to -56, and the other extends from positions -73 to -103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Ligação a DNA/fisiologia , Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Antígenos de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Citoplasma/metabolismo , Pegada de DNA , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli Enterotoxigênica/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Virulência/genéticaRESUMO
The expression of the inner membrane protein NlpA is repressed by the enterotoxigenic Escherichia coli (ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregative E. coli also repress expression of nlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon of nlpA, preventing RNA polymerase from forming an open complex at nlpAp. A second Rns binding site between positions -152 and -195 relative to the nlpA transcription start site is not required for repression. NlpA is not essential for growth of E. coli under laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression of nlpA may be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.