RESUMO
The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.
Assuntos
Pseudomonas aeruginosa , Pequeno RNA não Traduzido , Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA Mensageiro/genética , Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genéticaRESUMO
Previously, we reported that RelA protein facilitates Hfq-mediated mRNA-sRNA regulation by binding sRNAs carrying a Shine-Dalgarno-like GGAG sequence. In turn, sRNA-Hfq monomers are stabilized, enabling the attachment of more Hfq subunits to form a functional hexamer. Here, using CLIP-seq, we present a global analysis of RelA-bound RNAs showing that RelA interacts with sRNAs as well as with mRNAs carrying a GGAG motif. RelA binding of mRNAs carrying GGAG at position -7 relative to the initiation codon (AUG) inhibits translation by interfering with the binding of 30S ribosomes. The extent of inhibition depends on the distance of GGAG relative to the AUG, as shortening the spacing between GGAG and AUG abrogates RelA-mediated inhibition. Interestingly, RelA binding of target mRNAs carrying GGAG in the coding sequence or close to AUG facilitates target gene regulation by sRNA partners that lack GGAG. However, translation inhibition caused by RelA binding of mRNAs carrying GGAG at position -7 relative to the AUG renders sRNA-mRNA basepairing regulation ineffective. Our study indicates that by binding RNAs carrying GGAG the ribosome-associated RelA protein inhibits translation of specific newly synthesized incoming mRNAs or enables basepairing regulation by their respective sRNA partners, thereby introducing a new regulatory concept for the bacterial response.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , GTP Pirofosfoquinase/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Pareamento de Bases , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Motivos de Nucleotídeos , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismoRESUMO
Expression of cholera toxin (CT), the principal virulence factor of the cholera pathogen Vibrio cholerae, is positively modulated by the RNA polymerase binding unusual transcription factor DksA (DksAVc) of the stringent response pathway. Here we report that even though CT (encoded by the genes ctxAB) production is downregulated in the V. cholerae ΔdksA (ΔdksAVc) mutant, the expression of the ctxA gene as well as the genes encoding different virulence regulators, namely, AphA, TcpP and ToxT, were also upregulated. Since DksAVc positively regulates HapR, a known negative regulator of CT production, the increased expression of different virulence genes in ΔdksAVc was due most probably to downregulation of HapR. There was no secretion/transport-related defect in ΔdksAVc cells because whole cell lysates of the mutant showed a negligible amount of CT accumulation similar to WT cells. To understand further, the hapR gene was deleted in ΔdksAVc background, however, the double mutant failed to rescue the CT production defect suggesting strongly towards post-transcriptional/translational regulation by DksAVc. This hypothesis was further confirmed when the site-directed mutagenesis of each or both of the conserved aspartic acid residues at positions 68 and 71 of DksAVc, which are essential for transcription initiation during the stringent response, had no effect in the regulation of CT expression. Interestingly, progressive deletion analysis indicated that the C4-type Zn finger motif present in the C-terminus of DksAVc is essential for optimal CT production. Since this motif plays important roles in DNA/RNA binding, the present study indicates a novel complex post-transcriptional regulation of CT expression by DksAVc.
Assuntos
Proteínas de Bactérias/metabolismo , Toxina da Cólera/biossíntese , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Vibrio cholerae/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Vibrio cholerae/genéticaRESUMO
DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases/genética , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Vibrio cholerae/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metaloendopeptidases/metabolismo , Proteínas Repressoras/genética , Fator sigma/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1ß and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Metaloproteases/metabolismo , Sepse Neonatal/microbiologia , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Cromatografia em Gel , Cromatografia por Troca Iônica , Citocinas/metabolismo , Estabilidade Enzimática , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metaloproteases/química , Metaloproteases/isolamento & purificação , Camundongos , Especificidade por Substrato , Espectrometria de Massas em Tandem , Temperatura , Fatores de Virulência/química , Fatores de Virulência/isolamento & purificaçãoRESUMO
In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and guanosine 3',5'-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp(0) cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.
Assuntos
Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Ligases/genética , Ligases/metabolismo , Vibrio cholerae/enzimologia , Vibrio cholerae/genética , Substituição de Aminoácidos , Análise Mutacional de DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Deleção de SequênciaRESUMO
The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.
Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/química , GTP Pirofosfoquinase/genética , Fator Proteico 1 do Hospedeiro/química , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Deleção de SequênciaRESUMO
Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).