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1.
Mol Microbiol ; 96(2): 249-62, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25586884

RESUMO

Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1) and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.


Assuntos
Fímbrias Bacterianas/metabolismo , RNA Bacteriano/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , RNA Bacteriano/genética , Sorogrupo , Especificidade da Espécie , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Virulência
2.
Infect Immun ; 82(5): 1744-54, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24516115

RESUMO

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sorotipagem , Fatores de Tempo , Virulência , Fatores de Virulência/genética
3.
Infect Immun ; 81(1): 364-72, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23147037

RESUMO

The bacterial pathogen group A Streptococcus (GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α(2)-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given that grab and hasABC are also negatively regulated by the two-component system CovR/S (control of virulence), we tested whether RivR functions through CovR/S. A comparison of riv and cov single and double mutant strains showed that RivR requires CovR activity for grab and hasABC regulation. Analysis of the upstream region of rivR identified a novel promoter the deletion of which reduced rivR mRNA abundance by 70%. A rivR mutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. The rivR mutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.


Assuntos
Proteínas de Bactérias/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/genética , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte , Linhagem Celular , Feminino , Regulação Bacteriana da Expressão Gênica/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/metabolismo , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/imunologia , Virulência/genética , Virulência/imunologia , Fatores de Virulência/biossíntese , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
4.
Mol Microbiol ; 86(1): 140-54, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22882718

RESUMO

Bacterial pathogens use cell surface-associated adhesion molecules to promote host attachment and colonization, and the ability to modulate adhesion expression is critical to pathogen success. Here, we show that the human-specific pathogen the group A Streptococcus (GAS) uses a small regulatory RNA (sRNA) to regulate the expression of adhesive pili. The fibronectin/fibrinogen-binding/haemolytic-activity/streptokinase-regulator-X (FasX) sRNA, previously shown to positively regulate expression of the secreted virulence factor streptokinase (SKA), negatively regulates the production of pili on the GAS cell surface. FasX base pairs to the extreme 5' end of mRNA from the pilus biosynthesis operon, and this RNA:RNA interaction reduces the stability of the mRNA, while also inhibiting translation of at least the first gene in the pilus biosynthesis operon (cpa, which encodes a minor pilin protein). The negative regulation of pilus expression by FasX reduces the ability of GAS to adhere to human keratinocytes. Our findings cement FasX sRNA as an important regulator of virulence factor production in GAS and identify that FasX uses at least three distinct mechanisms, positive (ska mRNA) and negative (pilus operon mRNA) regulation of mRNA stability, and negative regulation of mRNA translation (cpa mRNA), to post-transcriptionally regulate target mRNAs during infection.


Assuntos
Aderência Bacteriana , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Regiões 5' não Traduzidas , Sequência de Bases , Linhagem Celular , Humanos , Queratinócitos/microbiologia , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon , Estabilidade de RNA , Pequeno RNA não Traduzido/genética , Virulência
5.
Mol Microbiol ; 78(6): 1332-47, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21143309

RESUMO

Small RNA molecules play key regulatory roles in many bacterial species. However, little mechanistic data exists for the action of small regulatory RNAs in the human pathogen group A Streptococcus (GAS). Here, we analysed the relationship between a putative GAS sRNA and production of the secreted virulence factor streptokinase (SKA). SKA promotes GAS dissemination by activating conversion of host plasminogen into the fibrin-degrading protease plasmin. Homologues of the putative sRNA-encoding gene fibronectin/fibrinogen-binding/haemolytic-activity/streptokinase-regulator-X (fasX) were identified in four different pyogenic streptococcal species. However, despite 79% fasX nucleotide identity, a fasX allele from the animal pathogen Streptococcus zooepidemicus failed to complement a GAS fasX mutant. Using a series of precisely constructed fasX alleles we discovered that FasX is a bona-fide sRNA that post-transcriptionally regulates SKA production in GAS. By base-pairing to the 5' end of ska mRNA, FasX enhances ska transcript stability, resulting in a ∼10-fold increase in SKA activity. Our data provide new insights into the mechanisms used by small regulatory RNAs to activate target mRNAs, and enhances our understanding of the regulation of a key GAS virulence factor.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Enzimológica da Expressão Gênica , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Streptococcus pyogenes/enzimologia , Estreptoquinase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Estreptoquinase/química , Estreptoquinase/genética , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
Microbiology (Reading) ; 156(Pt 5): 1342-1350, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20110295

RESUMO

The signal recognition particle (SRP) is a ribonucleoprotein complex that targets proteins for secretion in a co-translational manner. While originally thought to be essential in all bacteria, recent data show that the SRP is dispensable in at least some streptococcal species. The SRP from the human pathogen group A Streptococcus (GAS, Streptococcus pyogenes) is predicted to be composed of protein Ffh and 4.5S RNA. Deletion of ffh alters the secretion of several GAS proteins, and leads to a severe reduction in virulence. Here, we report that mutation of the gene encoding 4.5S RNA results in phenotypes both similar to and distinct from that observed following ffh mutation. Similarities include a reduction in secretion of the haemolysin streptolysin O, and attenuation of virulence as assessed by a murine soft tissue infection model. Differences include a reduction in transcript levels for the genes encoding streptolysin O and NAD-glycohydrolase, and the reduced secretion of the SpeB protease. Several differences in transcript abundance between the parental and mutant strain were shown to be dependent on the sensor-kinase-encoding gene covS. Using growth in human saliva as an ex vivo model of upper respiratory tract infection we identified that 4.5S RNA mutation leads to a 10-fold reduction in colony-forming units over time, consistent with the 4.5S RNA contributing to GAS growth and persistence during upper respiratory tract infections. Finally, we determined that the 4.5S RNA was essential for GAS to cause lethal infections in a murine bacteraemia model of infection. The data presented extend our knowledge of the contribution of the SRP to the virulence of an important Gram-positive pathogen.


Assuntos
RNA Bacteriano/fisiologia , Partícula de Reconhecimento de Sinal/fisiologia , Streptococcus pyogenes/patogenicidade , Animais , Bacteriemia/microbiologia , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Mutagênese , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , Saliva/microbiologia , Células-Tronco , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Virulência/genética
7.
Infect Immun ; 77(8): 3141-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19451242

RESUMO

To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections.


Assuntos
Proteínas de Bactérias/fisiologia , Regulação Bacteriana da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Repressoras/fisiologia , Streptococcus pyogenes/fisiologia , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/genética , Deleção de Genes , Perfilação da Expressão Gênica , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/genética , Saliva/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Virulência
8.
PLoS One ; 4(11): e7668, 2009 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-19888332

RESUMO

The coordinated regulation of gene expression is essential for pathogens to infect and cause disease. A recently appreciated mechanism of regulation is that afforded by small regulatory RNA (sRNA) molecules. Here, we set out to assess the prevalence of sRNAs in the human bacterial pathogen group A Streptococcus (GAS). Genome-wide identification of candidate GAS sRNAs was performed through a tiling Affymetrix microarray approach and identified 40 candidate sRNAs within the M1T1 GAS strain MGAS2221. Together with a previous bioinformatic approach this brings the number of novel candidate sRNAs in GAS to 75, a number that approximates the number of GAS transcription factors. Transcripts were confirmed by Northern blot analysis for 16 of 32 candidate sRNAs tested, and the abundance of several of these sRNAs were shown to be temporally regulated. Six sRNAs were selected for further study and the promoter, transcriptional start site, and Rho-independent terminator identified for each. Significant variation was observed between the six sRNAs with respect to their stability during growth, and with respect to their inter- and/or intra-serotype-specific levels of abundance. To start to assess the contribution of sRNAs to gene regulation in M1T1 GAS we deleted the previously described sRNA PEL from four clinical isolates. Data from genome-wide expression microarray, quantitative RT-PCR, and Western blot analyses are consistent with PEL having no regulatory function in M1T1 GAS. The finding that candidate sRNA molecules are prevalent throughout the GAS genome provides significant impetus to the study of this fundamental gene-regulatory mechanism in an important human pathogen.


Assuntos
Genoma Bacteriano , MicroRNAs/genética , Streptococcus pyogenes/genética , Northern Blotting , Western Blotting , Biologia Computacional/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Estudo de Associação Genômica Ampla , Análise de Sequência com Séries de Oligonucleotídeos , RNA Bacteriano , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
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