RESUMEN
In addition to providing a typing mechanism for group A Streptococcus (GAS) isolates (T typing), cell surface pilus production impacts GAS virulence characteristics, including adherence and immune evasion. The pilus biosynthesis genes are located in the fibronectin- and collagen-binding T-antigen (FCT) region of the genome, and nine different FCT types, encoding more than 20 different T types, have been described. GAS isolates are not uniform in their degree or pattern of pilus expression, as highlighted by pilus production being thermoregulated in isolates that harbor the FCT-type FCT-3 (e.g., M-types M3 and M49) but not in isolates that harbor FCT-2 (e.g., M-type M1). Here, we investigated the molecular basis underlying our previous finding that M3 GAS isolates produce pili in lower abundance than M1 or M49 isolates do. We discovered that, at least in part, the low pilus expression observed for M3 isolates is a consequence of the repression of pilus gene expression by the CovR/CovS two-component regulatory system and of an M3-specific mutation in the nra gene, encoding a positive regulator of pilus gene expression. We also discovered that the orthologous transcriptional regulators RofA and Nra, whose encoding genes are located within FCT-2 and FCT-3, respectively, are not functionally identical. Finally, we sequenced the genome of an M3 isolate that had naturally undergone recombinational replacement of the FCT region, changing the FCT and T types of this strain from FCT-3/T3 to FCT-2/T1. Our study furthers the understanding of strain- and type-specific variation in virulence factor production by an important human pathogen. IMPORTANCE Our ability to characterize how a pathogen infects and causes disease, and consequently our ability to devise approaches to prevent or attenuate such infections, is inhibited by the finding that isolates of a given pathogen often show phenotypic variability, for example, in their ability to adhere to host cells through modulation of cell surface adhesins. Such variability is observed between isolates of group A Streptococcus (GAS), and this study investigates the molecular basis for why some GAS isolates produce pili, cell wall-anchored adhesins, in lower abundance than other isolates do. Given that pili are being considered as potential antigens in formulations of future GAS vaccines, this study may inform vaccine design.
Asunto(s)
Infecciones Estreptocócicas , Humanos , Infecciones Estreptocócicas/metabolismo , Proteínas Bacterianas/metabolismo , Streptococcus pyogenes/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
The group A Streptococcus (GAS; Streptococcus pyogenes) causes an elaborate array of human diseases. In part, such variability in disease potential is a consequence of GAS manipulating the expression of a catalogue of virulence factors, with regulation occurring at both the transcriptional and posttranscriptional levels. The GAS small regulatory RNA (sRNA) FasX contributes to this regulatory activity, enhancing expression of the thrombolytic agent streptokinase, and reducing expression of collagen (pili) and fibronectin (PrtF1 and PrtF2) -binding adhesins. Here, we expand insight into the regulatory targets of FasX by identifying the M-related protein (Mrp), a fibrinogen-binding adhesin with anti-phagocytic activity, as a negatively-regulated target of FasX. Importantly, investigation of the consequences of FasX-mediated regulation led to the discovery that FasX is a major positive regulator of GAS survival and proliferation in non-immune whole human blood, with a 30-fold difference in GAS cell numbers between a fasX mutant strain and isogenic parental and complemented mutant strains. No difference in cell numbers were observed when these strains were grown in human serum, consistent with the protective phenotype associated with FasX occurring due to the inhibition of cell (e.g., neutrophil) - mediated GAS killing. The FasX-regulated factor/s responsible for the blood survival phenotype remain to be defined. In summary, we expand the known FasX regulon and identify a new phenotype associated with the regulatory activity of this key GAS sRNA. IMPORTANCE Small regulatory RNAs (sRNAs) represent a major class of regulatory molecule that promotes the ability of the group A Streptococcus (GAS) and other pathogens to regulate virulence factor expression. Despite FasX being the best-described sRNA in GAS, there remains much to be learned. Here, we highlight the importance of FasX, identifying for the first time that the loss of this sRNA results in a major reduction in the ability of GAS to survive in human blood, a phenotype critical to the ability of this human-specific pathogen to cause severe invasive infections. We also identified a novel regulatory target of FasX, thereby expanding the known regulon of this key sRNA.
Asunto(s)
ARN Pequeño no Traducido , Streptococcus pyogenes , Humanos , Streptococcus pyogenes/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Serotype M28 isolates of the group A Streptococcus (GAS; Streptococcus pyogenes) are nonrandomly associated with cases of puerperal sepsis, a potentially life-threatening infection that can occur in women following childbirth. Previously, we discovered that the 36.3-kb RD2 pathogenicity island, which is present in serotype M28 isolates but lacking from most other isolates, promotes the ability of M28 GAS to colonize the female reproductive tract. Here, we performed a gain-of-function study in which we introduced RD2 into representative serotype M1, M49, and M59 isolates and assessed the phenotypic consequences of RD2 acquisition. All RD2-containing derivatives colonized a higher percentage of mice, and at higher CFU levels, than did the parental isolates in a mouse vaginal colonization model. However, for two additional phenotypes, survival in heparinized whole human blood and adherence to two human vaginal epithelial cell lines, there were serotype-specific differences from RD2 acquisition. Using transcriptomic comparisons, we identified that such differences may be a consequence of RD2 altering the abundance of transcripts from select core genome genes along serotype-specific lines. Our study is the first that interrogates RD2 function in GAS serotypes other than M28 isolates, shedding light on variability in the phenotypic consequences of RD2 acquisition and informing on why this mobile genetic element is not ubiquitous in the GAS population.
Asunto(s)
Elementos Transponibles de ADN , Islas Genómicas , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Línea Celular , Mutación con Ganancia de Función , Humanos , Fenotipo , Sepsis/microbiología , Serogrupo , Streptococcus pyogenes/clasificaciónRESUMEN
The group A Streptococcus (GAS) causes diseases that range from mild (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). Strain- and serotype-specific differences influence the ability of isolates to cause individual diseases. At the center of this variability is the CovR/S two-component system and the accessory protein RocA. Through incompletely defined mechanisms, CovR/S and RocA repress the expression of more than a dozen immunomodulatory virulence factors. Alleviation of this repression is selected for during invasive infections, leading to the recovery of covR, covS or rocA mutant strains. Here, we investigated how RocA promotes CovR/S activity, identifying that RocA is a pseudokinase that interacts with CovS. Disruption of CovS kinase or phosphatase activities abolishes RocA function, consistent with RocA acting through the modulation of CovS activity. We also identified, in conflict with a previous study, that the RocA regulon includes the secreted protease-encoding gene speB. Finally, we discovered an inverse correlation between the virulence of wild-type, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model. Our data inform on mechanisms that control GAS disease potential and provide an explanation for observed strain- and serotype-specific variability in RocA function.
Asunto(s)
Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Proteínas Represoras/metabolismo , Streptococcus pyogenes/metabolismo , Transactivadores/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/patogenicidad , Virulencia , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: Bacterial infections following childbirth-so-called puerperal infections-cause morbidity in 5%-10% of all new mothers. At low frequency, the infection can spread to the blood, resulting in life-threatening sepsis known as puerperal sepsis. Pathogens causing puerperal sepsis include group A Streptococcus (GAS), and epidemiological analyses have identified isolates of a single serotype, M28, as being nonrandomly associated with cases of puerperal sepsis. The genomes of serotype M28 GAS isolates harbor a 36.3-kb mobile genetic element of apparent group B Streptococcus origin, termed region of difference 2 (RD2). METHODS: The phenotypic (determined via tissue culture and a vaginal colonization model) and regulatory (determined via RNA sequencing analysis) contributions of RD2 were assessed by comparing parental, RD2 deletion mutant, and complemented mutant serotype M28 GAS strains. RESULTS: RD2 affords serotype M28 isolates an enhanced ability to adhere to human vaginal epithelial cells and to colonize the female reproductive tract in a mouse model of infection. In addition, RD2 influences the abundance of messenger RNAs from >100 core chromosomal GAS genes. CONCLUSIONS: The data are consistent with RD2 directly, via encoded virulence factors, and indirectly, via encoded regulatory proteins, modifying the virulence potential of GAS and contributing to the decades-old association of serotype M28 isolates with cases of puerperal sepsis.
Asunto(s)
Secuencias Repetitivas Esparcidas/genética , Infección Puerperal/microbiología , Sepsis/microbiología , Serogrupo , Streptococcus pyogenes/genética , Streptococcus pyogenes/aislamiento & purificación , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/crecimiento & desarrollo , Factores de Transcripción , Transcriptoma , Vagina/microbiología , Factores de Virulencia/genéticaRESUMEN
Populations of a bacterial pathogen, whether recovered from a single patient or from a worldwide study, are often a heterogeneous mix of genetically and phenotypically divergent strains. Such heterogeneity is of value in changing environments and arises via mechanisms such as gene gain or gene mutation. Here, we identify an isolate of serotype M12 group A Streptococcus (GAS) (Streptococcus pyogenes) that has a natural mutation in rocA, which encodes an accessory protein to the virulence-regulating two-component system CovR/CovS (CovR/S). Disruption of RocA activity results in the differential expression of multiple GAS virulence factors, including the anti-phagocytic hyaluronic acid capsule and the chemokine protease SpyCEP. While some of our data regarding RocA-regulated genes overlaps with previous studies, which were performed with isolates of alternate GAS serotypes, some variability was also observed. Perhaps as a consequence of this alternate regulatory activity, we discovered that the contribution of RocA to the ability of the M12 isolate to survive and proliferate in human blood ex vivo is opposite that previously observed in M1, M3, and M18 GAS strains. Specifically, rocA mutation reduced, rather than enhanced, survival of the isolate. Finally, we also present data from an analysis of rocA transcription and show that rocA is transcribed in both mono- and polycistronic mRNAs. In aggregate, our data provide insight into the important regulatory role of RocA and into the mechanisms and consequences of GAS phenotypic heterogeneity.IMPORTANCE This study investigates the regulatory and phenotypic consequences of a naturally occurring mutation in a strain of the bacterial pathogen the group A Streptococcus (Streptococcus pyogenes). We show that this mutation, which occurs in a regulator-encoding gene, rocA, leads to altered virulence factor expression and reduces the ability of this isolate to survive in human blood. Critically, the blood survival phenotype and the assortment of genes regulated by RocA differ compared to previous studies into RocA activity. The data are consistent with there being strain- or serotype-specific variability in RocA function. Given that phenotypic variants can lead to treatment failures and escape from preventative regimes, our data provide information with regard to a mechanism of phenotypic variation in a prevalent Gram-positive pathogen.
Asunto(s)
Mutación , Serogrupo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Transactivadores/genética , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Histidina Quinasa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/metabolismo , ARN Mensajero/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Streptococcus pyogenes/aislamiento & purificación , Virulencia/genéticaRESUMEN
Isolates of a given bacterial pathogen often display phenotypic variation, and this can negatively impact public health, for example, by reducing the efficacy of preventative measures. Here, we identify that the human pathogen group A Streptococcus (GAS; Streptococcus pyogenes) expresses pili on its cell surface in a serotype-specific manner. Specifically, we show that serotype M3 GAS isolates, which are nonrandomly associated with causing particularly severe and lethal invasive infections, produce negligible amounts of pili relative to serotype M1 and M49 isolates. Performance of an interserotype transcriptome comparison (serotype M1 versus serotype M3) was instrumental in this discovery. We also identified that the transcriptional regulator Nra positively regulates pilus expression in M3 GAS isolates and that the low level of pilus expression of these isolates correlates with a low level of nra transcription. Finally, we discovered that the phenotypic consequences of low levels of pilus expression by M3 GAS isolates are a reduced ability to adhere to host cells and an increased ability to survive and proliferate in human blood. We propose that an enhanced ability to survive in human blood, in part due to reduced pilus expression, is a contributing factor in the association of serotype M3 isolates with highly invasive infections. In conclusion, our data show that GAS isolates express pili in a serotype-dependent manner and may inform vaccine development, given that pilus proteins are being discussed as possible GAS vaccine antigens.
Asunto(s)
Variación Biológica Poblacional , Fimbrias Bacterianas/metabolismo , Serogrupo , Streptococcus pyogenes/fisiología , Adhesión Bacteriana , Proteínas Bacterianas/biosíntesis , Actividad Bactericida de la Sangre , Fimbrias Bacterianas/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Humanos , Viabilidad Microbiana , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Factores de Transcripción/biosíntesisRESUMEN
Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg2+ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Transactivadores/genética , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Células Sanguíneas/inmunología , Células Sanguíneas/microbiología , Catelicidinas/farmacología , Cationes Bivalentes , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Humanos , Magnesio/farmacología , Viabilidad Microbiana , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ARN , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/metabolismo , Transactivadores/metabolismo , VirulenciaRESUMEN
Data from multiple bacterial pathogens are consistent with regulator-encoding genes having higher mutation frequencies than the genome average. Such mutations drive both strain- and type- (e.g., serotype, haplotype) specific phenotypic heterogeneity, and may challenge public health due to the potential of variants to circumvent established treatment and/or preventative regimes. Here, using the human bacterial pathogen the group A Streptococcus (GAS; S. pyogenes) as a model organism, we review the types and regulatory-, phenotypic-, and disease-specific consequences of naturally occurring regulatory gene mutations. Strain-specific regulator mutations that will be discussed include examples that transform isolates into hyper-invasive forms by enhancing expression of immunomodulatory virulence factors, and examples that promote asymptomatic carriage of the organism. The discussion of serotype-specific regulator mutations focuses on serotype M3 GAS isolates, and how the identified rewiring of regulatory networks in this serotype may be contributing to a decades old epidemiological association of M3 isolates with particularly severe invasive infections. We conclude that mutation plays an outsized role in GAS pathogenesis and has clinical relevance. Given the phenotypic variability associated with regulatory gene mutations, the rapid examination of these genes in infecting isolates may inform with respect to potential patient complications and treatment options.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Genes Reguladores/genética , Genoma Bacteriano/genética , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Humanos , Mutación , Serogrupo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/inmunología , Factores de Virulencia/genéticaRESUMEN
Group A Streptococcus (GAS) (Streptococcus pyogenes) causes more than 700 million human infections each year. The significant morbidity and mortality rates associated with GAS infections are in part a consequence of the ability of this pathogen to coordinately regulate virulence factor expression during infection. RofA-like protein IV (RivR) is a member of the Mga-like family of transcriptional regulators, and previously we reported that RivR negatively regulates transcription of the hasA and grab virulence factor-encoding genes. Here, we determined that RivR inhibits the ability of GAS to survive and to replicate in human blood. To begin to assess the biochemical basis of RivR activity, we investigated its ability to form multimers, which is a characteristic of Mga-like proteins. We found that RivR forms both dimers and a higher-molecular-mass multimer, which we hypothesize is a tetramer. As cysteine residues are known to contribute to the ability of proteins to dimerize, we created a library of expression plasmids in which each of the four cysteines in RivR was converted to serine. While the C68S RivR protein was essentially unaffected in its ability to dimerize, the C32S and C377S proteins were attenuated, while the C470S protein completely lacked the ability to dimerize. Consistent with dimerization being required for regulatory activity, the C470S RivR protein was unable to repress hasA and grab gene expression in a rivR mutant. Thus, multimer formation is a prerequisite for RivR activity, which supports recent data obtained for other Mga-like family members, suggesting a common regulatory mechanism. IMPORTANCE: The modulation of gene transcription is key to the ability of bacterial pathogens to infect hosts to cause disease. Here, we discovered that the group A Streptococcus transcription factor RivR negatively regulates the ability of this pathogen to survive in human blood, and we also began biochemical characterization of this protein. We determined that, in order for RivR to function, it must self-associate, forming both dimers (consisting of two RivR proteins) and higher-order complexes (consisting of more than two RivR proteins). This functional requirement for RivR is shared by other regulators in the same family of proteins, suggesting a common regulatory mechanism. Insight into how these transcription factors function may facilitate the development of novel therapeutic agents targeting their activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidad , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Mutación Puntual , Conformación Proteica , Streptococcus pyogenes/genética , Factores de Transcripción/genética , VirulenciaRESUMEN
UNLABELLED: The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. IMPORTANCE: More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small RNA FasX. In addition to identifying that FasX reduces the abundance of the cell surface-located fibronectin-binding proteins PrtF1/2, fibronectin is present in high abundance in human tissues, and we have determined the mechanism behind this regulation. Importantly, as FasX is the only mechanistically characterized regulatory RNA in GAS, it serves as a model RNA in this and related pathogens.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Streptococcus pyogenes/genética , Regiones no Traducidas 5' , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Fibronectinas/metabolismo , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Streptococcus pyogenes/metabolismoRESUMEN
Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non-randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD(+) hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13-fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two-component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two-component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.
Asunto(s)
Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Bacteriemia/microbiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Eliminación de Gen , Humanos , Ratones , Ratones Pelados , Datos de Secuencia Molecular , Mutación , Serogrupo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/clasificación , Transactivadores/genética , Transactivadores/metabolismo , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
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.
Asunto(s)
Fimbrias Bacterianas/metabolismo , ARN Bacteriano/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidad , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , ARN Bacteriano/genética , Serogrupo , Especificidad de la Especie , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/genética , VirulenciaRESUMEN
The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ÏC31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl(+) host but normal in a Pgl(-) strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl(+) cell by bacteriophage ÏC31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Toxinas Bacterianas/metabolismo , Bacteriófagos/crecimiento & desarrollo , Enzimas de Restricción del ADN/metabolismo , Interacciones Huésped-Parásitos , Proteínas Quinasas/metabolismo , Streptomyces coelicolor/virología , ADN Viral/metabolismo , Transducción de SeñalRESUMEN
Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Streptococcus pyogenes/genética , Transcriptoma , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Perfilación de la Expresión Génica , Histidina Quinasa , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Fosforilación , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Serogrupo , Streptococcus pyogenes/metabolismoRESUMEN
The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Ácido Hialurónico/biosíntesis , Operón , Elementos Reguladores de la Transcripción , Streptococcus pyogenes/genética , Transcripción Genética , Animales , Actividad Bactericida de la Sangre , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Femenino , Perfilación de la Expresión Génica , Genes Reporteros , Variación Genética , Humanos , Evasión Inmune , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la Polimerasa , Eliminación de Secuencia , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , VirulenciaRESUMEN
RNA-based mechanisms of regulation represent a ubiquitous class of regulators that are associated with diverse processes including nutrient sensing, stress response, modulation of horizontal gene transfer, and virulence factor expression. While better studied in Gram-negative bacteria, the literature is replete with examples of the importance of RNA-mediated regulatory mechanisms to the virulence and fitness of Gram-positives. Regulatory RNAs are classified as cis-acting, e.g. riboswitches, which modulate the transcription, translation, or stability of co-transcribed RNA, or trans-acting, e.g. small regulatory RNAs, which target separate mRNAs or proteins. The group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive bacterial pathogen from which several regulatory RNA mechanisms have been characterized. The study of RNA-mediated regulation in GAS has uncovered novel concepts with respect to how small regulatory RNAs may positively regulate target mRNA stability, and to how CRISPR RNAs are processed from longer precursors. This review provides an overview of RNA-mediated regulation in Gram-positive bacteria, and is highlighted with specific examples from GAS research. The key roles that these systems play in regulating bacterial virulence are discussed and future perspectives outlined.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Streptococcus pyogenes/genética , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Streptococcus pyogenes/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidad , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Eliminación de Gen , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serotipificación , Factores de Tiempo , Virulencia , Factores de Virulencia/genéticaRESUMEN
Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system's ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.