RESUMEN
The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases.
Asunto(s)
Regiones no Traducidas 5'/genética , Proteínas Bacterianas/genética , Exotoxinas/genética , Estabilidad del ARN , Streptococcus pyogenes/genética , Proteínas Bacterianas/química , Cisteína Endopeptidasas/metabolismo , Exotoxinas/química , Exotoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenotipo , Procesamiento Postranscripcional del ARN , Eliminación de Secuencia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidadRESUMEN
Microbial pathogens must evade the human immune system to survive, disseminate and cause disease. By proteome analysis of the bacterium Group A Streptococcus (GAS), we identified a secreted protein with homology to the alpha-subunit of Mac-1, a leukocyte beta2 integrin required for innate immunity to invading microbes. The GAS Mac-1-like protein (Mac) was secreted by most pathogenic strains, produced in log-phase and controlled by the covR-covS two-component gene regulatory system, which also regulates transcription of other GAS virulence factors. Patients with GAS infection had titers of antibody specific to Mac that correlated with the course of disease, demonstrating that Mac was produced in vivo. Mac bound to CD16 (FcgammaRIIIB) on the surface of human polymorphonuclear leukocytes and inhibited opsonophagocytosis and production of reactive oxygen species, which resulted in significantly decreased pathogen killing. Thus, by mimicking a host-cell receptor required for an innate immune response, the GAS Mac protein inhibits professional phagocyte function by a novel strategy that enhances pathogen survival, establishment of infection and dissemination.
Asunto(s)
Proteínas Bacterianas , Integrinas/metabolismo , Antígeno de Macrófago-1/farmacología , Proteínas Opsoninas , Fagocitosis/efectos de los fármacos , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/patogenicidad , Enfermedad Aguda , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/sangre , Sitios de Unión , Convalecencia , Integrinas/genética , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/metabolismo , Modelos Inmunológicos , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Faringitis/inmunología , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/metabolismo , Fiebre Reumática/inmunología , Homología de Secuencia de AminoácidoRESUMEN
A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209-219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for "control of virulence genes" in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.
Asunto(s)
Adhesinas Bacterianas , Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Proteínas Musculares , Proteínas de Mieloma , Operón , Streptococcus pyogenes/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Conectina , Farmacorresistencia Microbiana/genética , Endopeptidasas/genética , Genes Bacterianos , Prueba de Complementación Genética , Genoma Bacteriano , Humanos , Modelos Genéticos , Mutagénesis Insercional , Sistemas de Lectura Abierta , Serotipificación , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/patogenicidad , Estreptoquinasa/genética , Estreptolisinas/genética , Transcripción Genética , Virulencia/genéticaRESUMEN
We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.