RESUMEN
The ability to fine tune global gene expression in response to host environment is critical for the virulence of pathogenic bacteria. The host temperature is exploited by the bacteria as a cue for triggering virulence gene expression. However, little is known about the mechanism employed by Pseudomonas aeruginosa to response to host body temperature. CspA family proteins are RNA chaperones that modulate gene expression. Here we explored the functions of P. aeruginosa CspA family proteins and found that CspC (PA0456) controls the bacterial virulence. Combining transcriptomic analyses, RNA-immunoprecipitation and high-throughput sequencing (RIP-Seq), we demonstrated that CspC represses the type III secretion system (T3SS) by binding to the 5' untranslated region of the mRNA of exsA, which encodes the T3SS master regulatory protein. We further demonstrated that acetylation at K41 of the CspC reduces its affinity to nucleic acids. Shifting the culture temperature from 25°C to 37°C or infection of mouse lung increased the CspC acetylation, which derepressed the expression of the T3SS genes, resulting in elevated virulence. Overall, our results identified the regulatory targets of CspC and revealed a regulatory mechanism of the T3SS in response to temperature shift and host in vivo environment.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Pseudomonas aeruginosa/genética , Transactivadores/genética , Sistemas de Secreción Tipo III/genética , Células A549 , Acetilación , Animales , Proteínas Bacterianas/biosíntesis , Humanos , Ratones , Neumonía Bacteriana/microbiología , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Transactivadores/biosíntesis , VirulenciaRESUMEN
Carbohydrate metabolism plays essential roles in energy generation and providing carbon skeletons for amino acid syntheses. In addition, carbohydrate metabolism has been shown to influence bacterial susceptibility to antibiotics and virulence. In this study, we demonstrate that citrate synthase gltA mutation can increase the expression of the type III secretion system (T3SS) genes and antibiotic tolerance in Pseudomonas aeruginosa. The stringent response is activated in the gltA mutant, and deletion of the (p)ppGpp synthetase gene relA restores the antibiotic tolerance and expression of the T3SS genes to wild-type level. We further demonstrate that the intracellular level of cAMP is increased by the stringent response in the gltA mutant, which increases the expression of the T3SS master regulator gene exsA. Overall, our results reveal an essential role of GltA in metabolism, antibiotic tolerance, and virulence, as well as a novel regulatory mechanism of the stringent response-mediated regulation of the T3SS in P. aeruginosa. IMPORTANCE Rising antimicrobial resistance imposes a severe threat to human health. It is urgent to develop novel antimicrobial strategies by understanding bacterial regulation of virulence and antimicrobial resistance determinants. The stringent response plays an essential role in virulence and antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. The bacterium produces an arsenal of virulence factors and is highly resistant to a variety of antibiotics. In this study, we provide evidence that citrate synthase GltA plays a critical role in P. aeruginosa metabolism and influences the antibiotic tolerance and virulence. We further reveal a role of the stringent response in the regulation of the antibiotic tolerance and virulence. The significance of this work is in elucidation of novel regulatory pathways that control both antibiotic tolerance and virulence in P. aeruginosa.
Asunto(s)
Infecciones por Pseudomonas , Sistemas de Secreción Tipo III , Humanos , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Pseudomonas aeruginosa/metabolismo , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Factores de Virulencia/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Infecciones por Pseudomonas/microbiologíaRESUMEN
Pseudomonas aeruginosa is a ubiquitous pathogenic bacterium that can adapt to a variety environments. The ability to effectively sense and respond to host local nutrients is critical for the infection of P. aeruginosa. However, the mechanisms employed by the bacterium to respond to nutrients remain to be explored. CspA family proteins are RNA binding proteins that are involved in gene regulation. We previously demonstrated that the P. aeruginosa CspA family protein CspC regulates the type III secretion system in response to temperature shift. In this study, we found that CspC regulates the quorum-sensing (QS) systems by repressing the translation of a QS negative regulatory gene, rsaL. Through RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) and electrophoretic mobility shift assays (EMSAs), we found that CspC binds to the 5' untranslated region of the rsaL mRNA. Unlike glucose, itaconate (a metabolite generated by macrophages during infection) reduces the acetylation of CspC, which increases the affinity between CspC and the rsaL mRNA, leading to upregulation of the QS systems. Our results revealed a novel regulatory mechanism of the QS systems in response to a host-generated metabolite. IMPORTANCE Bacterial infectious diseases impose a severe threat to human health. The ability to orchestrate virulence determinant in response to the host environment is critical for the pathogenesis of bacterial pathogens. Pseudomonas aeruginosa is a leading pathogen that causes various infections in humans. In P. aeruginosa, the quorum-sensing (QS) systems play an important role in regulating the production of virulence factors. In this study, we find that a small RNA binding protein, CspC, regulates the QS systems by repressing the expression of a QS negative regulator. We further demonstrate that CspC is acetylated in response to a host-derived metabolite, itaconate, which alters the function of CspC in regulating the QS system. The importance of this work is in elucidation of a novel regulatory pathway that regulates virulence determinants in P. aeruginosa in response to a host signal.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa , Acetilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum/genética , ARN Mensajero/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
OBJECTIVE: To identify the role of LGALS3BP/Gal-3 in the process of human periodontal ligament stem cells (hPDLSCs) differentiating into osteoblasts. METHODS: IP-WB experiments were carried out to examine the binding of LGALS3BP and Gal-3. Western blot was performed to detect the expressions of LGALS3BP and Gal-3 in hPDLSCs with or without osteogenic differentiation inducement. The expressions of differentiation-related Oct4, Sox2 and Runx2 were also detected by western blot. Alkaline Phosphatase (ALP) Assay Kit was used to measure ALP activity in hPDLSCs. The mineralization ability of hPDLSCs was observed by staining with Alizarin Red S solution. RESULTS: LGALS3BP bound with Gal-3 in hPDLSCs, and the expression of LGALS3BP and Gal-3 was improved after osteogenic differentiation of hPDLSCs. Recombinant GAL-3 promoted the expression of differentiation-related proteins Oct4 and Sox2 and Runx2 in osteogenic differentiation-induced hPDLSCs. Recombinant GAL-3 also promoted the differentiation of osteogenesis-induced hPDLSCs. Furthermore, LGALS3BP had a facilitating effect on differentiation-related protein expression, while it could be reversed by shGal-3. LGALS3BP also promoted osteogenic capacity of hPDLSCs, and shGal-3 could reverse this effect. CONCLUSION: LGALS3BP binds to Gal-3, producing a promoting effect on the osteogenic differentiation of human periodontal ligament stem cells.