RESUMO
The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.
Assuntos
Proteínas de Bactérias , Macrófagos , Infecções Estreptocócicas , Streptococcus pyogenes , Estreptolisinas , Fatores de Virulência , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Streptococcus pyogenes/imunologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/mortalidade , Humanos , Macrófagos/microbiologia , Macrófagos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estreptolisinas/genética , Estreptolisinas/metabolismo , Fatores de Virulência/genética , Mutação , Interações Hospedeiro-Patógeno/imunologia , Virulência/genética , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/imunologia , Viabilidade Microbiana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Camundongos , Regulação Bacteriana da Expressão Gênica , Proteínas de TransporteRESUMO
The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence amongst emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. Through the creation and analysis of isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates relative to the historic strains. Via creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found high ratio of mucosal (i.e., pharyngeal) relative to invasive infections amongst emm4 GAS. Inasmuch as ever-increasing virulence is unlikely to be evolutionary advantageous for a microbial pathogen, our data furthers understanding of the well described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.
RESUMO
Group A streptococcal (GAS) strains causing severe, invasive infections often have mutations in the control of virulence two-component regulatory system (CovRS) which represses capsule production, and high-level capsule production is considered critical to the GAS hypervirulent phenotype. Additionally, based on studies in emm1 GAS, hyperencapsulation is thought to limit transmission of CovRS-mutated strains by reducing GAS adherence to mucosal surfaces. It has recently been identified that about 30% of invasive GAS strains lacks capsule, but there are limited data regarding the impact of CovS inactivation in such acapsular strains. Using publicly available complete genomes (n = 2,455) of invasive GAS strains, we identified similar rates of CovRS inactivation and limited evidence for transmission of CovRS-mutated isolates for both encapsulated and acapsular emm types. Relative to encapsulated GAS, CovS transcriptomes of the prevalent acapsular emm types emm28, emm87, and emm89 revealed unique impacts such as increased transcript levels of genes in the emm/mga region along with decreased transcript levels of pilus operon-encoding genes and the streptokinase-encoding gene ska. CovS inactivation in emm87 and emm89 strains, but not emm28, increased GAS survival in human blood. Moreover, CovS inactivation in acapsular GAS reduced adherence to host epithelial cells. These data suggest that the hypervirulence induced by CovS inactivation in acapsular GAS follows distinct pathways from the better studied encapsulated strains and that factors other than hyperencapsulation may account for the lack of transmission of CovRS-mutated strains. IMPORTANCE Devastating infections due to group A streptococci (GAS) tend to occur sporadically and are often caused by strains that contain mutations in the control of virulence regulatory system (CovRS). In well-studied emm1 GAS, the increased production of capsule induced by CovRS mutation is considered key to both hypervirulence and limited transmissibility by interfering with proteins that mediate attachment to eukaryotic cells. Herein, we show that the rates of covRS mutations and genetic clustering of CovRS-mutated isolates are independent of capsule status. Moreover, we found that CovS inactivation in multiple acapsular GAS emm types results in dramatically altered transcript levels of a diverse array of cell-surface protein-encoding genes and a unique transcriptome relative to encapsulated GAS. These data provide new insights into how a major human pathogen achieves hypervirulence and indicate that factors other than hyperencapsulation likely account for the sporadic nature of the severe GAS disease.
Assuntos
Proteínas de Bactérias , Transcriptoma , Humanos , Transcriptoma/genética , Proteínas de Bactérias/genética , Virulência/genética , Mutação/genética , Fenótipo , Streptococcus pyogenes/genéticaRESUMO
Although prokaryotic DNA methylation investigations have long focused on immunity against exogenous DNA, it has been recently recognized that DNA methylation impacts gene expression and phase variation in Streptococcus pneumoniae and Streptococcus suis. A comprehensive analysis of DNA methylation is lacking for beta-hemolytic streptococci, and thus we sought to examine DNA methylation in the major human pathogen group A Streptococcus (GAS). Using a database of 224 GAS genomes encompassing 80 emm types, we found that nearly all GAS strains encode a type I restriction modification (RM) system that lacks the hsdS' alleles responsible for impacting gene expression in S. pneumoniae and S. suis. The GAS type I system is located on the core chromosome, while sporadically present type II orphan methyltransferases were identified on prophages. By combining single-molecule real-time (SMRT) analyses of 10 distinct emm types along with phylogenomics of 224 strains, we were able to assign 13 methylation patterns to the GAS population. Inactivation of the type I RM system, occurring either naturally through phage insertion or through laboratory-induced gene deletion, abrogated DNA methylation detectable via either SMRT or MinION sequencing. Contrary to a previous report, inactivation of the type I system did not impact transcript levels of the gene (mga) encoding the key multigene activator protein (Mga) or Mga-regulated genes. Inactivation of the type I system significantly increased plasmid transformation rates. These data delineate the breadth of the core chromosomal type I RM system in the GAS population and clarify its role in immunity rather than impacting Mga regulon expression. IMPORTANCE The advent of whole-genome approaches capable of detecting DNA methylation has markedly expanded appreciation of the diverse roles of epigenetic modification in prokaryotic physiology. For example, recent studies have suggested that DNA methylation impacts gene expression in some streptococci. The data described herein are from the first systematic analysis of DNA methylation in a beta-hemolytic streptococcus and one of the few analyses to comprehensively characterize DNA methylation across hundreds of strains of the same bacterial species. We clarify that DNA methylation in group A Streptococcus (GAS) is primarily due to a type I restriction modification (RM) system present in the core genome and does not impact mga-regulated virulence gene expression, but does impact immunity against exogenous DNA. The identification of the DNA motifs recognized by each type I RM system may assist with optimizing methods for GAS genetic manipulation and help us understand how bacterial pathogens acquire exogenous DNA elements.
Assuntos
Proteínas de Bactérias/genética , Metilação de DNA , Enzimas de Restrição-Modificação do DNA/genética , Streptococcus pyogenes/genética , DNA Bacteriano/genética , Deleção de Genes , Humanos , Variação de Fase , Regulon , Streptococcus pneumoniae/genética , Virulência/genéticaRESUMO
Clonal replacement is a major driver for changes in bacterial disease epidemiology. Recently, it has been proposed that episodic emergence of novel, hypervirulent clones of group A Streptococcus (GAS) results from acquisition of a 36-kb DNA region leading to increased expression of the cytotoxins Nga (NADase) and SLO (streptolysin O). We previously described a gene fusion event involving the gene encoding the GAS M protein (emm) and an adjacent M-like protein (enn) in the emm4 GAS population, a GAS emm type that lacks the hyaluronic acid capsule. Using whole-genome sequencing of a temporally and geographically diverse set of 1,126 isolates, we discovered that the North American emm4 GAS population has undergone clonal replacement with emergent GAS strains completely replacing historical isolates by 2017. Emergent emm4 GAS strains contained a handful of small genetic variations, including the emm-enn gene fusion, and showed a marked in vitro growth defect compared to historical strains. In contrast to other previously described GAS clonal replacement events, emergent emm4 GAS strains were not defined by acquisition of exogenous DNA and had no significant increase in transcript levels of nga and slo toxin genes via RNA sequencing and quantitative real-time PCR analysis relative to historic strains. Despite the in vitro growth differences, emergent emm4 GAS strains were hypervirulent in mice and ex vivo growth in human blood compared to historical strains. Thus, these data detail the emergence and dissemination of a hypervirulent acapsular GAS clone defined by small, endogenous genetic variation, thereby defining a novel model for GAS strain replacement. IMPORTANCE Severe invasive infections caused by group A Streptococcus (GAS) result in substantial morbidity and mortality in children and adults worldwide. Previously, GAS clonal strain replacement has been attributed to acquisition of exogenous DNA leading to novel virulence gene acquisition or increased virulence gene expression. Our study of type emm4 GAS identified emergence of a hypervirulent GAS clade defined by variation in endogenous DNA content and lacking augmented toxin gene expression relative to replaced strains. These findings expand our understanding of the molecular mechanisms underlying bacterial clonal emergence.
RESUMO
Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Streptococcus pyogenes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/metabolismo , Cromatina/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Exotoxinas/genética , Genoma Bacteriano/genética , Ligação Proteica/fisiologia , RNA-Seq , Proteínas Repressoras/metabolismo , Serina Endopeptidases/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Estreptolisinas/genética , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.
Assuntos
Streptococcus pyogenes/patogenicidade , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Feminino , Genes Bacterianos , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Regulon , Proteínas Repressoras/genética , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/ultraestrutura , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.
Assuntos
Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nasofaringe/microbiologia , Monoéster Fosfórico Hidrolases/metabolismo , Pele/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Pelados , Nasofaringe/enzimologia , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Sorogrupo , Pele/enzimologia , Infecções Estreptocócicas/enzimologia , Streptococcus pyogenes/enzimologia , VirulênciaRESUMO
Group A Streptococcus (GAS) is classified on the basis of the sequence of the gene encoding the M protein (emm) and the patterns into which emm types are grouped. We discovered a novel emm pattern in emm4 GAS, historically considered pattern E, arising from a fusion event between emm and the adjacent enn gene. We identified the emm-enn fusion event in 51 out of 52 emm4 GAS strains isolated by national surveillance in 2015. GAS isolates with an emm-enn fusion event completely replaced pattern E emm4 strains over a 4-year span in Houston (2013-2017). The novel emm-enn gene fusion and new emm pattern has potential vaccine implications.
Assuntos
Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/genética , Fusão Gênica , Streptococcus pyogenes/genética , Proteínas de Bactérias/genética , Humanos , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain.IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms' ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis, namely, that loss of this metabolic capacity made it a better colonizer.
Assuntos
Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Etanolaminas/metabolismo , Trato Gastrointestinal/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/genética , Microbioma Gastrointestinal , Trato Gastrointestinal/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Positivas/metabolismo , Humanos , Polipeptídeo PancreáticoRESUMO
Background: Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid. Methods: Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction. Results: Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use. Conclusions: The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Linezolida/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/genética , Gestão de Antimicrobianos , Proteínas de Bactérias/genética , Evolução Molecular , Fezes/microbiologia , Humanos , Microbiota , Staphylococcus epidermidis/efeitos dos fármacos , Sequenciamento Completo do GenomaRESUMO
Catabolite control protein A (CcpA) is a highly conserved, master regulator of carbon source utilization in gram-positive bacteria, but the CcpA regulon remains ill-defined. In this study we aimed to clarify the CcpA regulon by determining the impact of CcpA-inactivation on the virulence and transcriptome of three distinct serotypes of the major human pathogen Group A Streptococcus (GAS). CcpA-inactivation significantly decreased GAS virulence in a broad array of animal challenge models consistent with the idea that CcpA is critical to gram-positive bacterial pathogenesis. Via comparative transcriptomics, we established that the GAS CcpA core regulon is enriched for highly conserved CcpA binding motifs (i.e. cre sites). Conversely, strain-specific differences in the CcpA transcriptome seems to consist primarily of affected secondary networks. Refinement of cre site composition via analysis of the core regulon facilitated development of a modified cre consensus that shows promise for improved prediction of CcpA targets in other medically relevant gram-positive pathogens.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Regulon , Proteínas Repressoras/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Transcriptoma , Animais , Proteínas de Bactérias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Motivos de Nucleotídeos , Proteínas Repressoras/metabolismo , Sorogrupo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/mortalidade , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/metabolismo , Análise de Sobrevida , VirulênciaRESUMO
The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding-response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.
Assuntos
Cobamidas/metabolismo , Enterococcus faecalis/genética , Etanolamina/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro/metabolismo , Elementos de Resposta , Riboswitch/fisiologia , Transcrição Gênica , Sequência de Bases , Enterococcus faecalis/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Riboswitch/genéticaRESUMO
ANTAR proteins are widespread bacterial regulatory proteins that have RNA-binding output domains and utilize antitermination to control gene expression at the post-initiation level. An ANTAR protein, EutV, regulates the ethanolamine-utilization genes (eut) in Enterococcus faecalis. Using this system, we present genetic and biochemical evidence of a general mechanism of antitermination used by ANTARs, including details of the antiterminator structure. The novel antiterminator structure consists of two small hairpins with highly conserved terminal loop residues, both features being essential for successful antitermination. The ANTAR protein dimerizes and associates with its substrate RNA in response to signal-induced phosphorylation. Furthermore, bioinformatic searches using this conserved antiterminator motif identified many new ANTAR target RNAs in phylogenetically diverse bacterial species, some comprising complex regulons. Despite the unrelatedness of the species in which they are found, the majority of the ANTAR-associated genes are thematically related to nitrogen management. These data suggest that the central tenets for gene regulation by ANTAR antitermination occur widely in nature to specifically control nitrogen metabolism.
Assuntos
Enterococcus faecalis , Nitrogênio/metabolismo , Proteínas de Ligação a RNA/genética , RNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Fosforilação , Filogenia , Multimerização Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/genética , Dobramento de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transdução de SinaisRESUMO
Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.
Assuntos
Enterococcus faecalis/genética , Técnicas Genéticas , Genoma Bacteriano , Mutagênese Insercional , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Infecção Hospitalar/microbiologia , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , CamundongosRESUMO
The type II secretion (T2S) system of Legionella pneumophila is required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages, lsp mutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, IL-8, IL-1ß, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complemented proA mutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system of L. pneumophila dampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, an lspF mutant, but not the proA mutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.
Assuntos
Sistemas de Secreção Bacterianos/imunologia , Citocinas/imunologia , Doença dos Legionários/imunologia , Macrófagos/imunologia , Mucosa Respiratória/imunologia , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Humanos , Legionella pneumophila/imunologia , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Mucosa Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite's interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.
Assuntos
Genoma de Protozoário , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Proteínas de Protozoários/metabolismo , Algoritmos , Animais , Clonagem Molecular , Cricetinae , Humanos , Soros Imunes/imunologia , Immunoblotting , Leishmania infantum/genética , Leishmania infantum/imunologia , Masculino , Mesocricetus , Microscopia Confocal , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/biossínteseRESUMO
Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70-75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had approximately 75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.
Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Membranas Intracelulares/metabolismo , Legionella pneumophila/fisiologia , Doença dos Legionários/metabolismo , Doença dos Legionários/microbiologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Doença dos Legionários/genética , Camundongos , Mutação/genéticaRESUMO
The type II secretion system of Legionella pneumophila promotes pathogenesis. Among the Legionella type II-dependent exoenzymes is a p-nitrophenol phosphorylcholine (p-NPPC) hydrolase whose activity is only partially explained by the PlcA phospholipase C. In a screen to identify other factors that promote secreted hydrolase activity, we isolated a mip mutant. L. pneumophila Mip is a surface-exposed, FK506-binding protein that is needed for optimal infection and has peptidylproline cis-trans-isomerase (PPIase) activity. Since the molecular target of Mip was undefined, we investigated a possible relationship between Mip and the secreted p-NPPC hydrolase activity. In the mip mutant there was a 40 to 70% reduction in secreted activity that was successfully complemented by providing mip on a plasmid. A similar phenotype was observed when we examined four other independently derived mip mutants, and in all cases the defect was complemented by reintroduction of mip. Thus, mip promotes the presence of a p-NPPC hydrolase activity in culture supernatants. We also found that the C terminus of Mip is required for this effect. When supernatants were examined by anion-exchange chromatography, the p-NPPC hydrolase activity associated with Mip proved to be type II dependent but distinct from PlcA. This conclusion was supported by the phenotype of a newly constructed mip plcA double mutant. Thus, Mip promotes the elaboration of a new type II exoprotein. These data provide both the first evidence for a target for Mip and the first indication that a surface PPIase is involved in the secretion or activation of proteins beyond the outer membrane.
Assuntos
Imunofilinas/metabolismo , Legionella pneumophila/enzimologia , Proteínas de Membrana/metabolismo , Peptidilprolil Isomerase/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas de Bactérias , Imunofilinas/genética , Legionella pneumophila/genética , Proteínas de Membrana/genética , Mutação , Peptidilprolil Isomerase/genética , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Transporte ProteicoRESUMO
Plants have evolved a powerful immune system to defend against infection by most microbial organisms. However, successful pathogens, such as Pseudomonas syringae, have developed countermeasures and inject virulence proteins into the host plant cell to suppress immunity and cause devastating diseases. Despite intensive research efforts, the molecular targets of bacterial virulence proteins that are important for plant disease development have remained obscure. Here, we show that a conserved P. syringae virulence protein, HopM1, targets an immunity-associated protein, AtMIN7, in Arabidopsis thaliana. HopM1 mediates the destruction of AtMIN7 via the host proteasome. Our results illustrate a strategy by which a bacterial pathogen exploits the host proteasome to subvert host immunity and causes infection in plants.