RESUMO
Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases.
Assuntos
Proteínas de Bactérias/fisiologia , Redes Reguladoras de Genes , Streptococcus agalactiae/patogenicidade , Fatores de Virulência/fisiologia , Virulência/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos , Genes Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Regiões Promotoras Genéticas , Prófagos/genética , Streptococcus agalactiae/genética , Transcrição Gênica/fisiologia , Fatores de Virulência/genéticaRESUMO
Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Osmorregulação/fisiologia , Streptococcus agalactiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Homeostase/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Mutação , Osmorregulação/genética , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Potássio/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/crescimento & desenvolvimentoRESUMO
Binding of microbial pathogens to host vitronectin (Vtn) is a common theme in the pathogenesis of invasive infections. In this study, we characterized the role of Vtn in the invasion of mucosal epithelial cells by Streptococcus agalactiae (i.e. group B streptococcus or GBS), a frequent human pathogen. Moreover, we identified PbsP, a previously described plasminogen-binding protein of GBS, as a dual adhesin that can also interact with human Vtn through its streptococcal surface repeat (SSURE) domains. Deletion of the pbsP gene decreases both bacterial adhesion to Vtn-coated inert surfaces and the ability of GBS to interact with epithelial cells. Bacterial adherence to and invasion of epithelial cells were either inhibited or enhanced by cell pretreatment with, respectively, anti-Vtn antibodies or Vtn, confirming the role of Vtn as a GBS ligand on host cells. Finally, antibodies directed against the integrin αv subunit inhibited Vtn-dependent cell invasion by GBS. Collectively, these results indicate that Vtn acts as a bridge between the SSURE domains of PbsP on the GBS surface and host integrins to promote bacterial invasion of epithelial cells. Therefore, inhibition of interactions between PbsP and extracellular matrix components could represent a viable strategy to prevent colonization and invasive disease by GBS.
Assuntos
Proteínas de Bactérias/metabolismo , Integrina alfaV/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidade , Vitronectina/metabolismo , Células A549 , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Células CACO-2 , Parede Celular/metabolismo , Células Epiteliais/microbiologia , Humanos , Integrina alfaV/genética , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus agalactiae/genética , Vitronectina/genéticaRESUMO
Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine-rich region, and a LPXTG cell wall-anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface-bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non-CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.
Assuntos
Adesinas Bacterianas/metabolismo , Plasminogênio/metabolismo , Streptococcus agalactiae/metabolismo , Sequência de Aminoácidos , Animais , Aderência Bacteriana/fisiologia , Parede Celular/metabolismo , Células Endoteliais/metabolismo , Fibrinolisina/metabolismo , Humanos , Camundongos , Ligação Proteica , Infecções Estreptocócicas/microbiologia , Streptococcus/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , VirulênciaRESUMO
Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The ß-hemolysin/cytolysin (ß-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for ß-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the ß-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, ß-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.
Assuntos
Proteínas Hemolisinas/análise , Pigmentos Biológicos/análise , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificação , Adulto , Técnicas Bacteriológicas , Meios de Cultura/química , França/epidemiologia , Deleção de Genes , Estudos de Associação Genética , Teste de Complementação Genética , Genoma Bacteriano , Humanos , Recém-Nascido , Análise de Sequência de DNA , Infecções Estreptocócicas/epidemiologia , Streptococcus agalactiae/classificaçãoRESUMO
Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (â¼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.
Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Parede Celular/fisiologia , Proteínas Fúngicas/fisiologia , Proteoma/fisiologia , Candida albicans/citologia , Adesão Celular/fisiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Fenótipo , Proteoma/genética , Resistência ao Cisalhamento/fisiologia , Transcriptoma/fisiologiaRESUMO
The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida glabrata/genética , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida glabrata/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , Caspofungina , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Proteínas Fúngicas/genética , Deleção de Genes , Técnicas de Inativação de Genes , Biblioteca Gênica , Lipopeptídeos , Testes de Sensibilidade Microbiana , Pressão Osmótica , FenótipoRESUMO
Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.
Assuntos
Adenosina/metabolismo , Viabilidade Microbiana , N-Glicosil Hidrolases/metabolismo , Streptococcus agalactiae/enzimologia , Adenosina/genética , Motivos de Aminoácidos , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/imunologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/imunologiaRESUMO
Streptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Fibrinogênio/imunologia , Interações Hospedeiro-Patógeno/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia , Fatores de Virulência/imunologia , Animais , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Proteínas de Bactérias/genética , Células CACO-2 , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Fibrinogênio/genética , Humanos , Camundongos , Ligação Proteica/genética , Ligação Proteica/imunologia , Infecções Estreptocócicas/genética , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/imunologia , Fatores de Virulência/genéticaRESUMO
The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Hemolisinas/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Macrófagos/metabolismo , RNA Bacteriano/metabolismo , Streptococcus agalactiae/fisiologia , Animais , Humanos , Interleucina-1beta/metabolismo , Lisossomos/metabolismo , Lisossomos/microbiologia , Macrófagos/citologia , Macrófagos/microbiologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagossomos/metabolismo , Fagossomos/microbiologia , Streptococcus agalactiae/metabolismoRESUMO
Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Epistasia Genética , Feminino , Perfilação da Expressão Gênica , Hemólise , Histidina Quinase , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Pigmentos Biológicos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Streptococcus agalactiae/metabolismo , Streptococcus agalactiae/patogenicidade , Virulência/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: SUN proteins are involved in yeast morphogenesis, but their function is unknown. RESULTS: SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. CONCLUSION: Both Candida albicans and Aspergillus fumigatus sun proteins show a ß-(1,3)-glucanase activity. SIGNIFICANCE: The mode of action of SUN proteins on ß-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on ß-(1,3)-oligomers from dimer up to insoluble ß-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Hifas/enzimologia , Morfogênese , Esporos Fúngicos/enzimologia , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Candida albicans/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosilação , Hidrólise , Hifas/genética , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Oligossacarídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.
Assuntos
Alanina/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Parede Celular/metabolismo , Resistência Microbiana a Medicamentos , Lipopolissacarídeos/metabolismo , Streptococcus/efeitos dos fármacos , Ácidos Teicoicos/metabolismo , Alanina/farmacologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Concentração Osmolar , Processamento de Proteína Pós-Traducional/fisiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/metabolismo , Streptococcus/ultraestrutura , Propriedades de Superfície , Ácidos Teicoicos/farmacologiaRESUMO
Group B Streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis. A major virulence factor is a pigmented beta-haemolytic/cyto-lysin (ß-h/c) toxin with an ornithine rhamnolipid structure. We initially observed that absence of MprF enzyme altered pigmentation and haemolytic activity in GBS. Next, we showed that MprF-dependent lipid lysination contributes to the retention of the ornithine rhamnolipid within GBS membrane. Furthermore, cationic lipidation by MprF altered membrane properties contributing to resistance to the cyclic lipopeptide daptomycin and to acidic pH. This study highlights the importance of cationic lipids in cell envelope homeostasis and in modulating ß-h/c activity.
RESUMO
Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is not known whether specific mechanisms constrain the emergence of resistance. In this study, we first report ß-lactam tolerance due to the inactivation of the c-di-AMP phosphodiesterase GdpP. Mechanistically, tolerance depends on antagonistic regulation by the repressor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility through the BusAB osmolyte transporter and the AmaP/Asp23/GlsB cell envelope stress complex. The BusR transcriptional response is synergistic with the simultaneous allosteric inhibition of potassium and osmolyte transporters by c-di-AMP, which individually contribute to low-level ß-lactam tolerance. Genome-wide transposon mutagenesis confirms the role of GdpP and highlights functional interactions between a lysozyme-like hydrolase, the KhpAB RNA chaperone and the protein S immunomodulator in the response of GBS to ß-lactam. Overall, we demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response at the transcriptional and post-translational levels to cell wall weakening caused by ß-lactam activity, and reveal additional mechanisms that could foster resistance.
RESUMO
Group B Streptococcus (GBS) is a pathobiont responsible for invasive infections in neonates and the elderly. The transition from a commensal to an invasive pathogen relies on the timely regulation of virulence factors. In this study, we characterized the role of the SaeRS two-component system in GBS pathogenesis. Loss-of-function mutations in the SaeR response regulator decrease virulence in mouse models of invasive infection by hindering the ability of bacteria to persist at the inoculation site and to spread to distant organs. Transcriptome and in vivo analysis reveal a specialized regulatory system specifically activated during infection to control the expression of only two virulence factors: the PbsP adhesin and the BvaP secreted protein. The in vivo surge in SaeRS-regulated genes is complemented by fine-tuning mediated by the repressor of virulence CovRS system to establish a coordinated response. Constitutive activation of the SaeRS regulatory pathway increases PbsP-dependent adhesion and invasion of epithelial and endothelial barriers, though at the cost of reduced virulence. In conclusion, SaeRS is a dynamic, highly specialized regulatory system enabling GBS to express a restricted set of virulence factors that promote invasion of host barriers and allow these bacteria to persist inside the host during lethal infection. IMPORTANCE: Group B Streptococcus (or GBS) is a normal inhabitant of the human gastrointestinal and genital tracts that can also cause deadly infections in newborns and elderly people. The transition from a harmless commensal to a dangerous pathogen relies on the timely expression of bacterial molecules necessary for causing disease. In this study, we characterize the two-component system SaeRS as a key regulator of such virulence factors. Our analysis reveals a specialized regulatory system that is activated only during infection to dynamically adjust the production of two virulence factors involved in interactions with host cells. Overall, our findings highlight the critical role of SaeRS in GBS infections and suggest that targeting this system may be useful for developing new antibacterial drugs.
RESUMO
Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.
Assuntos
Proteínas de Transporte , Streptococcus agalactiae , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo , Guanosina Pentafosfato , Guanosina Tetrafosfato , Proteínas de Bactérias/metabolismo , AMP Cíclico , Fosfatos de Dinucleosídeos/metabolismo , Potássio/metabolismoRESUMO
Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 µg/ml) were found to harbor truncated tet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 µg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet(M). Expression in Enterococcus faecalis of a tet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet(M) and by alterations of the upstream transcriptional attenuator.
Assuntos
Antibacterianos/farmacologia , Oligossacarídeos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Acinetobacter/efeitos dos fármacos , Acinetobacter/metabolismo , Acinetobacter/ultraestrutura , Alginatos/química , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Burkholderia/efeitos dos fármacos , Burkholderia/metabolismo , Burkholderia/ultraestrutura , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Microscopia de Força Atômica , Microscopia Confocal , Oligossacarídeos/síntese química , Oligossacarídeos/química , Pseudomonas/efeitos dos fármacos , Pseudomonas/metabolismo , Pseudomonas/ultraestrutura , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestrutura , Resistência a TetraciclinaRESUMO
Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.