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1.
Infect Immun ; 91(4): e0029622, 2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-36877045

RESUMO

Pneumococcal Ser/Thr kinase (StkP) and its cognate phosphatase (PhpP) play a crucial role in bacterial cytokinesis. However, their individual and reciprocal metabolic and virulence regulation-related functions have yet to be adequately investigated in encapsulated pneumococci. Here, we demonstrate that the encapsulated pneumococcal strain D39-derived D39ΔPhpP and D39ΔStkP mutants displayed differential cell division defects and growth patterns when grown in chemically defined media supplemented with glucose or nonglucose sugars as the sole carbon source. Microscopic and biochemical analyses supported by RNA-seq-based global transcriptomic analyses of these mutants revealed significantly down- and upregulated polysaccharide capsule formation and cps2 genes in D39ΔPhpP and D39ΔStkP mutants, respectively. While StkP and PhpP individually regulated several unique genes, they also participated in sharing the regulation of the same set of differentially regulated genes. Cps2 genes were reciprocally regulated in part by the StkP/PhpP-mediated reversible phosphorylation but independent of the MapZ-regulated cell division process. StkP-mediated dose-dependent phosphorylation of CcpA proportionately inhibited CcpA-binding to Pcps2A, supporting increased cps2 gene expression and capsule formation in D39ΔStkP. While the attenuation of the D39ΔPhpP mutant in two mouse infection models corroborated with several downregulated capsules-, virulence-, and phosphotransferase systems (PTS)-related genes, the D39ΔStkP mutant with increased amounts of polysaccharide capsules displayed significantly decreased virulence in mice compared to the D39 wild-type, but more virulence compared to D39ΔPhpP. NanoString technology-based inflammation-related gene expression and Meso Scale Discovery-based multiplex chemokine analysis of human lung cells cocultured with these mutants confirmed their distinct virulence phenotypes. StkP and PhpP may, therefore, serve as critical therapeutic targets.


Assuntos
Monoéster Fosfórico Hidrolases , Proteínas Serina-Treonina Quinases , Humanos , Animais , Camundongos , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Cápsulas/metabolismo , Virulência , Processamento de Proteína Pós-Traducional , Streptococcus pneumoniae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica
3.
Mol Oncol ; 16(7): 1508-1522, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-33969603

RESUMO

The role of commensal bacterial microbiota in the pathogenesis of human malignancies has been a research field of incomparable progress in recent years. Although breast tissue is commonly assumed to be sterile, recent studies suggest that human breast tissue may contain a bacterial microbiota. In this study, we used an immune-competent orthotopic breast cancer mouse model to explore the existence of a unique and independent bacterial microbiota in breast tumors. We observed some similarities in breast cancer microbiota with skin; however, breast tumor microbiota was mainly enriched with Gram-negative bacteria, serving as a primary source of lipopolysaccharide (LPS). In addition, dextran sulfate sodium (DSS) treatment in late-stage tumor lesions increased LPS levels in the breast tissue environment. We also discovered an increased expression of S100A7 and low level of TLR4 in late-stage tumors with or without DSS as compared to early-stage tumor lesions. The treatment of breast cancer cells with LPS increased the expression of S100A7 in breast cancer cells in vitro. Furthermore, S100A7 overexpression downregulated TLR4 and upregulated RAGE expression in breast cancer cells. Analysis of human breast cancer samples also highlighted the inverse correlation between S100A7 and TLR4 expression. Overall, these findings suggest that the commensal microbiota of breast tissue may enhance breast tumor burden through a novel LPS/S100A7/TLR4/RAGE signaling axis.


Assuntos
Neoplasias da Mama , Microbiota , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Proteína A7 Ligante de Cálcio S100/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo
4.
Front Microbiol ; 12: 689246, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34950110

RESUMO

Streptococcus pyogenes (Group A Streptococcus, GAS) genomes do not contain a gene encoding a typical bacterial-type tyrosine kinase (BY-kinase) but contain an orphan gene-encoding protein Tyr-phosphatase (SP-PTP). Hence, the importance of Tyr-phosphorylation is underappreciated and not recognized for its role in GAS pathophysiology and pathogenesis. The fact that SP-PTP dephosphorylates Abl-tyrosine kinase-phosphorylated myelin basic protein (MBP), and SP-STK (S. pyogenes Ser/Thr kinase) also autophosphorylates its Tyr101-residue prompted us to identify a putative tyrosine kinase and Tyr-phosphorylation in GAS. Upon a genome-wide search of kinases possessing a classical Walker motif, we identified a non-canonical tyrosine kinase M5005_Spy_1476, a ∼17 kDa protein (153 aa) (SP-TyK). The purified recombinant SP-TyK autophosphorylated in the presence of ATP. In vitro and in vivo phosphoproteomic analyses revealed two key phosphorylated tyrosine residues located within the catalytic domain of SP-TyK. An isogenic mutant lacking SP-TyK derived from the M1T1 strain showed a retarded growth pattern. It displayed defective cell division and long chains with multiple parallel septa, often resulting in aggregates. Transcriptomic analysis of the mutant revealed 287 differentially expressed genes responsible for GAS pathophysiology and pathogenesis. SP-TyK also phosphorylated GAS CovR, WalR, SP-STP, and SDH/GAPDH proteins with dual specificity targeting their Tyr/Ser/Thr residues as revealed by biochemical and mass-spectrometric-based phosphoproteomic analyses. SP-TyK-phosphorylated CovR bound to PcovR efficiently. The mutant displayed sustained release of IL-6 compared to TNF-α during co-culturing with A549 lung cell lines, attenuation in mice sepsis model, and significantly reduced ability to adhere to and invade A549 lung cells and form biofilms on abiotic surfaces. SP-TyK, thus, plays a critical role in fine-tuning the regulation of key cellular functions essential for GAS pathophysiology and pathogenesis through post-translational modifications and hence, may serve as a promising target for future therapeutic developments.

5.
EMBO Rep ; 20(12): e48109, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31637841

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a growing health concern due to increasing resistance to antibiotics. As a facultative intracellular pathogen, MRSA is capable of persisting within professional phagocytes including macrophages. Here, we identify a role for CASP11 in facilitating MRSA survival within murine macrophages. We show that MRSA actively prevents the recruitment of mitochondria to the vicinity of the vacuoles they reside in to avoid intracellular demise. This process requires CASP11 since its deficiency allows increased association of MRSA-containing vacuoles with mitochondria. The induction of mitochondrial superoxide by antimycin A (Ant A) improves MRSA eradication in casp11-/- cells, where mitochondria remain in the vicinity of the bacterium. In WT macrophages, Ant A does not affect MRSA persistence. When mitochondrial dissociation is prevented by the actin depolymerizing agent cytochalasin D, Ant A effectively reduces MRSA numbers. Moreover, the absence of CASP11 leads to reduced cleavage of CASP1, IL-1ß, and CASP7, as well as to reduced production of CXCL1/KC. Our study provides a new role for CASP11 in promoting the persistence of Gram-positive bacteria.


Assuntos
Caspases Iniciadoras/metabolismo , Macrófagos/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/imunologia , Animais , Antibacterianos/farmacologia , Antimicina A/farmacologia , Caspases Iniciadoras/genética , Células Cultivadas , Macrófagos/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/microbiologia , Vacúolos/metabolismo
6.
Microbiol Spectr ; 7(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30767846

RESUMO

In the past decade, the field of the cellular microbiology of group A Streptococcus (S. pyogenes) infection has made tremendous advances and touched upon several important aspects of pathogenesis, including receptor biology, invasive and evasive phenomena, inflammasome activation, strain-specific autophagic bacterial killing, and virulence factor-mediated programmed cell death. The noteworthy aspect of S. pyogenes-mediated cell signaling is the recognition of the role of M protein in a variety of signaling events, starting with the targeting of specific receptors on the cell surface and on through the induction and evasion of NETosis, inflammasome, and autophagy/xenophagy to pyroptosis and apoptosis. Variations in reports on S. pyogenes-mediated signaling events highlight the complex mechanism of pathogenesis and underscore the importance of the host cell and S. pyogenes strain specificity, as well as in vitro/in vivo experimental parameters. The severity of S. pyogenes infection is, therefore, dependent on the virulence gene expression repertoire in the host environment and on host-specific dynamic signaling events in response to infection. Commonly known as an extracellular pathogen, S. pyogenes finds host macrophages as safe havens wherein it survives and even multiplies. The fact that endothelial cells are inherently deficient in autophagic machinery compared to epithelial cells and macrophages underscores the invasive nature of S. pyogenes and its ability to cause severe systemic diseases. S. pyogenes is still one of the top 10 causes of infectious mortality. Understanding the orchestration of dynamic host signaling networks will provide a better understanding of the increasingly complex mechanism of S. pyogenes diseases and novel ways of therapeutically intervening to thwart severe and often fatal infections.


Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Transdução de Sinais/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Animais , Antígenos de Bactérias/metabolismo , Apoptose/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Piroptose/fisiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Sci Rep ; 7(1): 5067, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28698584

RESUMO

Ser/Thr protein kinase (STK1) plays a critical role in cell wall biosynthesis of and drug resistance in methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains lacking STK1 become susceptible to failing cephalosporins, such as Ceftriaxone and Cefotaxime. STK1, despite being nonessential protein for MRSA survival, it can serve as an important therapeutic agent for combination therapy. Here, we report a novel small molecule quinazoline compound, Inh2-B1, which specifically inhibits STK1 activity by directly binding to its ATP-binding catalytic domain. Functional analyses encompassing in vitro growth inhibition of MRSA, and in vivo protection studies in mice against the lethal MRSA challenge indicated that at high concentration neither Inh2-B1 nor Ceftriaxone or Cefotaxime alone was able to inhibit the growth of bacteria or protect the challenged mice. However, the growth of MRSA was inhibited, and a significant protection in mice against the bacterial challenge was observed at a micromolar concentration of Ceftriaxone or Cefotaxime in the presence of Inh2-B1. Cell-dependent minimal to no toxicity of Inh2-B1, and its abilities to down-regulate cell wall hydrolase genes and disrupt the biofilm formation of MRSA clearly indicated that Inh2-B1 serves as a therapeutically important "antibiotic-resistance-breaker," which enhances the bactericidal activity of Ceftriaxone/Cefotaxime against highly pathogenic MRSA infection.


Assuntos
Resistência Microbiana a Medicamentos/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Virulência/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Sítios de Ligação , Biofilmes/efeitos dos fármacos , Domínio Catalítico , Cefotaxima/farmacologia , Cefotaxima/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Linhagem Celular , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Genes Bacterianos , Humanos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Camundongos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sepse/tratamento farmacológico , Sepse/microbiologia , Sepse/patologia , Bibliotecas de Moléculas Pequenas/química , Fatores de Virulência/química , Fatores de Virulência/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-27790410

RESUMO

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Parede Celular/química , Hemeproteínas/genética , Hemeproteínas/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Antibacterianos/farmacologia , Aderência Bacteriana , Farmacorresistência Bacteriana , Deleção de Genes , Glicosídeos/análise , Heme/metabolismo , Proteínas Ligantes de Grupo Heme , N-Acetil-Muramil-L-Alanina Amidase/análise , Peptidoglicano/análise , Ligação Proteica , Multimerização Proteica , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/fisiologia , beta-Lactamas/farmacologia
9.
Mol Microbiol ; 97(3): 515-40, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25939957

RESUMO

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP-PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP-PTP is, therefore, questionable. Here, we demonstrate that SP-PTP possesses dual phosphatase specificity for Tyr- and Ser/Thr-phosphorylated GAS proteins, such as Ser/Thr kinase (SP-STK) and the SP-STK-phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP-PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP-PTP displayed increased Ser-/Thr-/Tyr-phosphorylation. SP-PTP also differentially regulates the expression of ∼50% of the total GAS genes, including several virulence genes potentially through the two-component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP-PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr-phosphorylation in GAS and the relevance of SP-PTP as an important therapeutic target.


Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Tirosina Fosfatases/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Fatores de Virulência/biossíntese , Animais , Divisão Celular , Linhagem Celular , Células Epiteliais/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases/genética , Análise de Sequência de DNA , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/fisiologia , Análise de Sobrevida , Virulência
10.
Infect Immun ; 80(4): 1361-72, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22311926

RESUMO

Streptococcus pneumoniae exploits a battery of virulence factors to colonize the host. Although the eukaryote-like Ser/Thr kinase of S. pneumoniae (StkP) has been implicated in physiology and virulence, the role of its cotranscribing phosphatase (PhpP) has remained elusive. The construction of nonpolar markerless phpP knockout mutants (ΔphpP) in two pathogenic strains, D39 (type 2) and 6A-EF3114 (type 6A), indicated that PhpP is not indispensable for pneumococcal survival. Further, PhpP also participates in the regulation of cell wall biosynthesis/division, adherence, and biofilm formation in a strain-specific manner. Additionally, we provide hitherto-unknown in vitro and in vivo evidence of a physiologically relevant biochemical link between the StkP/PhpP-mediated cognate regulation and the two-component regulatory system TCS06 (RR06/HK06) that regulates the expression of the gene encoding an important pneumococcal surface adhesin, CbpA, which was found to be significantly upregulated in ΔphpP mutants. In particular, StkP (threonine)-phosphorylated RR06 bound to the cbpA promoter with high efficiency even in the absence of the HK06-responsive and catalytically active aspartate 51 residue. Together, our findings unravel the significant contributions of PhpP in pneumococcal physiology and adherence.


Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Streptococcus pneumoniae/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Fosfoproteínas Fosfatases/genética , Transdução de Sinais , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Streptococcus pneumoniae/fisiologia
11.
J Biol Chem ; 287(12): 9147-67, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22262847

RESUMO

This investigation illustrates an important property of eukaryote-type serine/threonine phosphatase (SP-STP) of group A Streptococcus (GAS) in causing programmed cell death of human pharyngeal cells. The secretory nature of SP-STP, its elevated expression in the intracellular GAS, and the ability of wild-type GAS but not the GAS mutant devoid of SP-STP to cause apoptosis of the host cell both in vitro and in vivo suggest that GAS deploys SP-STP as an important virulence determinant to exploit host cell machinery for its own advantage during infection. The exogenously added SP-STP is able to enter the cytoplasm and subsequently traverses into the nucleus in a temporal fashion to cause apoptosis of the pharyngeal cells. The programmed cell death induced by SP-STP, which requires active transcription and de novo protein synthesis, is also caspase-dependent. Furthermore, the entry of SP-STP into the cytoplasm is dependent on its secondary structure as the catalytically inactive SP-STP with an altered structure is unable to internalize and cause apoptosis. The ectopically expressed wild-type SP-STP was found to be in the nucleus and conferred apoptosis of Detroit 562 pharyngeal cells. However, the catalytically inactive SP-STP was unable to cause apoptosis even when intracellularly expressed. The ability of SP-STP to activate pro-apoptotic signaling cascades both in the cytoplasm and in the nucleus resulted in mitochondrial dysfunctioning and perturbation in the phosphorylation status of histones in the nucleus. SP-STP thus not only functions as a virulence regulator but also as an important factor responsible for host-related pathogenesis.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Bactérias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/fisiopatologia , Streptococcus pyogenes/enzimologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Bactérias/genética , Linhagem Celular , Humanos , Camundongos , Doenças Faríngeas/microbiologia , Doenças Faríngeas/fisiopatologia , Fosfoproteínas Fosfatases/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Virulência
12.
J Biol Chem ; 286(48): 41368-41380, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21917918

RESUMO

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Mutação , Fosfoproteínas Fosfatases/genética , Fosforilação/fisiologia , Fatores de Virulência/genética
13.
mBio ; 2(3): e00068-11, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21628503

RESUMO

UNLABELLED: Streptococcal surface dehydrogenase (SDH) (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) is an anchorless major multifunctional surface protein in group A Streptococcus (GAS) with the ability to bind important mammalian proteins, including plasmin(ogen). Although several biological properties of SDH are suggestive of its possible role in GAS virulence, its direct role in GAS pathogenesis has not been ascertained because it is essential for GAS survival. Thus, it has remained enigmatic as to "how and why" SDH/GAPDH is exported onto the bacterial surface. The present investigation highlights "why" SDH is exported onto the GAS surface. Differential microarray-based genome-wide transcript abundance analysis was carried out using a specific mutant, which was created by inserting a hydrophobic tail at the C-terminal end of SDH (M1-SDH(HBtail)) and thus preventing its exportation onto the GAS surface. This analysis revealed downregulation of the majority of genes involved in GAS virulence and genes belonging to carbohydrate and amino acid metabolism and upregulation of those related to lipid metabolism. The complete attenuation of this mutant for virulence in the mouse model and the decreased and increased virulence of the wild-type and mutant strains postcomplementation with SDH(HBtail) and SDH, respectively, indicated that the SDH surface export indeed regulates GAS virulence. M1-SDH(HBtail) also displayed unaltered growth patterns, increased intracellular ATP concentration and Hpr double phosphorylation, and significantly reduced pH tolerance, streptolysin S, and SpeB activities. These phenotypic and physiological changes observed in the mutant despite the unaltered expression levels of established transcriptional regulators further highlight the fact that SDH interfaces with many regulators and its surface exportation is essential for GAS virulence. IMPORTANCE: Streptococcal surface dehydrogenase (SDH), a classical anchorless cytoplasmically localized glycolytic enzyme, is exported onto the group A Streptococcus (GAS) surface through a hitherto unknown mechanism(s). It has not been known why GAS or other prokaryotes should export this protein onto the surface. By genetic manipulations, we created a novel GAS mutant strain expressing SDH with a 12-amino-acid hydrophobic tail at its C-terminal end and thus were able to prevent its surface exportation without altering its enzymatic activity or growth pattern. Interestingly, the mutant was completely attenuated for virulence in a mouse peritonitis model. The global gene expression profiles of this mutant reveal that the surface exportation of SDH is mandatory to maintain GAS virulence. The ability of GAS as a successful pathogen to localize SDH in the cytoplasm as well as on the surface is physiologically relevant and dynamically obligatory to fine-tune the functions of many transcriptional regulators and also to exploit its virulence properties for infection.


Assuntos
Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Redes e Vias Metabólicas/genética , Camundongos , Análise em Microsséries , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Doenças dos Roedores/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Virulência , Fatores de Virulência/genética
14.
J Biol Chem ; 285(40): 30861-74, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20643653

RESUMO

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/enzimologia , Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Escherichia coli/genética , Deleção de Genes , Humanos , Hidrolases/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Serina-Treonina Quinases/genética , Staphylococcus aureus/genética , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/terapia , Streptococcus pyogenes/genética , Fatores de Virulência/genética
15.
PLoS One ; 5(1): e8810, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20098674

RESUMO

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO(4), a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO(4) dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.


Assuntos
Fosfopiruvato Hidratase/metabolismo , Streptococcus pyogenes/enzimologia , Dicroísmo Circular , Dimerização , Cinética , Modelos Moleculares , Mutação , Fosfopiruvato Hidratase/antagonistas & inibidores , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/genética , Ligação Proteica , Conformação Proteica , Ultracentrifugação
16.
Infect Immun ; 77(12): 5519-27, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19752027

RESUMO

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is a low-grade opportunistic pathogen which is considered to be associated with colon cancer. It is thought that colon polyps or tumors are the main portal of entry for this bacterium and that heparan sulfate proteoglycans (HSPGs) at the colon tumor cell surface are involved in bacterial adherence during the first stages of infection. In this study, we have shown that the histone-like protein A (HlpA) of S. gallolyticus is a genuine anchorless bacterial surface protein that binds to lipoteichoic acid (LTA) of the gram-positive cell wall in a growth phase-dependent manner. In addition, HlpA was shown to be one of the major heparin-binding proteins of S. gallolyticus able to bind to the HSPG-expressing colon tumor cell lines HCT116 and HT-29. Strikingly, although wild-type levels of HlpA appeared to contribute to adherence, coating of additional HlpA at the bacterial surface resulted in reduced binding to colon tumor cells. This may be explained by the fact that heparan sulfate and LTA compete for the same binding site in HlpA. Altogether, this study implies that HlpA serves as a fine-tuning factor for bacterial adherence.


Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Proteínas de Bactérias/fisiologia , Proteínas de Membrana/fisiologia , Streptococcus bovis/patogenicidade , Linhagem Celular Tumoral , Heparina/metabolismo , Humanos , Ligação Proteica
17.
J Biol Chem ; 284(25): 17129-17137, 2009 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-19363026

RESUMO

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae alpha-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN(K252 + 255A), SEN(K304A), SEN(K334A), SEN(K344E), SEN(K435L), and SEN(Delta434-435)) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN(K344E), as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN(K344E) had lost alpha-enolase activity, which was also reduced in SEN(K334A) and SEN(Delta434-435). Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN(K252 + 255A), SEN(K435L), and SEN(Delta434-435). The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Streptococcus pyogenes/enzimologia , Proteínas de Bactérias/genética , Estabilidade Enzimática , Humanos , Técnicas In Vitro , Lisina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfopiruvato Hidratase/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Ressonância de Plasmônio de Superfície
18.
J Bacteriol ; 191(9): 3095-107, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19270100

RESUMO

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.


Assuntos
Aeromonas hydrophila/enzimologia , Aeromonas hydrophila/patogenicidade , Proteínas de Bactérias/metabolismo , Fosfopiruvato Hidratase/metabolismo , Fatores de Virulência/metabolismo , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/isolamento & purificação , Animais , Vacinas Bacterianas/imunologia , Sítios de Ligação , Contagem de Colônia Microbiana , Diarreia/microbiologia , Fibrinolisina/metabolismo , Deleção de Genes , Genes Bacterianos , Genes Essenciais , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Fígado/microbiologia , Camundongos , Viabilidade Microbiana , Mutação de Sentido Incorreto , Fosfopiruvato Hidratase/imunologia , Plasminogênio/metabolismo , Ligação Proteica , Baço/microbiologia , Análise de Sobrevida
19.
Infect Immun ; 77(4): 1406-16, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19188361

RESUMO

It is well established that prokaryotes and eukaryotes alike utilize phosphotransfer to regulate cellular functions. One method by which this occurs is via eukaryote-like serine/threonine kinase (ESTK)- and phosphatase (ESTP)-regulated pathways. The role of these enzymes in Staphylococcus aureus has not yet been examined. This resilient organism is a common cause of hospital-acquired and community-associated infections, infecting immunocompromised and immunocompetent hosts alike. In this study, we have characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of cell wall structure and susceptibility to cell wall-acting beta-lactam antibiotics. By utilizing gene knockout strategies, we created S. aureus N315 mutants lacking STP and/or STK. The strain lacking both STP and STK displayed notable cell division defects, including multiple and incomplete septa, bulging, and irregular cell size, as observed by transmission electron microscopy. Mutants lacking STP alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, compared to the wild type. Additionally, mutant strains lacking STK or both STK and STP displayed increased sensitivity to cell wall-acting cephalosporin and carbapenem antibiotics. Together, these results indicate that S. aureus STK- and STP-mediated reversible phosphorylation reactions play a critical role in proper cell wall architecture, and thus the modulation of antimicrobial resistance, in S. aureus.


Assuntos
Antibacterianos/farmacologia , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Eucarióticas/enzimologia , Regulação Bacteriana da Expressão Gênica , Humanos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Mutação , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/genética , beta-Lactamas/farmacologia
20.
J Bacteriol ; 190(10): 3658-69, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18310335

RESUMO

OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.


Assuntos
Biofilmes/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrogênio/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/metabolismo , Microscopia Eletrônica de Transmissão , Peptidoglicano/metabolismo , Plâncton/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Transativadores/genética
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