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1.
Am J Pathol ; 190(4): 862-873, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32200972

RESUMO

Group A streptococcus (GAS) is a major pathogen that impacts health and economic affairs worldwide. Although the oropharynx is the primary site of infection, GAS can colonize the female genital tract and cause severe diseases, such as puerperal sepsis, neonatal infections, and necrotizing myometritis. Our understanding of how GAS genes contribute to interaction with the primate female genital tract is limited by the lack of relevant animal models. Using two genome-wide transposon mutagenesis screens, we identified 69 GAS genes required for colonization of the primate vaginal mucosa in vivo and 96 genes required for infection of the uterine wall ex vivo. We discovered a common set of 39 genes important for GAS fitness in both environments. They include genes encoding transporters, surface proteins, transcriptional regulators, and metabolic pathways. Notably, the genes that encode the surface-exclusion protein (SpyAD) and the immunogenic secreted protein 2 (Isp2) were found to be crucial for GAS fitness in the female primate genital tract. Targeted gene deletion confirmed that isogenic mutant strains ΔspyAD and Δisp2 are significantly impaired in ability to colonize the primate genital tract and cause uterine wall pathologic findings. Our studies identified novel GAS genes that contribute to female reproductive tract interaction that warrant translational research investigation.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Doenças Vaginais/microbiologia , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Regulação Bacteriana da Expressão Gênica , Macaca fascicularis , Proteínas de Membrana/genética , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Doenças Vaginais/patologia , Virulência
3.
PLoS Biol ; 17(10): e3000496, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603896

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been harnessed as powerful genome editing tools in diverse organisms. However, the off-target effects and the protospacer adjacent motif (PAM) compatibility restrict the therapeutic applications of these systems. Recently, a Streptococcus pyogenes Cas9 (SpCas9) variant, xCas9, was evolved to possess both broad PAM compatibility and high DNA fidelity. Through determination of multiple xCas9 structures, which are all in complex with single-guide RNA (sgRNA) and double-stranded DNA containing different PAM sequences (TGG, CGG, TGA, and TGC), we decipher the molecular mechanisms of the PAM expansion and fidelity enhancement of xCas9. xCas9 follows a unique two-mode PAM recognition mechanism. For non-NGG PAM recognition, xCas9 triggers a notable structural rearrangement in the DNA recognition domains and a rotation in the key PAM-interacting residue R1335; such mechanism has not been observed in the wild-type (WT) SpCas9. For NGG PAM recognition, xCas9 applies a strategy similar to WT SpCas9. Moreover, biochemical and cell-based genome editing experiments pinpointed the critical roles of the E1219V mutation for PAM expansion and the R324L, S409I, and M694I mutations for fidelity enhancement. The molecular-level characterizations of the xCas9 nuclease provide critical insights into the mechanisms of the PAM expansion and fidelity enhancement of xCas9 and could further facilitate the engineering of SpCas9 and other Cas9 orthologs.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , RNA Guia/genética , Substituição de Aminoácidos , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Engenharia de Proteínas/métodos , RNA Guia/química , RNA Guia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
4.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470538

RESUMO

Protein conjugations at post-translational levels are known to be essential to protein stability and function. Recently, it has been proven that the split protein CnaB2 (SpyTag/SpyCatcher, ST/SC) from Streptococcus pyogenes can induce covalent conjugation rapidly and efficiently under various conditions. The protein of interest fused with the split protein SC/ST could be assembled spontaneously. In light of this finding, we introduced the ST/SC protein coupling concept into the silkworm-bacmid protein expression system (SpyBEVS). As a proof of concept, we first examined and confirmed that a competent ligation occurred between ST/SC-fused protein partners in vitro in cultured silkworm cells and in vivo in silkworm larvae by co-infection of several recombinant baculoviruses. The protein conjugation could be also achieved sufficiently by a simple one-step mixture of purified ST/SC-tagged peptide-protein pairs in vitro. Given the flexibility and robustness of silkworm-BEVS, our results on SpyBEVS show an alternative method for enabling the production of protein decorations in vitro and inside of silkworms.


Assuntos
Bombyx/genética , Vetores Genéticos/genética , Proteínas de Insetos/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bombyx/citologia , Bombyx/metabolismo , Células Cultivadas , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Engenharia de Proteínas/métodos , Estabilidade Proteica , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo
5.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527126

RESUMO

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Clindamicina/farmacologia , Histidina Quinase/metabolismo , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/metabolismo , Estreptolisinas/biossíntese , Proteínas de Bactérias/biossíntese , Histidina Quinase/antagonistas & inibidores , Histidina Quinase/genética , Magnésio/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Streptococcus pyogenes/patogenicidade
6.
Bull Exp Biol Med ; 167(3): 367-370, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31346882

RESUMO

We analyzed cytokine profile in the sera of CBA mice in 1, 5, and 24 h after intraperitoneal injection of supernatants of broth cultures of group A Streptococcus types 1M and 3M and Dochez NY5 type 10M strain. The increase of the cytokine content was observed in response to supernatants of all three types, but the highest values were recorded after injection of supernatant of strain Dochez-NY5. The level of IL-2 increased most drastically (by 51 times) and the level of IL-5 increased by 8.9 times in comparison with the control. The level of IL-2 also increased after injection of supernatants of type 1M and type 3M, but to a lesser extent (by 5 and 2.3 times, respectively). The content of proinflammatory cytokines IL-1ß, TNFα, and IFNγ in mouse sera increased to a lesser extent than IL-2 after administration of all three supernatants.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Citocinas/sangue , Streptococcus pyogenes/metabolismo , Animais , Interferon gama/sangue , Interleucina-1beta/sangue , Interleucina-2/sangue , Interleucina-5/sangue , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Masculino , Camundongos , Camundongos Endogâmicos CBA
7.
PLoS Pathog ; 15(6): e1007841, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206562

RESUMO

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (ΔRSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ΔRSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ΔRSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ΔRSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Metilação de DNA , Enzimas de Restrição-Modificação do DNA , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Streptococcus pyogenes , Animais , Proteínas de Bactérias/genética , Citocinas/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Fasciite Necrosante/genética , Fasciite Necrosante/metabolismo , Fasciite Necrosante/patologia , Feminino , Humanos , Camundongos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade
8.
Nucleic Acids Res ; 47(14): 7476-7493, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31188450

RESUMO

Pathogenic bacteria encounter host-imposed manganese (Mn) limitation during infection. Herein we report that in the human pathogen Streptococcus pyogenes, the adaptive response to Mn limitation is controlled by a DtxR family metalloregulator, MtsR. Genes upregulated by MtsR during Mn limitation include Mn (mtsABC) and Fe acquisition systems (sia operon), and a metal-independent DNA synthesis enzyme (nrdFEI.2). To elucidate the mechanism of metal sensing and gene regulation by MtsR, we determined the crystal structure of MtsR. MtsR employs two Mn-sensing sites to monitor metal availability, and metal occupancy at each site influences MtsR regulatory activity. The site 1 acts as the primary Mn sensing site, and loss of metal at site 1 causes robust upregulation of mtsABC. The vacant site 2 causes partial induction of mtsABC, indicating that site 2 functions as secondary Mn sensing site. Furthermore, we show that the C-terminal FeoA domains of adjacent dimers participate in the oligomerization of MtsR on DNA, and multimerization is critical for MtsR regulatory activity. Finally, the mtsR mutant strains defective in metal sensing and oligomerization are attenuated for virulence in a mouse model of invasive infection, indicating that Mn sensing and gene regulation by MtsR are critical processes during S. pyogenes infection.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Manganês/metabolismo , Streptococcus pyogenes/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Manganês/química , Camundongos , Modelos Moleculares , Mutação , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Virulência/genética
9.
Nat Commun ; 10(1): 2727, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227708

RESUMO

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.


Assuntos
Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Cromatografia de Afinidade , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Mapeamento de Epitopos , Voluntários Saudáveis , Humanos , Espectrometria de Massas , Proteínas Opsonizantes/imunologia , Proteínas Opsonizantes/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/imunologia , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo
10.
mBio ; 10(3)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239377

RESUMO

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro, affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS.IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae, binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization.


Assuntos
Adesinas Bacterianas/genética , Ferro/metabolismo , Membrana Mucosa/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Vagina/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homeostase , Camundongos , Regulon/genética , Streptococcus pyogenes/crescimento & desenvolvimento
11.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052154

RESUMO

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.


Assuntos
Toxinas Bacterianas/química , Engenharia de Proteínas/métodos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Nanopartículas/química , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismo
12.
Curr Microbiol ; 76(6): 698-705, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30955044

RESUMO

Group A streptococcus (GAS) is an important human pathogen whose clinical isolates differ in their ability to produce hydrogen peroxide (H2O2). H2O2 is primarily produced by the enzyme lactate oxidase (LctO), an in depth in silico research revealed that all genome-sequenced GAS possess the required gene lctO. The importance of lctO for GAS is underlined by its highly conserved catabolite control element (cre box) as well as its perfect promotor sequence in comparison to the known consensus sequences of the Gram-positive model organism Bacillus subtilis. In this study, we provide further insight in the function and regulation of lactate oxidase by analyzing a large group of clinical GAS isolates. We found that H2O2 production increased over time in the late stationary phase; after 4 days of incubation, 5.4% of the isolates showed a positive result at 37 °C, while the rate increased to 16.4% at 20 °C. This correlation between H2O2 production and low temperatures suggests additional regulatory mechanisms for lctO besides catabolite control protein A (CcpA) and indicates that lctO might play a role for GAS energy metabolism at sub-body temperatures. Furthermore, we could identify that H2O2 production was different among clinical isolates; we could correlate H2O2 production to emm-types, indicating that emm-types 6 and 75 had the highest rate of H2O2 production. The emm-type- and temperature-dependent H2O2 production of clinical GAS isolates might contribute to their different survival strategies.


Assuntos
Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa/análise , Proteínas de Transporte/análise , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxidantes/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/efeitos da radiação , Proteínas de Bactérias/metabolismo , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica , Humanos , Proteínas Repressoras/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Temperatura
13.
Biochem Biophys Res Commun ; 510(4): 539-544, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30737033

RESUMO

Streptococcus pyogenes (group A Streptococcus, GAS) has caused a wide variety of human diseases. Its multifunctional surface dehydrogenase (SDH) is crucial for GAS life cycle. Furthermore, GAS infection into human pharyngeal cells has been previously shown to be mediated by the interaction between SDH and host urokinase-type plasminogen activator receptor (uPAR). However, the structural information of SDH remains to be elucidated and there are few detailed studies to characterize its interaction with uPAR. In-depth research on these issues will provide potential targets and strategies for combating GAS. Here, we prepared recombinant SDH tetramer in Escherichia coli BL21 (DE3) cells. After purification and crystallization, we determined its crystal structure at 1.74 Å. The unique characteristics might be potentially explored as drug targets or vaccine immunogen. We subsequently performed gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and in vitro pull-down analyses. The results showed that their interaction was too weak to form stable complexes and the role of uPAR involved in GAS infection needs further demonstration. Altogether the current work provides the first view of SDH and deepens the knowledge of GAS infection.


Assuntos
Proteínas de Bactérias/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus pyogenes/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Mapas de Interação de Proteínas , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/química
14.
Mol Cell Proteomics ; 18(4): 806-817, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705124

RESUMO

Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) is widely used for proteomics analysis given its high throughput and reproducibility, but ensuring consistent quantification of analytes across large-scale studies of heterogeneous samples such as human plasma remains challenging. Heterogeneity in large-scale studies can be caused by large time intervals between data acquisition, acquisition by different operators or instruments, and intermittent repair or replacement of parts, such as the liquid chromatography column, all of which affect retention time (RT) reproducibility and, successively, performance of SWATH-MS data analysis. Here, we present a novel algorithm for RT alignment of SWATH-MS data based on direct alignment of raw MS2 chromatograms using a hybrid dynamic programming approach. The algorithm does not impose a chronological order of elution and allows for alignment of elution-order-swapped peaks. Furthermore, allowing RT mapping in a certain window around a coarse global fit makes it robust against noise. On a manually validated dataset, this strategy outperformed the current state-of-the-art approaches. In addition, on real-world clinical data, our approach outperformed global alignment methods by mapping 98% of peaks compared with 67% cumulatively. DIAlignR reduced alignment error up to 30-fold for extremely distant runs. The robustness of technical parameters used in this pairwise alignment strategy is also demonstrated. The source code is released under the BSD license at https://github.com/Roestlab/DIAlignR.


Assuntos
Proteômica/métodos , Alinhamento de Sequência/métodos , Software , Algoritmos , Bases de Dados de Proteínas , Humanos , Peptídeos/metabolismo , Reprodutibilidade dos Testes , Streptococcus pyogenes/metabolismo , Fatores de Tempo
15.
Biochem J ; 476(3): 595-611, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30670571

RESUMO

Bacterial pathogens encounter a variety of adverse physiological conditions during infection, including metal starvation, metal overload and oxidative stress. Here, we demonstrate that group A Streptococcus (GAS) utilises Mn(II) import via MtsABC during conditions of hydrogen peroxide stress to optimally metallate the superoxide dismutase, SodA, with Mn. MtsABC expression is controlled by the DtxR family metalloregulator MtsR, which also regulates the expression of Fe uptake systems in GAS. Our results indicate that the SodA in GAS requires Mn for full activity and has lower activity when it contains Fe. As a consequence, under conditions of hydrogen peroxide stress where Fe is elevated, we observed that the PerR-regulated Fe(II) efflux system PmtA was required to reduce intracellular Fe, thus protecting SodA from becoming mismetallated. Our findings demonstrate the co-ordinate action of MtsR-regulated Mn(II) import by MtsABC and PerR-regulated Fe(II) efflux by PmtA to ensure appropriate Mn(II) metallation of SodA for optimal superoxide dismutase function.


Assuntos
Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/farmacologia , Manganês/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Streptococcus pyogenes/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Bactérias/genética , Ferro/metabolismo , Estresse Oxidativo/genética , Streptococcus pyogenes/genética , Superóxido Dismutase/genética
16.
J Clin Invest ; 129(2): 887-901, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30667377

RESUMO

Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon-directed insertion-site sequencing (TraDIS), we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins and exporters for toxins, quorum-sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding 5 transporters confirmed that each isogenic mutant strain was significantly (P < 0.05) impaired in causing necrotizing myositis in NHPs. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis showed that these 5 genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface exposed, suggesting that our findings have translational research implications.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Fasciite Necrosante , Regulação Bacteriana da Expressão Gênica , Miosite , Streptococcus pyogenes , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular , Modelos Animais de Doenças , Fasciite Necrosante/genética , Fasciite Necrosante/metabolismo , Fasciite Necrosante/patologia , Humanos , Camundongos , Miosite/genética , Miosite/metabolismo , Miosite/microbiologia , Miosite/patologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade
17.
PLoS One ; 14(1): e0206338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30653526

RESUMO

The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Cães , Modelos Moleculares , Fosfopiruvato Hidratase/química , Ligação Proteica , Multimerização Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Streptococcus pyogenes/química
18.
APMIS ; 127(2): 87-92, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30698306

RESUMO

Streptococcal toxic shock syndrome (STSS) is primarily caused by Streptococcus pyogenes, but it may also be caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE). The analyses of S. pyogenes have revealed the important roles of NAD+ -glycohydrolase (Nga) and CovR/CovS, a two-component regulatory system. We examined these factors in SDSE by analyzing mainly two isogenic SDSE strains (12-10-1 and 12-10-3) from the blood of a patient with STSS. The Nga activities were measured and the nucleotide sequences of covR and covS genes were determined. We detected one nucleotide difference between the covR gene of 12-10-1 and that of 12-10-3, and the Nga activity of 12-10-1 was approximately 6.8-fold more than that of 12-10-3. The introduction of covR of 12-10-3 into 12-10-1 significantly reduced the Nga activity, but the introduction of 12-10-1 covR into itself had only a little effect. In addition, the knockout of covR or covS of 12-10-3 remarkably increased the Nga activity. We are the first to report that strains with wild-type and mutated covR were isolated simultaneously from an SDSE STSS patient and that the CovR/CovS two-component regulatory system is involved in the Nga activity in SDSE as well as in S. pyogenes.


Assuntos
Proteínas de Bactérias/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , NAD+ Nucleosidase/metabolismo , Proteínas Repressoras/genética , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/metabolismo , Proteínas de Bactérias/metabolismo , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Repressoras/metabolismo , Choque Séptico/microbiologia , Choque Séptico/patologia , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação
19.
J Bacteriol ; 201(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30478086

RESUMO

CovR/CovS is a two-component regulatory system in group A Streptococcus and primarily acts as a transcriptional repressor. The D53 residue of CovR (CovRD53) is phosphorylated by the sensor kinase CovS, and the phosphorylated CovRD53 protein binds to the intergenic region of rgg-speB to inhibit speB transcription. Nonetheless, the transcription of rgg and speB is suppressed in covS mutants. The T65 residue of CovR is phosphorylated in a CovS-independent manner, and phosphorylation at the D53 and T65 residues of CovR is mutually exclusive. Therefore, how phosphorylation at the D53 and T65 residues of CovR contributes to the regulation of rgg and speB expression was elucidated. The transcription of rgg and speB was suppressed in the strain that cannot phosphorylate the D53 residue of CovR (CovRD53A mutant) but restored to levels similar to those of the wild-type strain in the CovRT65A mutant. Nonetheless, inactivation of the T65 residue phosphorylation in the CovRD53A mutant cannot derepress the rgg and speB transcription, indicating that phosphorylation at the T65 residue of CovR is not required for repressing rgg and speB transcription. Furthermore, trans complementation of the CovRD53A protein in the strain that expresses the phosphorylated CovRD53 resulted in the repression of rgg and speB transcription. Unlike the direct binding of the phosphorylated CovRD53 protein and its inhibition of speB transcription demonstrated previously, the present study showed that inactivation of phosphorylation at the D53 residue of CovR contributes dominantly in suppressing rgg and speB transcription.IMPORTANCE CovR/CovS is a two-component regulatory system in group A Streptococcus (GAS). The D53 residue of CovR is phosphorylated by CovS, and the phosphorylated CovRD53 binds to the rgg-speB intergenic region and acts as the transcriptional repressor. Nonetheless, the transcription of rgg and Rgg-controlled speB is upregulated in the covR mutant but inhibited in the covS mutant. The present study showed that nonphosphorylated CovRD53 protein inhibits rgg and speB transcription in the presence of the phosphorylated CovRD53 in vivo, indicating that nonphosphorylated CovRD53 has a dominant role in suppressing rgg transcription. These results reveal the roles of nonphosphorylated CovRD53 in regulating rgg transcription, which could contribute significantly to invasive phenotypes of covS mutants.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Exotoxinas/biossíntese , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/metabolismo , Transativadores/biossíntese , Transcrição Genética , Proteínas de Bactérias/genética , Exotoxinas/genética , Genótipo , Fosforilação , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Transativadores/genética
20.
Cell Microbiol ; 21(1): e12956, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30239106

RESUMO

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotising fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions along with the host-pathogen interactions that might influence biofilm formation. Here, we establish and characterise an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a mouse model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a 3D architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) that induce endoplasmic reticulum (ER) stress in the host. In an in vivo mouse model, the streptolysin null mutant is attenuated in both microcolony formation and bacterial spread, but pretreatment of soft-tissue with an ER stressor restores the ability of the mutant to form wild-type-like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.


Assuntos
Biofilmes/crescimento & desenvolvimento , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fasciite Necrosante/microbiologia , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/metabolismo , Estreptolisinas/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Modelos Teóricos
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