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1.
mSphere ; 6(1)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627509

RESUMO

Lipoteichoic acid (LTA) is a Gram-positive bacterial cell surface polymer that participates in host-microbe interactions. It was previously reported that the major human pathogen Streptococcus pneumoniae and the closely related oral commensals S. mitis and S. oralis produce type IV LTAs. Herein, using liquid chromatography/mass spectrometry-based lipidomic analysis, we found that in addition to type IV LTA biosynthetic precursors, S. mitis, S. oralis, and S. pneumoniae also produce glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a biosynthetic precursor of type I LTA. cdsA and pgsA mutants produce DHDAG but lack (Gro-P)-DHDAG, indicating that the Gro-P moiety is derived from phosphatidylglycerol (PG), whose biosynthesis requires these genes. S. mitis, but not S. pneumoniae or S. oralis, encodes an ortholog of the PG-dependent type I LTA synthase, ltaS By heterologous expression analyses, we confirmed that S. mitisltaS confers poly(Gro-P) synthesis in both Escherichia coli and Staphylococcus aureus and that S. mitisltaS can rescue the growth defect of an S. aureusltaS mutant. However, we do not detect a poly(Gro-P) polymer in S. mitis using an anti-type I LTA antibody. Moreover, Gro-P-linked DHDAG is still synthesized by an S. mitisltaS mutant, demonstrating that S. mitis LtaS does not catalyze Gro-P transfer to DHDAG. Finally, an S. mitisltaS mutant has increased sensitivity to human serum, demonstrating that ltaS confers a beneficial but currently undefined function in S. mitis Overall, our results demonstrate that S. mitis, S. pneumoniae, and S. oralis produce a Gro-P-linked glycolipid via a PG-dependent, ltaS-independent mechanism.IMPORTANCE The cell wall is a critical structural component of bacterial cells that confers important physiological functions. For pathogens, it is a site of host-pathogen interactions. In this work, we analyze the glycolipids synthesized by the mitis group streptococcal species, S. pneumoniae, S. oralis, and S. mitis We find that all produce the glycolipid, glycerophosphate (Gro-P)-linked dihexosyl (DH)-diacylglycerol (DAG), which is a precursor for the cell wall polymer type I lipoteichoic acid in other bacteria. We investigate whether the known enzyme for type I LTA synthesis, LtaS, plays a role in synthesizing this molecule in S. mitis Our results indicate that a novel mechanism is responsible. Our results are significant because they identify a novel feature of S. pneumoniae, S. oralis, and S. mitis glycolipid biology.


Assuntos
Glicolipídeos/biossíntese , Glicolipídeos/genética , Streptococcus mitis/química , Streptococcus oralis/química , Streptococcus pneumoniae/química , Glicerofosfatos/biossíntese , Glicerofosfatos/genética , Glicolipídeos/química , Glicolipídeos/metabolismo , Lipopolissacarídeos , Fosfatidilgliceróis/biossíntese , Fosfatidilgliceróis/genética , Streptococcus mitis/genética , Streptococcus mitis/metabolismo , Streptococcus oralis/genética , Streptococcus oralis/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Ácidos Teicoicos
2.
Infect Genet Evol ; 85: 104483, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32731044

RESUMO

Streptococcus mitis strain Nm-65 secretes an atypical 5-domain-type cholesterol-dependent cytolysin (CDC) called S. mitis-derived human platelet aggregation factor (Sm-hPAF) originally described as a platelet aggregation factor. Sm-hPAF belongs to Group III CDC that recognize both membrane cholesterol and human CD59 as the receptors, and shows preferential activity towards human cells. Draft genome analyses have shown that the Nm-65 strain also harbors a gene encoding another CDC called mitilysin (MLY). This CDC belongs to Group I CDC that recognizes only membrane cholesterol as a receptor, and it is a homolog of the pneumococcal CDC, pneumolysin. The genes encoding each CDC are located about 20 kb apart on the Nm-65 genome. Analysis of the genomic locus of these CDC-encoding genes in silico showed that the gene encoding Sm-hPAF and the region including the gene encoding MLY were both inserted into a specific locus of the S. mitis genome. The results obtained using deletion mutants of the gene(s) encoding CDC in Nm-65 indicated that each CDC contributes to both hemolysis and cytotoxicity, and that MLY is the major hemolysin/cytolysin in Nm-65. The present study aimed to determine the potential pathogenicity of an S. mitis strain that produces two CDC with different receptor recognition properties and secretion modes.


Assuntos
Toxinas Bacterianas/genética , Citotoxinas/genética , Citotoxinas/toxicidade , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Streptococcus mitis/genética , Sequência de Aminoácidos , Antígenos CD59/isolamento & purificação , Colesterol , Variação Genética , Genótipo , Humanos , Mutação , Inibidores da Agregação Plaquetária/isolamento & purificação , Streptococcus mitis/química
3.
ACS Nano ; 14(1): 1070-1083, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31854972

RESUMO

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule which facilitates immune evasion and determines virulence. Recent data have shown that the closely related Streptococcus mitis also expresses polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We utilized atomic force microscopy (AFM) techniques to investigate the biophysical properties of S. mitis and S. pneumoniae strains expressing the same capsular serotypes that might relate to differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes, S. mitis strains were susceptible to neutrophil killing, and electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the center and edge areas of encapsulated streptococcal strains. This "edge effect" was not observed in unencapsulated bacteria and therefore was a direct representation of the mechanical properties of the bacterial capsule. When two strains of S. mitis and S. pneumoniae expressed an identical capsular serotype, they presented similar biomechanical characteristics. This infers a potential relationship between capsule biochemistry and nanomechanics, independent of bacterial strain. Overall, this study demonstrates that it is possible to investigate reproducibly the mechanistic, structural, and mechanical properties of both the capsule and the body of individual living bacterial cells and relate the data to virulence phenotypes. We have demonstrated that using nanomechanics to investigate individual bacterial cells we can now begin to identify the surface properties bacterial pathogens require to avoid host-mediated immunity.


Assuntos
Cápsulas Bacterianas/química , Polissacarídeos/química , Streptococcus mitis/química , Streptococcus pneumoniae/química , Tamanho da Partícula , Fenótipo , Streptococcus mitis/patogenicidade , Streptococcus pneumoniae/patogenicidade , Propriedades de Superfície , Virulência
4.
J Clin Microbiol ; 55(3): 914-922, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28053215

RESUMO

Reliable distinction of Streptococcus pneumoniae and viridans group streptococci is important because of the different pathogenic properties of these organisms. Differentiation between S. pneumoniae and closely related Sreptococcusmitis species group streptococci has always been challenging, even when using such modern methods as 16S rRNA gene sequencing or matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. In this study, a novel algorithm combined with an enhanced database was evaluated for differentiation between S. pneumoniae and S. mitis species group streptococci. One hundred one clinical S. mitis species group streptococcal strains and 188 clinical S. pneumoniae strains were identified by both the standard MALDI Biotyper database alone and that combined with a novel algorithm. The database update from 4,613 strains to 5,627 strains drastically improved the differentiation of S. pneumoniae and S. mitis species group streptococci: when the new database version containing 5,627 strains was used, only one of the 101 S. mitis species group isolates was misidentified as S. pneumoniae, whereas 66 of them were misidentified as S. pneumoniae when the earlier 4,613-strain MALDI Biotyper database version was used. The updated MALDI Biotyper database combined with the novel algorithm showed even better performance, producing no misidentifications of the S. mitis species group strains as S. pneumoniae All S. pneumoniae strains were correctly identified as S. pneumoniae with both the standard MALDI Biotyper database and the standard MALDI Biotyper database combined with the novel algorithm. This new algorithm thus enables reliable differentiation between pneumococci and other S. mitis species group streptococci with the MALDI Biotyper.


Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Streptococcus mitis/classificação , Streptococcus pneumoniae/classificação , Algoritmos , Bases de Dados de Compostos Químicos , Humanos , Infecções Estreptocócicas/microbiologia , Streptococcus mitis/química , Streptococcus mitis/isolamento & purificação , Streptococcus pneumoniae/química , Streptococcus pneumoniae/isolamento & purificação
5.
Microbiol Immunol ; 58(3): 155-71, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24401114

RESUMO

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59.


Assuntos
Toxinas Bacterianas/metabolismo , Colesterol/metabolismo , Citotoxinas/metabolismo , Receptores de Superfície Celular/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus intermedius/metabolismo , Streptococcus mitis/metabolismo , Toxinas Bacterianas/química , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/microbiologia , Colesterol/química , Citotoxinas/química , Humanos , Cinética , Ligação Proteica , Receptores de Superfície Celular/química , Infecções Estreptocócicas/microbiologia , Streptococcus intermedius/química , Streptococcus mitis/química
6.
J Clin Microbiol ; 51(9): 3079-82, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23784130

RESUMO

The Vitek MS v2.0 matrix-assisted laser desorption ionization-time of flight mass spectrometry system accurately distinguished Streptococcus pneumoniae from nonpneumococcal S. mitis group species. Only 1 of 116 nonpneumococcal isolates (<1%) was misidentified as S. pneumoniae. None of 95 pneumococcal isolates was misidentified. This method provides a rapid, simple means of discriminating among these challenging organisms.


Assuntos
Erros de Diagnóstico/estatística & dados numéricos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Infecções Estreptocócicas/microbiologia , Streptococcus mitis/classificação , Streptococcus mitis/isolamento & purificação , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/isolamento & purificação , Humanos , Infecções Estreptocócicas/diagnóstico , Streptococcus mitis/química , Streptococcus pneumoniae/química
7.
Clin Microbiol Infect ; 19(11): 1066-71, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23331578

RESUMO

Accurate species-level identification of alpha-hemolytic (viridans) streptococci (VGS) is very important for understanding their pathogenicity and virulence. However, an extremely high level of similarity between VGS within the mitis group (S. pneumoniae, S. mitis, S. oralis and S. pseudopneumoniae) often results in misidentification of these organisms. Earlier, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a tool for the rapid identification of S. pneumoniae. However, by using Biotyper 3.0 (Bruker) or Vitek MS (bioMérieux) databases, Streptococcus mitis/oralis species can be erroneously identified as S. pneumoniae. ClinProTools 2.1 software was used for the discrimination of MALDI-TOF mass spectra of 25 S. pneumoniae isolates, 34 S. mitis and three S. oralis. Phenotypical tests and multilocus gene typing schemes for the S. pneumoniae (http://spneumoniae.mlst.net/) and viridans streptococci (http://viridans.emlsa.net/) were used for the identification of isolates included in the study. The classifying model was generated based on different algorithms (Genetic Algorithm, Supervised Neural Network and QuickClassifier). In all cases, values of sensitivity and specificity were found to be equal or close to 100%, allowing discrimination of mass spectra of different species. Three peaks (6949, 9876 and 9975 m/z) were determined conferring the maximal statistical weight onto each model built. We find this approach to be promising for viridans streptococci discrimination.


Assuntos
Técnicas Bacteriológicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Streptococcus mitis/química , Streptococcus mitis/classificação , Streptococcus pneumoniae/química , Streptococcus pneumoniae/classificação , Algoritmos , Sensibilidade e Especificidade , Software
8.
Appl Environ Microbiol ; 74(19): 5891-7, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18708515

RESUMO

Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.


Assuntos
Conjuntivite/microbiologia , Surtos de Doenças , Infecções Pneumocócicas/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Streptococcus pneumoniae/química , Streptococcus pneumoniae/classificação , Proteínas de Bactérias/análise , Técnicas de Tipagem Bacteriana/métodos , Análise por Conglomerados , Conjuntivite/epidemiologia , Enterococcus faecalis/química , Escherichia coli/química , Humanos , Epidemiologia Molecular/métodos , Peso Molecular , Infecções Pneumocócicas/epidemiologia , Proteoma/análise , Staphylococcus aureus/química , Streptococcus mitis/química , Streptococcus oralis/química , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pyogenes/química
9.
J Biol Chem ; 283(27): 18636-45, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18434320

RESUMO

We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316-12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.


Assuntos
Anti-Infecciosos/farmacologia , Proteínas de Bactérias/farmacologia , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Peptídeos/farmacologia , Streptococcus mitis/química , Motivos de Aminoácidos/genética , Substituição de Aminoácidos , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/toxicidade , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Eritrócitos/citologia , Cavalos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Mutação de Sentido Incorreto , Peptídeos/síntese química , Peptídeos/genética , Peptídeos/toxicidade , Streptococcus mitis/genética , Relação Estrutura-Atividade
10.
Colloids Surf B Biointerfaces ; 44(1): 41-8, 2005 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-16023949

RESUMO

Bacterial adhesion to silica sand was related to variations in system Gibbs energy DeltaG(adh). Two typical Gram-positive bacterial strains of Streptococcus mitis and Lactobacillus casei were used as the model bacteria in this research. Impacts of solution chemistry and goethite coating of silica sand on bacterial adhesion were also explored. S. mitis and L. casei had negative DeltaG(adh) with both uncoated and goethite-coated silica sand, demonstrating their adhesion potentials to these substrate. After goethite coating, DeltaG(adh) decreased (negatively increased) for both S. mitis and L. casei. In the presence of rhamnolipid biosurfactant, DeltaG(adh) increased (negatively decreased) in answer to the increase of the rhamnolipid biosurfactant concentration. Bacterial percentage adhesion to silica sand corresponded to DeltaG(adh). This study demonstrated that bacterial adhesion to substrate could be explained in terms of bacterial, substratum and intervening medium physicochemical surface properties, which can be independently determined based on contact angle measurements.


Assuntos
Aderência Bacteriana/fisiologia , Lacticaseibacillus casei/química , Dióxido de Silício/química , Streptococcus mitis/química , Aderência Bacteriana/efeitos dos fármacos , Glicolipídeos/farmacologia , Compostos de Ferro/química , Lacticaseibacillus casei/metabolismo , Minerais , Dióxido de Silício/metabolismo , Soluções/química , Streptococcus mitis/metabolismo , Propriedades de Superfície , Tensão Superficial , Tensoativos/farmacologia , Termodinâmica
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