RESUMO
Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.
Assuntos
Cápsulas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Polissacarídeos Bacterianos , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Glicolipídeos/química , Glicolipídeos/metabolismo , Hidrólise , Microscopia de Fluorescência , Modelos Moleculares , Polissacarídeos Bacterianos/metabolismo , Polissacarídeos Bacterianos/química , Especificidade por SubstratoRESUMO
The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
Assuntos
Cápsulas Bacterianas/virologia , Bacteriófagos/enzimologia , Klebsiella pneumoniae/virologia , Polissacarídeo-Liases/metabolismo , Proteínas Virais/metabolismo , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestrutura , Bacteriófagos/genética , Bacteriófagos/fisiologia , Microscopia Crioeletrônica , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/ultraestrutura , Polissacarídeo-Liases/genética , Proteínas Virais/genéticaRESUMO
An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM) together with mechanical characterization by force spectroscopic measurements, has been carried out. Three meningococcal strains were used: the encapsulated serogroup B strain B1940, and the isogenic mutants B1940 siaD(+C) (lacking capsule), and B1940 cps (lacking both capsule and lipooligosaccharide outer core). AFM experiments with the encapsulated strain B1940 provided unprecedented images of the meningococcal capsule, which seems to be characterized by protrusions ("bumps") with the lateral dimensions of about 30 nm. Measurement of the Young's modulus provided quantitative assessment of the property of the capsule to confer resistance to mechanical stress. Moreover, Raman spectroscopy gave a fingerprint by which it was possible to identify the specific molecular species of the three strains analyzed, and to highlight major differences between them.
Assuntos
Cápsulas Bacterianas/ultraestrutura , Membrana Externa Bacteriana/ultraestrutura , Neisseria meningitidis Sorogrupo B/ultraestrutura , Cápsulas Bacterianas/química , Cápsulas Bacterianas/fisiologia , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/fisiologia , Módulo de Elasticidade , Microscopia de Força Atômica , Neisseria meningitidis Sorogrupo B/química , Neisseria meningitidis Sorogrupo B/genética , Polissacarídeos Bacterianos/química , Análise Espectral Raman , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.
Assuntos
Cápsulas Bacterianas/imunologia , Clostridiales/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade/imunologia , Doenças Inflamatórias Intestinais/imunologia , Polissacarídeos/imunologia , Adulto , Animais , Antibacterianos/farmacologia , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Células Cultivadas , Criança , Clostridiales/classificação , Clostridiales/genética , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Humanos , Íleo/imunologia , Íleo/metabolismo , Íleo/microbiologia , Doenças Inflamatórias Intestinais/microbiologia , Camundongos Endogâmicos C57BL , Família Multigênica/genética , FilogeniaRESUMO
The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.
Assuntos
Cápsulas Bacterianas/metabolismo , Carbono/metabolismo , Polissacarídeos Bacterianos/metabolismo , Streptococcus pneumoniae/metabolismo , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Frutose/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Humanos , Infecções Pneumocócicas/microbiologia , Polissacarídeos Bacterianos/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/ultraestrutura , Sacarose/metabolismoRESUMO
The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
Assuntos
Técnicas Bacteriológicas/métodos , Parede Celular/química , Parede Celular/ultraestrutura , Bactérias Gram-Positivas/ultraestrutura , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Congelamento , Imageamento Tridimensional/métodos , Lipopolissacarídeos , Microscopia Eletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Mycobacterium/ultraestrutura , Peptidoglicano/biossíntese , Ácidos TeicoicosRESUMO
Streptococcus suis is an economically important pathogen of pigs as well as a zoonotic cause of human disease. Serotyping is used for further characterization of isolates; some serotypes seem to be more virulent and more widely spread than others. This study characterizes a collection of German field isolates of Streptococcus suis from pigs dating from 1996 to 2016 with respect to capsular genes (cps) specific for individual serotypes and pathotype by multiplex PCR and relates results to the clinical background of these isolates. The most prominent finding was the reduction in prevalence of serotype-2/serotype-1/2 among invasive isolates during this sampling period, which might be attributed to widely implemented autogenous vaccination programs in swine against serotype 2 in Germany. In diseased pigs (systemically ill; respiratory disease) isolates of serotype-1/serotype-14, serotype-2/serotype-1/2, serotype 3 to 5 and 7 to 9 were most frequent while in carrier isolates a greater variety of cps types was found. Serotype-1/serotype-14 seemed to be preferentially located in joints, serotype 4 and serotype 3 in the central nervous system, respectively. The virulence associated extracellular protein factor was almost exclusively associated with invasive serotype-1/serotype-14 and serotype-2/serotype-1/2 isolates. In contrast, lung isolates of serotype-2/serotype-1/2 mainly harbored the gene for muramidase-released protein. Serotype 4 and serotype 9 isolates from clinically diseased pigs most frequently carried the muramidase-released protein gene and the suilysin gene. When examined by transmission electron microscopy all but one of the isolates which were non-typable by molecular and serological methods showed various amounts of capsular material indicating potentially new serotypes among these isolates. Given the variety of cps types/serotypes detected in pigs, not only veterinarians but also medical doctors should consider other serotypes than just serotype 2 when investigating potential human cases of Streptococcus suis infection.
Assuntos
Infecções Estreptocócicas/veterinária , Streptococcus suis/classificação , Sus scrofa/microbiologia , Doenças dos Suínos/microbiologia , Animais , Antígenos de Bactérias/genética , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Portador Sadio/virologia , Genes Bacterianos , Alemanha/epidemiologia , Humanos , Microscopia Eletrônica de Transmissão , Tipagem Molecular , Sorotipagem , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/isolamento & purificação , Suínos , Doenças dos Suínos/epidemiologia , Zoonoses/microbiologiaRESUMO
Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated-a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.
Assuntos
Streptococcus pyogenes/patogenicidade , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Feminino , Genes Bacterianos , Histidina Quinase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Regulon , Proteínas Repressoras/genética , Sorogrupo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/ultraestrutura , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Streptococcus pneumoniae is the major bacterial cause of community-acquired pneumonia, and the leading agent of childhood pneumonia deaths worldwide. Nasal colonization is an essential step prior to infection. The cytokine IL-17 protects against such colonization and vaccines that enhance IL-17 responses to pneumococcal colonization are being developed. The role of IL-17 in host defence against pneumonia is not known. To address this issue, we have utilized a murine model of pneumococcal pneumonia in which the gene for the IL-17 cytokine family receptor, Il17ra, has been inactivated. Using this model, we show that IL-17 produced predominantly from γδ T cells protects mice against death from the invasive TIGR4 strain (serotype 4) which expresses a relatively thin capsule. However, in pneumonia produced by two heavily encapsulated strains with low invasive potential (serotypes 3 and 6B), IL-17 significantly enhanced mortality. Neutrophil uptake and killing of the serotype 3 strain was significantly impaired compared to the serotype 4 strain and depletion of neutrophils with antibody enhanced survival of mice infected with the highly encapsulated SRL1 strain. These data strongly suggest that IL-17 mediated neutrophil recruitment to the lungs clears infection from the invasive TIGR4 strain but that lung neutrophils exacerbate disease caused by the highly encapsulated pneumococcal strains. Thus, whilst augmenting IL-17 immune responses against pneumococci may decrease nasal colonization, this may worsen outcome during pneumonia caused by some strains.
Assuntos
Interleucina-17/imunologia , Pneumonia Pneumocócica/imunologia , Receptores de Interleucina-17/genética , Streptococcus pneumoniae/imunologia , Animais , Bacteriemia/imunologia , Bacteriemia/microbiologia , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/ultraestrutura , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Modelos Animais de Doenças , Pulmão/citologia , Pulmão/enzimologia , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nasofaringe/microbiologia , Neutrófilos/citologia , Neutrófilos/imunologia , Peroxidase/metabolismo , Fagocitose , Pneumonia Pneumocócica/mortalidade , Pneumonia Pneumocócica/prevenção & controle , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Organismos Livres de Patógenos Específicos , Streptococcus pneumoniae/ultraestruturaRESUMO
AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.
Assuntos
Cápsulas Bacterianas/química , Cryptococcus neoformans/metabolismo , Fósforo/análise , Cápsulas Bacterianas/metabolismo , Cápsulas Bacterianas/ultraestrutura , Cryptococcus neoformans/genética , Cryptococcus neoformans/ultraestrutura , Microscopia Eletrônica de Varredura , Fósforo/metabolismoRESUMO
Direct interaction between pathogens and host cells often is a prerequisite for colonization, infection and dissemination. Regulated production of capsular polysaccharide (CPS), which is made of hyaluronic acid, is essential for the pathogenicity of Streptococcus equi subsp. Zooepidemicus (SEZ). Here, we constructed a CPS-deleted mutant and analyzed it along with the parental wild-type strain in attachment and invasion of mammalian epithelial and endothelial cell lines. The CPS-deleted mutant exhibited significant increase in adherence and invasion by several orders of magnitude compared with the wild-type strain through quantitative analysis and electron microscopy observation. After the wild-type strain was recovered from invaded cells, its morphology was analyzed by visual methods and scanning electron microscopy, which revealed that its capsule was almost completely absent. Capsule measurements showed a similar result in which CPS production was nearly attenuated to the same extent as in the CPS-deleted mutant. qPCR assays revealed a marked reduction in the transcriptional levels of the CPS biosynthesis genes, has operon. Moreover, the repression in capsular production was stable inheritance. Our findings indicate that SEZ is a facultative intracellular bacterium, capsule attenuation in SEZ contributes to attachment and invasion in interactions with host cells, and the active regulation of capsule breakdown is controlled by SEZ during internalization.
Assuntos
Aderência Bacteriana , Cápsulas Bacterianas/fisiologia , Células Endoteliais/microbiologia , Células Epiteliais/microbiologia , Streptococcus equi/genética , Streptococcus equi/fisiologia , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Células CACO-2 , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Ácido Hialurônico , Microscopia Eletrônica de Varredura , Óperon , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Streptococcus equi/citologia , Streptococcus equi/patogenicidadeRESUMO
Oenococcus oeni (O. oeni), which is the main species that drives malolactic fermentation (FML), an essential step for wine microbial stabilization and quality improvement, is known to produce exopolysaccharides (EPS). Depending on the strain, these EPS can be soluble, remain attached to the cell or both. In the present study, fourteen strains were examined for eps gene content and EPS production capacities. Cell-linked and soluble heteropolysaccharides made of glucose, galactose and rhamnose, soluble ß-glucan, and soluble dextran or levan were found, depending on the strain. The protective potential of either cell-linked heteropolysaccharides or dextrans produced was then studied during freeze drying of the bacterial strains.
Assuntos
Oenococcus/química , Oenococcus/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Cápsulas Bacterianas/química , Cápsulas Bacterianas/ultraestrutura , Fermentação/fisiologia , Liofilização , Genômica , Microscopia Eletrônica de Transmissão , Fenótipo , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/isolamento & purificação , Vinho/microbiologiaRESUMO
BACKGROUND: Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. It has been reported that S. suis infection in humans is mostly caused by serotype 2. However, human cases caused by other serotypes have rarely been reported. This is the first report of a human case of infection with S. suis serotype 31 in Thailand. CASE PRESENTATION: A 55-year-old male alcohol misuser with liver cirrhosis was admitted with sepsis to a hospital in the Central Region of Thailand. He had consumed a homemade, raw pork product prior to the onset of illness. He was alive after treatment with ceftriaxone and no complication occurred. An isolate from blood culture at the hospital was suspected as viridans group Streptococcus. It was confirmed at a reference laboratory as S. suis serotype 31 by biochemical tests, 16S rDNA sequencing, and multiplex polymerase chain reaction for serotyping, but it was untypable by the co-agglutination test with antisera against recognized S. suis serotypes, suggesting loss of capsular material. The absence of a capsule was confirmed by transmission electron microscopy. The isolate was confirmed to be sequence type 221, with 13 putative virulence genes that are usually found in serotype 2 strains. CONCLUSION: We should be aware of the emergence of S. suis infections caused by uncommon serotypes in patients with predisposing conditions. Laboratory capacity to identify S. suis in the hospital is needed in developing countries, which can contribute to enhanced surveillance, epidemiological control, and prevention strategies in the prevalent area.
Assuntos
DNA Ribossômico/genética , Alimentos Crus/microbiologia , Carne Vermelha/microbiologia , Sepse/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Testes de Aglutinação , Animais , Cápsulas Bacterianas/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Sorogrupo , Sorotipagem , Streptococcus suis/isolamento & purificação , Streptococcus suis/ultraestrutura , Suínos/microbiologia , Tailândia , Virulência/genéticaRESUMO
OBJECTIVES: In this study we focused on the mechanism of colistin resistance in Klebsiella pneumoniae. METHODS: We used two strains of K. pneumoniae: a colistin-susceptible strain (K. pneumoniae ATCC 700603, KpATCC) and its colistin-resistant derivative (KpATCCm, MIC of colistin 16 mg/L). We performed a genotypic analysis based on the expression of genes involved in LPS synthesis and L-Ara4N moiety addition. We also explored the status of the mgrB gene. Then, a phenotypic analysis was performed using atomic force microscopy (AFM). The Young modulus was extracted from force curves fitted using the Hertz model, and stiffness values were extracted from force curves fitted using the Hooke model. RESULTS: We failed to observe any variation in the expression of genes implicated in LPS synthesis or L-Ara4N moiety addition in KpATCCm, in the absence of colistin or under colistin pressure (versus KpATCC). This led us to identify an insertional inactivation/mutation in the mgrB gene of KpATCCm. In addition, morphology results obtained by AFM showed that colistin removed the capsule from the susceptible strain, but not from the resistant strain. Nanomechanical data on the resistant strain showed that colistin increased the Young modulus of the capsule. Extend force curves recorded on top of the cells allowed us to make the following hypothesis about the nanoarchitecture of the capsule of the two strains: KpATCC has a soft capsule consisting of one layer, whereas the KpATCCm capsule is harder and organized in several layers. CONCLUSIONS: We hypothesize that capsular polysaccharides might be implicated in the mechanism of colistin resistance in K. pneumoniae, depending on its genotype.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Klebsiella pneumoniae/efeitos dos fármacos , Microscopia de Força Atômica , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/ultraestrutura , Testes de Sensibilidade MicrobianaRESUMO
Streptococcus suis is an important pathogen of pigs and may cause serious disease in humans. Serotyping is one of the important diagnostic tools and is used for the epidemiological study of S. suis. Nontypeable S. suis strains have been reported in many studies; however, the capsular polysaccharide (CPS) synthesis cps loci of nontypeable strains have not been analyzed. In this study, we investigated the genetic characteristics of cps loci in 78 nontypeable strains isolated from healthy pigs. Eight novel cps loci (NCLs) were found, and all of them were located between the orfZ-orfX region and the glf gene. All NCLs possess the wzy and wzx genes, strongly suggesting that the CPSs of these NCLs were synthesized using the Wzx/Wzy-dependent pathway. The cps genes found in the 78 isolates were assigned to 96 homology groups (HGs), 55 of which were NCL specific. The encapsulation of the 78 isolates was also examined using transmission electron microscopy. Fifty-three isolates were found to have a capsule, and these were of varied thicknesses. Our data enhance our understanding of the cps gene cluster diversity of nontypeable S. suis strains and provide insight into the evolution of the S. suis capsular genes.
Assuntos
Cápsulas Bacterianas/química , Genes Bacterianos , Loci Gênicos , Polissacarídeos Bacterianos/biossíntese , Polissacarídeos Bacterianos/genética , Streptococcus suis/genética , Animais , Cápsulas Bacterianas/ultraestrutura , Técnicas de Tipagem Bacteriana , Biologia Computacional , Dados de Sequência Molecular , Família Multigênica , Filogenia , Análise de Sequência de DNA , Streptococcus suis/classificação , Streptococcus suis/metabolismo , Streptococcus suis/ultraestrutura , SuínosRESUMO
The production of capsular polysaccharides (CPS) or secreted exopolysaccharides is ubiquitous in bacteria, and the Wzy pathway constitutes a prototypical mechanism to produce these structures. Despite the differences in polysaccharide composition among species, a group of proteins involved in this pathway is well conserved. Streptococcus agalactiae (group B Streptococcus; GBS) produces a CPS that represents the main virulence factor of the bacterium and is a prime target in current vaccine development. We used this human pathogen to investigate the roles and potential interdependencies of the conserved proteins CpsABCD encoded in the cps operon, by developing knock-out and functional mutant strains. The mutant strains were examined for CPS quantity, size, and attachment to the cell surface as well as CpsD phosphorylation. We observed that CpsB, -C, and -D compose a phosphoregulatory system where the CpsD autokinase phosphorylates its C-terminal tyrosines in a CpsC-dependent manner. These Tyr residues are also the target of the cognate CpsB phosphatase. An interaction between CpsD and CpsC was observed, and the phosphorylation state of CpsD influenced the subsequent action of CpsC. The CpsC extracellular domain appeared necessary for the production of high molecular weight polysaccharides by influencing CpsA-mediated attachment of the CPS to the bacterial cell surface. In conclusion, although having no impact on cps transcription or the synthesis of the basal repeating unit, we suggest that these proteins are fine-tuning the last steps of CPS biosynthesis (i.e. the balance between polymerization and attachment to the cell wall).
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Óperon , Polímeros/metabolismo , Streptococcus agalactiae/metabolismo , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Camundongos , Microscopia Imunoeletrônica , Mutação , Fosforilação , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Tirosina/genética , Tirosina/metabolismo , Fatores de Virulência/genéticaRESUMO
Vibrio mimicus is a bacterium that causes gastroenteritis; it is closely related to Vibrio cholerae, and can cause acute diarrhea like cholera- or dysentery-type diarrhea. It is distributed worldwide. Factors associated with virulence (such as hemolysins, enterotoxins, proteases, phospholipases, aerobactin, and hemagglutinin) have been identified; however, its pathogenicity mechanism is still unknown. In pathogenic Vibrio species such as V. cholerae, Vibrio. parahaemolyticus and Vibrio vulnificus, capsule, biofilms, lateral flagellum, and type IV pili are structures described as essential for pathogenicity. These structures had not been described in V. mimicus until this work. We used 20 V. mimicus strains isolated from water (6), oyster (9), and fish (5) samples and we were able to identify the capsule, biofilm, lateral flagellum, and type IV pili through phenotypic tests, electron microscopy, PCR, and sequencing. In all tested strains, we observed and identified the presence of capsular exopolysaccharide, biofilm formation in an in vitro model, as well as swarming, multiple flagellation, and pili. In addition, we identified homologous genes to those described in other bacteria of the genus in which these structures have been found. Identification of these structures in V. mimicus is a contribution to the biology of this organism and can help to reveal its pathogenic behavior.
Assuntos
Cápsulas Bacterianas/ultraestrutura , Biofilmes/crescimento & desenvolvimento , Fímbrias Bacterianas/ultraestrutura , Flagelos/fisiologia , Vibrio mimicus/fisiologia , Vibrio mimicus/ultraestrutura , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , Peixes/microbiologia , Locomoção , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ostreidae/microbiologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Vibrio mimicus/isolamento & purificação , Vibrio mimicus/patogenicidade , Microbiologia da ÁguaRESUMO
Streptococcus suis serotype 2 is known to cause severe infections in pigs, including meningitis, endocarditis and pneumonia. Furthermore, this bacterium is considered an emerging zoonotic agent. Recently, increased antibiotic resistance in S. suis has been reported worldwide. The objective of this study was to evaluate the potential of nisin, a bacteriocin of the lantibiotic class, as an antibacterial agent against the pathogen S. suis serotype 2. In addition, the synergistic activity of nisin in combination with conventional antibiotics was assessed. Using a plate assay, the nisin-producing strain Lactococcus lactis ATCC 11454 proved to be capable of inhibiting the growth of S. suis (n=18) belonging to either sequence type (ST)1, ST25, or ST28. In a microdilution broth assay, the minimum inhibitory concentration (MIC) of purified nisin ranged between 1.25 and 5 µg/mL while the minimum bactericidal concentration (MBC) was between 5 and 10 µg/mL toward S. suis. The use of a capsule-deficient mutant of S. suis indicated that the presence of this polysaccharidic structure has no marked impact on susceptibility to nisin. Following treatment of S. suis with nisin, transmission electron microscopy observations revealed lysis of bacteria resulting from breakdown of the cell membrane. A time-killing curve showed a rapid bactericidal activity of nisin. Lastly, synergistic effects of nisin were observed in combination with several antibiotics, including penicillin, amoxicillin, tetracycline, streptomycin and ceftiofur. This study brought clear evidence supporting the potential of nisin for the prevention and treatment of S. suis infections in pigs.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Nisina/farmacologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/efeitos dos fármacos , Amoxicilina/farmacologia , Animais , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/ultraestrutura , Técnicas de Tipagem Bacteriana , Membrana Celular/ultraestrutura , Cefalosporinas/farmacologia , Combinação de Medicamentos , Resistência Microbiana a Medicamentos , Lactococcus lactis/fisiologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Penicilinas/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus suis/crescimento & desenvolvimento , Streptococcus suis/isolamento & purificação , Streptococcus suis/ultraestrutura , Estreptomicina/farmacologia , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologia , Tetraciclina/farmacologiaRESUMO
AIM: Selection of high-mucoid morphotype of Streptococcus equi subsp. zooepidemicus (Streptococcus zooepidemicus) and study of its morphological, physiological, biochemical and technological characteristics for providing increased secretion of hyaluronic acid (HA). MATERIALS AND METHODS: Submerged cultivation was performed in 100 ml glass flasks without baffles or in 1.5 or 10 1 laboratory bioreactors. LB and MRS media were used for cultivation. Mutagenesis was carried out by UV exposure with consequent selection of mucoid phenotype. HA was determined by carbazole method or after exhaustive acid hydrolysis by reaction of N-acetylglucosamine with Morgan-Elson reagent. Total hyaluronidase activity was evaluated by viscosimeter. Determination of cell and capsule size, ability to ferment carbohydrates and other microbiological, physiological and biochemical tests were performed by standard techniques. RESULTS: Instability of capsule phenotype of S. zooepidemicus B-8014 strain was revealed that is explained most probably by formation under certain conditions of bacterial hyaluronidase. This is confirmed by a reduction of HA concentration in cultural medium at pre- and stationary growth phases. Mucoid strain S. zooepidemicus KB-04 was obtained by mutagenesis with subsequent selection that is characterized by increased capsules. The strain was studied for HA formation. Optimization of growth medium composition, physical-chemical conditions and modes of cultivation allowed to significantly increase HA yield. CONCLUSION: The studies of morphologic, physiologic, biochemical and technological characteristics of the high-mucoid S. zooepidemicus KB-04 strain obtained by mutagenesis with consequent selection were performed, conditions of its cultivation and composition of growth mediu by carbon source and content of bivalent metal ions were optimized.
Assuntos
Cápsulas Bacterianas/efeitos da radiação , Ácido Hialurônico/biossíntese , Streptococcus equi/metabolismo , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/metabolismo , Reatores Biológicos , Cálcio/metabolismo , Meios de Cultura , Fermentação , Glucose/metabolismo , Ácido Hialurônico/isolamento & purificação , Hialuronoglucosaminidase/metabolismo , Magnésio/metabolismo , Mutagênese , Seleção Genética , Streptococcus equi/genética , Streptococcus equi/crescimento & desenvolvimento , Streptococcus equi/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. RESULTS: Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 µm in diameter and 5-50 µm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. CONCLUSION: The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal nanowires for electrical devices. The novelty of this work is the use of filamentous E. coli templates and the use of S-layer proteins in a new material construct.