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1.
J Dairy Sci ; 103(1): 840-845, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733844

RESUMO

This study investigated the antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) isolated from cases of subclinical bovine mastitis in China, as well as resistance mechanisms and virulence genes encoding adhesins and toxins. We determined antimicrobial susceptibility using the disk diffusion method, and analyzed resistance, adhesin, and toxin genes using PCR. We confirmed MRSA in 73 of 498 (14.7%) Staph. aureus isolates recovered from subclinical mastitic milk samples. All isolates were positive for mecA. The MRSA isolates showed high resistance to penicillin (100.0%), gentamicin (100.0%), and tetracycline (98.6%). All MRSA isolates harbored resistance genes blaZ (penicillin), aacA/aphD (gentamicin), and tetM (alone or in combination with tetK, tetracycline). Moreover, all isolates carried the adhesin genes fnbpA, clfA, clfB, cna, sdrE, and map/eap, and most carried sdrC (98.6%), sdrD (95.9%), bbp (94.5%), and ebpS (80.8%). The toxin genes seh, hla, and hld were present in all isolates, and most isolates carried sea (71.2%), seg (84.9%), sei (82.2%), lukE-lukD (97.3%), and hlg (72.6%). These findings of high-level resistance to antimicrobials commonly used in dairy cattle should lead to calls for antibiogram analysis before antimicrobial therapy. The high frequency of adhesin and toxin genes in MRSA indicates their potential virulence in bovine mastitis in China.


Assuntos
Mastite Bovina/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/veterinária , Adesinas Bacterianas/genética , Animais , Antibacterianos/farmacologia , Bovinos , China , Feminino , Gentamicinas/farmacologia , Mastite Bovina/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase/veterinária , Infecções Estafilocócicas/microbiologia , Tetraciclina/farmacologia , Virulência/genética
2.
World J Microbiol Biotechnol ; 35(12): 185, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31728760

RESUMO

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0 mg/L) and GSH (17.3 mg/L) was achieved after 17 h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.


Assuntos
Glutationa/metabolismo , Lactococcus lactis/metabolismo , Engenharia Metabólica/métodos , S-Adenosilmetionina/metabolismo , Adesinas Bacterianas/genética , Vias Biossintéticas , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Metionina Adenosiltransferase/genética , Nisina/metabolismo , Probióticos
3.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591167

RESUMO

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.


Assuntos
Adesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade , Adesinas Bacterianas/genética , Cápsulas Bacterianas/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Polissacarídeos Bacterianos/metabolismo , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Virulência/genética
4.
Pak J Pharm Sci ; 32(4): 1485-1494, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31608866

RESUMO

This study sheds the light on the presence of (some) food-borne pathogens in raw market milk in Mansoura city, (Egypt) using several techniques for isolation and identification including serology and PCR. It determines, further, the susceptibility of the isolated pathogens to some antimicrobial agents and natural oils, including watercress, basil, parsley, and hot green pepper oils. From 100 milk samples, 22 Escherichia coli isolates harboured stx1, stx2 and/or eae genes. Additionally, 17 Listeria monocytogenes (L. monocytogenes) isolates harboured hylA gene. Moreover, other related pathogens such as Shigella flexneri and Klebsiella pneumoniae were also detected. Antimicrobial susceptibility testing showed that E. coli strains were (completely) resistant to amoxicillin and sulfamethoxazole-trimethoprim but highly sensitive to gentamicin. L. monocytogenes strains showed complete resistance against oxytetracycline while the highest percentage of sensitivity was observed against norfloxacin. This study has also proved the following: L. monocytogenes was susceptible to all of the investigated oils, Klebsiella pneumoniae was sensitive to two types of oils, but E. coli and Shigella flexneri were resistant to all oils. In conclusion, it is risky to consume unpasteurized milk. Further, some natural oils (e.g. parsley and hot green pepper oils) can successfully be used as food additives to control the presence of some pathogens in milk.


Assuntos
Antibacterianos/farmacologia , Microbiologia de Alimentos , Leite/microbiologia , Óleos Vegetais/farmacologia , Adesinas Bacterianas/genética , Animais , Farmacorresistência Bacteriana/efeitos dos fármacos , Egito , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Doenças Transmitidas por Alimentos/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Testes de Sensibilidade Microbiana , Toxina Shiga I/genética , Toxina Shiga II/genética , Shigella flexneri/efeitos dos fármacos
5.
BMC Infect Dis ; 19(1): 812, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533652

RESUMO

BACKGROUND: Invasive group B Streptococcus (GBS) disease in Chinese infants has gradually gained attention in recent years, but the molecular epidemiology of the pathogen is still not well known. METHODS: This multicenter study retrospectively investigated distribution of capsular serotypes, sequence types (STs), and hypervirulent GBS adhesin gene (hvgA) in clinical GBS isolates that caused invasive disease in infants aged < 3 months of age in southern mainland China between January 2013 and June 2016. Genes for antibiotic resistance to tetracycline, erythromycin, and clindamycin were also examined. RESULTS: From a total of 93 GBS isolates taken from 34 early-onset disease (EOD, 0-6 days after birth) and 59 late-onset disease (LOD, 7-89 days after birth) cases, four serotypes were identified: serotypes III (79.6%), Ib (12.9%), Ia (4.3%), and V (3.2%). Serotype III accounted for 73.5% of EOD and 83.1% of LOD and was responsible for 75.5% of cases involving meningitis. Fifteen STs were found, with the majority being ST17 (61.3%), ST12 (7.5%), ST19 (7.5%), and others (23.7%). 96.8% of STs belonged to only five clonal complexes (CCs): CC17 (64.5%), CC10 (12.9%), CC19 (9.7%), CC23 (6.5%), and CC1 (3.2%). The hvgA gene was detected in 66.7% of GBS isolates and 95% of CC17 isolates, all of which were serotype III except one serotype Ib/CC17 isolate. A large proportion of GBS isolates were found to be resistant to tetracycline (93.5%), clindamycin (65.5%), and erythromycin (60.2%). Genes of tetO (74.7%) and tetM (46.0%) were found in tetracycline resistant isolates, linB (24.6%) in clindamycin resistant isolates, and ermB (87.5%) and mefA (3.6%) in erythromycin resistant isolates. CONCLUSION: Our results reveal higher prevalence of serotype III, ST17, CC17, hvgA expressing, and antibiotic resistant GBS isolates than previously reported in southern mainland China. This study provides guidance for appropriate measures of prevention and control to be taken in the future.


Assuntos
Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/isolamento & purificação , Adesinas Bacterianas/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , China/epidemiologia , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Lactente , Recém-Nascido , Tipagem de Sequências Multilocus , Prevalência , Estudos Retrospectivos , Sorogrupo , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/patologia , Streptococcus agalactiae/genética
6.
World J Gastroenterol ; 25(33): 4870-4884, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543679

RESUMO

Helicobacter pylori (H. pylori) is one of the most important human pathogens, infecting approximately half of the global population. Despite its high prevalence, only a subset of H. pylori infected individuals develop serious gastroduodenal pathology. The pathogenesis of H. pylori infection and disease outcome is thus thought to be mediated by an intricate interplay between host, environmental and bacterial virulence factors. H. pylori has adapted to the harsh milieu of the human stomach through possession of various virulence genes that enable survival of the bacteria in the acidic environment, movement towards the gastric epithelium, and attachment to gastric epithelial cells. These virulence factors enable successful colonization of the gastric mucosa and sustain persistent H. pylori infection, causing chronic inflammation and tissue damage, which may eventually lead to the development of peptic ulcers and gastric cancer. Numerous studies have focused on the prevalence and role of putative H. pylori virulence genes in disease pathogenesis. While several virulence factors with various functions have been identified, disease associations appear to be less evident, especially among different study populations. This review presents key findings on the most important H. pylori virulence genes, including several bacterial adhesins and toxins, in children and adults, and focuses on their prevalence, clinical significance and potential relationships.


Assuntos
Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Fatores de Virulência/genética , Virulência/genética , Adesinas Bacterianas/genética , Mucosa Gástrica/patologia , Genes Bacterianos , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Prevalência
7.
Vet Microbiol ; 235: 301-309, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383317

RESUMO

We have previously demonstrated that prophage phiv142-3 enhances the colonization ability of avian pathogenic Escherichia coli (APEC) strain DE142. However, the mechanism of this action remains unknown. In this study, we demonstrate that deletion of phiv142-3 orf20 leads to a decrease in the survival ability in chicken serum, adhesion, and ability to invade DF-1 cells of mutant strain DE142Δorf20 compared with that of wild type (WT). Avian infection assays showed that bacterial loads in lungs and hearts of chickens challenged with the mutant are decreased to 7% and 8.3% compared with those challenged with the WT. The number of flagella and I fimbriae of the mutant are decreased and the mutant exhibits filamentation. However, protein ORF20 shows no adhesion ability to DF-1 cells in adherence inhibition experiments, indicating that it does not directly participate in adhesion. qRT-PCR revealed that the deletion of orf20 leads to reduction in the expression of nine genes related to the exportation of flagellar protein and two I-fimbriae-related genes (fimA and fimH), but does not affect genes related to the synthesis of flagella and other adhesins. Compared with the WT, the transcription level of the cell-division-associated genes minC and minD was increased 1.4-fold and 2.5-fold in mutant DE142Δorf20, respectively, indicating that orf20 affects the morphology of DE142 by regulating expression of minC and minD. Thus, our study revealed that orf20 in prophage phiv142-3 played a role in flagellar exportation, cell morphology, and I fimbriae synthesis.


Assuntos
Aderência Bacteriana , Escherichia coli/fisiologia , Escherichia coli/virologia , Fímbrias Bacterianas/metabolismo , Flagelos/metabolismo , Prófagos/genética , Adesinas Bacterianas/genética , Animais , Carga Bacteriana , Galinhas/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/genética , Proteínas de Membrana/genética , Mutação , Fatores de Virulência/genética
8.
Food Microbiol ; 84: 103273, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31421766

RESUMO

Shiga toxin-producing Escherichia coli (STEC) are important pathogens transmitted by food that may cause severe illness in human beings. Thus, systems for STEC detection in food should have increasingly higher sensitivity and specificity. Here we compared six commercial systems for non-O157 STEC detection in meat and vegetables and determined their sensitivity, specificity and repeatability. A total of 46 samples (meat n = 23; chard n = 23) were experimentally contaminated with strains O26:H11, O45:H-, O103:H2, O111:NM, O121:H19 and O145:NM isolated in Argentina. Strain detection was confirmed by isolation according to ISO 13136:2012. Detection of the stx and eae genes in meat samples was highly satisfactory with all commercial kits, but only five had 100% sensitivity and specificity in chard. Of four kits evaluated for serogroup detection, three had 100% sensitivity and specificity, and one had 93.7% sensitivity and 100% specificity. All kits were adequate to analyze meat but not vegetable samples, and were not therefore validated for the latter matrix. The challenge for microbiology laboratories is to identify the advantages and disadvantages of the available kits for STEC detection in food based on a clear knowledge of the particular needs of each laboratory.


Assuntos
Contaminação de Alimentos/análise , Microbiologia de Alimentos/métodos , Carne/microbiologia , Sorotipagem/normas , Escherichia coli Shiga Toxigênica/isolamento & purificação , Verduras/microbiologia , Adesinas Bacterianas/genética , Microbiologia de Alimentos/normas , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade , Sorogrupo , Sorotipagem/métodos , Toxina Shiga/genética
9.
J Vet Sci ; 20(4): e35, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31364320

RESUMO

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21-42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.


Assuntos
Vacinas contra Adenovirus/administração & dosagem , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Imunização/veterinária , Doenças dos Suínos/prevenção & controle , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Feminino , Proteínas Recombinantes/imunologia , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Vacinas Sintéticas/administração & dosagem , Yersinia pseudotuberculosis/genética
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 533-539, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292058

RESUMO

Objective To screen aptamers that specifically bind to adhesin HpaA from Helicobacter pylori (H. pylori) by systematic evolution of ligands by exponential enrichment (SELEX) and identify the binding properties of aptamers. Methods The prokaryotic expression recombinant plasmid pET28a/HpaA was constructed and the HpaA protein was expressed and purified with IPTG. With the recombinant HpaA protein as target, we screened aptamers with high affinity and specificity binding force by SELEX. The binding force between aptamers and H. pylori in vitro and the performance of aptamers in H. pylori detection from the biopsy of gastric mucosa were examined using the aptamers we had screened. Results We extracted genome from H. pylori ATCC26695 strains and amplified 699 bp HpaA gene using PCR. The recombinant plasmid pET28a/HpaA was constructed successfully. The recombinant HpaA was expressed and purified up to 98% as target for aptamer screening. The six highest affinity aptamers were obtained and named HA1 to HA6 through 10-round positive screening and five-round negative screening by SELEX. The full-length aptamer HA6 and the core sequence of HA6 showed highest affinity and specificity in H. pylori detection in vitro. In view of this, the FAM-labelled aptamer HA6 was used to detect H. pylori in gastric mucosa from 166 patients. The aptamer HA6 showed a higher detection rate (94.58%) than URT (87.95%) in the same batch of clinical samples. Conclusion The aptamers that specifically bind to HpaA may be applied for the detection of H. pylori in gastric mucosa as a novel method.


Assuntos
Adesinas Bacterianas/genética , Aptâmeros de Nucleotídeos/genética , Mucosa Gástrica/microbiologia , Helicobacter pylori/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-31263685

RESUMO

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Mycoplasma hyopneumoniae/enzimologia , Fosfopiruvato Hidratase/química , Fosfopiruvato Hidratase/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/isolamento & purificação , Animais , Cristalografia por Raios X , Células Epiteliais/microbiologia , Modelos Moleculares , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma hyopneumoniae/patogenicidade , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Plasminogênio/metabolismo , Pneumonia Suína Micoplasmática/microbiologia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Suínos , Fatores de Virulência
12.
Biomed Res Int ; 2019: 9297129, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360728

RESUMO

Rickettsia heilongjiangensis is an obligate intracellular bacterium that is responsible for far-eastern spotted fever. Surface-exposed proteins (SEPs) play important roles in its pathogenesis. Previous work identified a ribosomal protein RpsB as an SEP by biotin-avidin affinity, a seroreactive antigen, and a diagnostic candidate protein, indicating that it might play an important role in the pathogenesis of rickettsiae. However, in the absence of other evidence, its subcellular location of being surface-exposed was puzzling because ribosomal proteins are located in the cytoplasm. In the present study, the subcellular location of RpsB was analyzed with bioinformatics tools coupled with immunoelectron microscopy. The adhesion ability of RpsB was evaluated by protein microarray and cellular ELISA. Consequently, different bioinformatics tools gave different location predication results. Thus, RpsB was found in the cytoplasma and inner and outer membranes of R. heilongjiangensis by transmission electron microscopy. Protein microarray and cellular ELISA showed that RpsB binds to the host cell surface and its adhesion ability was even stronger than the known adhesin Adr1. In conclusion, RpsB was visually and directly shown for the time to be an SEP of rickettsiae and might be an important ligand and adhesin of rickettsiae. Its roles in pathogenesis warrant further study.


Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Rickettsia/ultraestrutura , Rickettsiose do Grupo da Febre Maculosa/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Humanos , Microscopia Eletrônica de Transmissão , Análise Serial de Proteínas , Proteínas Ribossômicas/genética , Rickettsia/genética , Rickettsia/patogenicidade , Rickettsiose do Grupo da Febre Maculosa/microbiologia
13.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31358567

RESUMO

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.


Assuntos
Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Plasmídeos/química , Sistemas de Secreção Tipo III/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Criança , Mapeamento Cromossômico , Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Filogenia , Plasmídeos/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo V/genética , Sistemas de Secreção Tipo V/metabolismo
14.
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
15.
Pol J Microbiol ; 68(2): 233-246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250594

RESUMO

The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67-72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67-72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67-72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67­72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67­72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67­72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.


Assuntos
Adesinas Bacterianas/genética , Antígenos de Bactérias/genética , Corynebacterium diphtheriae/genética , Toxoide Diftérico/genética , Difteria/prevenção & controle , Variação Genética , Proteínas de Membrana/genética , Adesinas Bacterianas/imunologia , Antígenos de Bactérias/imunologia , Biologia Computacional , Sequência Conservada , Corynebacterium diphtheriae/imunologia , Toxoide Diftérico/imunologia , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Proteínas de Membrana/imunologia , Reação em Cadeia da Polimerase , Ligação Proteica , Análise de Sequência de DNA
16.
Microbiol Res ; 223-225: 79-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178055

RESUMO

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors. After having been alerted by an anonymous reader the authors found out that in order to substantiate one of their conclusions (DeltaToxR-reduced killing activity is mediated via T6SS2) more experiments are needed. To avoid any potentially wrong conclusions being published, the authors decided to retract the article and to resubmit their manuscript once the additional experiments have been completed. The Editor-in-Chief agreed to the retraction. The authors wish to apologize for any inconvenience caused.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Vibrio parahaemolyticus/metabolismo , Adesinas Bacterianas/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Ácidos Graxos/metabolismo , Percepção de Quorum , Metabolismo Secundário , Análise de Sequência de RNA , Deleção de Sequência , Fatores de Transcrição/genética , Transcriptoma , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , Virulência/genética
17.
Microbiol Res ; 223-225: 88-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178056

RESUMO

CodY and (p)ppGpp synthetases are two important global regulators of bacteria. In some pathogens, such as Listeria monocytogenes, the GTP pool links these two regulatory systems, and introducing a codY mutant into the ΔrelA strain restored the pathogenicity of the attenuated ΔrelA mutant. In previous studies, we identified the (p)ppGpp synthetases (RelA and RelQ) and CodY of Streptococcus suis. To understand the interrelationships between these two regulators in S. suis, a ΔrelAΔrelQΔcodY mutant was constructed, and its growth, morphology, and pathogenicity were evaluated. Compared with ΔrelAΔrelQ, ΔcodY, its growth was very slow, but its chain length was partly restored to the wild-type length and its capsule became thick and rough. The adherence, invasion ability, and resistance to whole-blood killing in vitro of ΔrelAΔrelQΔcodY and its lethality and colonization ability in mice were clearly reduced, which differs from the effects of these mutations in L. monocytogenes. An analysis of gene expression showed that CodY interacted with the relA promoter in a GTP-independent manner to positively regulate the expression of relA. The introduction of a codY mutant into the ΔrelAΔrelQ strain further reduced the expression of virulence factors, which suggests a novel interaction between the (p)ppGpp synthetases and CodY. This study extends our understanding of the relationship between the (p)ppGpp-mediated stringent response and the regulation of CodY in S. suis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Ligases/metabolismo , Streptococcus suis/citologia , Streptococcus suis/metabolismo , Streptococcus suis/patogenicidade , Fatores de Transcrição/metabolismo , Transcriptoma , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Guanosina Trifosfato/metabolismo , Ligases/genética , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Camundongos , Mutação , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Fatores de Transcrição/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
19.
Methods Mol Biol ; 1997: 233-266, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119628

RESUMO

Modern DNA recombinant techniques and major advances in genetic engineering have resulted in the development of bacterial expression systems that guarantee an unlimited supply of valuable proteins that have potential clinical or industrial use, but which are often limited by their low natural availability. This chapter provides the reader with a general scheme to clone, express, and purify native histidine (His)-tagged proteins in the desired quantity and quality required for its intended use, and reviews the most important factors affecting the production of recombinant proteins in a soluble form. Alternative methods for purification of insoluble recombinant proteins under denaturing conditions are also discussed. An optimized protocol to successfully purify native Neisseria gonorrhoeae Adhesin Complex Protein (Ng-ACP; NGO1981) is used as a technical example for the processes, which could potentially be applied to any gonococcal recombinant protein of interest.


Assuntos
Adesinas Bacterianas/genética , Clonagem Molecular/métodos , Neisseria gonorrhoeae/genética , Adesinas Bacterianas/química , Adesinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Vetores Genéticos/genética , Neisseria gonorrhoeae/química , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Transformação Bacteriana
20.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 377-384, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045567

RESUMO

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.


Assuntos
Adesinas Bacterianas/química , Proteínas de Bactérias/química , Cálcio/química , Gelo/análise , Marinomonas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Organismos Aquáticos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cátions Bivalentes , Clonagem Molecular , Temperatura Baixa , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
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