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1.
Mikrobiyol Bul ; 54(1): 144-153, 2020 Jan.
Artigo em Turco | MEDLINE | ID: mdl-32050885

RESUMO

Avian chlamydiosis, is a highly contagious, systemic disease occuring in domestic and wild birds. Chlamydia psittaci, the causative agent of the disease, is a gram-negative bacterium in the Chlamydiaceae family that can only live within the cell. The agent can be transmitted directly to humans by contact with infected animals or feces of infected animals. It can also be transmitted by inhalation of fecal dust. Since the disease has a zoonotic character, it is also important in terms of public health. By using the monoclonal antibodies against cell wall proteins (OMP) of C.psittaci, six (A-F) and two (WC and M56) serotypes were determined in mammals. The aim of this study was to investigate and genotype the presence of C.psittaci ompA gene in domestic pigeon feces grown in family management style in ten different districts in Ankara in winter and summer seasons. Within the scope of the study, 100 pigeon stool samples were collected from birdhouses in 10 different districts of Ankara (Beypazari, Haymana, Kizilcahamam, Cubuk, Pursaklar, Bala, Cankaya, Polatli, Golbasi and city center) in two different seasons. DNA extraction from fecal samples was performed by classical methods. The presence of the agent in the extracted DNA samples was investigated by polymerase chain reaction (PCR) analysis of the ompA gene. Two-way sequence analysis of the ompA gene was performed with the primers used in the study from the target DNA products amplified by PCR. The results of sequence analysis were compared with the international database and serotyping/genotyping was performed. In the study, C.psittaci ompA gene was detected in 6 (6%) samples of 100 pigeon stool samples. Among these positive samples, two were from Bala (one sample from winter, one sample from summer), two were from Haymana (one sample from winter, one sample from summer) and two were from Golbasi (one sample from winter, one sample from summer); where the same agent was isolated in the same aviaries in different seasons. In this study, no difference was found between the presence of C.psittaci in pigeon droppings and season. In addition when the sequence analysis of the isolated samples were compared with the World database; all isolates were found to be 100% genotype B and 99% genotype E. In this study, the sequence analysis of the ompA gene of C.psittaci from domestic pigeon feces was determined for the first time in Turkey. Although the presence of C.psittaci in domestic pigeons is low, it is a zoonotic bacterium and is important for the public health.


Assuntos
Proteínas da Membrana Bacteriana Externa , Doenças das Aves , Chlamydophila psittaci , Columbidae , Fezes , Psitacose , Animais , Proteínas da Membrana Bacteriana Externa/genética , Doenças das Aves/microbiologia , Chlamydophila psittaci/genética , Columbidae/microbiologia , Fezes/microbiologia , Genótipo , Psitacose/microbiologia , Turquia
2.
J Basic Microbiol ; 60(1): 72-81, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31737922

RESUMO

Salmonella Enteritidis is an important foodborne pathogen that can infect a wide range of animal species including human beings, resulting in great losses to commercial husbandry and human health. CirA is an outer membrane receptor involved in iron uptake and colicin1A/B-mediated competitive killing. Although iron uptake is crucial to bacterial virulence, limited literature is available about the role of CirA in infection. In the present work, we aimed to evaluate the role of CirA during S. Enteritidis infection. For this purpose, we generated a CirA-deficient mutant of the S. Enteritidis strain C50336 and examined its biological characteristics. The results showed that cirA gene inactivation caused sharply decreased biofilm formation and apparently impaired antibiotic resistance. Furthermore, the cirA gene deletion mutant showed markedly reduced adhesion and invasion to human epithelial cell line Caco-2 cells and decreased proliferation in mouse macrophage cell line RAW264.7 cells. Moreover, attenuated virulence was determined by a mouse model, with an LD50 increase of approximately 1,000-fold. These data indicated that CirA plays critical roles in the S. Enteritidis infection process.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Receptores de Superfície Celular/metabolismo , Salmonella enteritidis/fisiologia , Salmonella enteritidis/patogenicidade , Virulência/genética , Animais , Antibacterianos/farmacologia , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Biofilmes/crescimento & desenvolvimento , Células CACO-2 , Colicinas , Farmacorresistência Bacteriana , Humanos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Células RAW 264.7 , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Salmonelose Animal/microbiologia , Salmonella enteritidis/efeitos dos fármacos
3.
Microbiol Res ; 230: 126350, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31629270

RESUMO

Outer membrane protein U (OmpU) is a major porin from Vibrio alginolyticus and has been considered a vaccine candidate against infection by V. alginolyticus. After pre-incubated with polyclonal antibody against rOmpU, V. alginolyticus showed a 78% decrease in extracellular iron level, suggesting that interruption of OmpU could increase intracellular iron level. The mRNA expression of ompU under iron-limited conditions was determined using real-time reverse transcriptase PCR. The mRNA level of ompU was downregulated to 0.27-, 0.036- and 0.019-fold after the addition of the iron chelator 2,2'-bipyridyl for 10, 30 and 60 min, respectively. In addition, the promoter of ompU contained a ferric uptake regulator (Fur) binding site, which revealed the potential regulation of ompU by Fur and iron. Fur from V. alginolyticus was purified and used for electrophoretic mobility shift assay. The result showed that in the absence of Fe2+, purified recombinant Fur could specifically bind to the promoter DNA of ompU, while in the presence of Fe2+, the binding of Fur and the promoter DNA was suppressed. Our study preliminarily explored the function of OmpU in iron balance in V. alginolyticus, and these findings were helpful in understanding iron metabolism in V. alginolyticus.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Ferro/metabolismo , Vibrio alginolyticus/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Vibrio alginolyticus/genética
4.
PLoS Negl Trop Dis ; 13(12): e0007883, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790395

RESUMO

BACKGROUND: Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes.


Assuntos
Aedes/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Biofilmes/crescimento & desenvolvimento , Enterobacteriaceae/genética , Trato Gastrointestinal/microbiologia , Deleção de Genes , Técnicas de Inativação de Genes , Animais , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Enterobacteriaceae/crescimento & desenvolvimento , Simbiose
5.
World J Microbiol Biotechnol ; 36(1): 9, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31858269

RESUMO

Campylobacter jejuni is the one of the leading cause of bacterial food borne gastroenteritis. PglB, a glycosyltransferase, plays a crucial role of mediating glycosylation of numerous periplasmic proteins. It catalyzes N-glycosylation at the sequon D/E-X1-N-X2-S/T in its substrate proteins. Here we report that the PglB itself is a glycoprotein which self-glycosylates at N534 site in its DYNQS sequon by its own catalytic WWDYG motif. Site-directed mutagenesis, lectin Immunoblot, and mobility shift assays confirmed that the DYNQS is an N-glycosylation motif. PglB's N-glycosylation motif is structurally and functionally similar to its widely studied glycosylation substrate, the OMPH1. Its DYNQS motif forms a solvent-exposed crest. This motif is close to a cluster of polar and hydrophilic residues, which form a loop flanked by two α helices. This arrangement extremely apposite for auto-glycosylation at N534. This self-glycosylation ability of PglB could mediate C. jejuni's ability to colonize the intestinal epithelium. Further this capability may also bear significance for the development of novel conjugated vaccines and diagnostic tests.


Assuntos
Campylobacter jejuni/enzimologia , Glicoproteínas/química , Hexosiltransferases/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Glicosilação , Hexosiltransferases/genética , Hexosiltransferases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , Vacinas
6.
PLoS Negl Trop Dis ; 13(12): e0007990, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31877135

RESUMO

BACKGROUND: Leptospirosis is a widespread zoonotic disease that causes reproductive losses and/or hepatorenal failure in a number of animal species. Wild reservoirs of the disease, such as rodents, harbor the causative bacterium, Leptospira spp., in their kidneys and contaminate the environment by excreting infected urine. In this study, we tested small wild mammals, environmental water, and livestock in the Cumberland Gap region of southeastern Appalachia for the presence of pathogenic Leptospira or leptospiral antibodies. METHODS/RESULTS: Small wild mammals (n = 101) and environmental water samples (n = 89) were screened by a real time quantitative PCR that targets the pathogenic Leptospira-specific lipl32 gene. Kidneys from 63 small wild mammals (62.37%) and two water sources (2.25%) tested positive for leptospiral DNA. To identify the infecting leptospiral species in qPCR-positive water and kidney samples, a fragment of leptospiral rpoB gene was PCR amplified and sequenced. L. kirschneri and L. interrogans were the leptospiral species carried by small wild mammals. Furthermore, sera from livestock (n = 52; cattle and horses) were screened for leptospiral antibodies using microscopic agglutination test (MAT). Twenty sera (38.46%) from livestock had antibodies to one or more serovars of pathogenic Leptospira spp. CONCLUSIONS: In conclusion, results from our study show exposure to leptospiral infection in farm animals and the presence of this zoonotic pathogen in the environmental water and kidneys of a significant number of small wild mammals. The public health implications of these findings remain to be assessed.


Assuntos
Animais Domésticos , Leptospira/isolamento & purificação , Leptospirose/veterinária , Roedores , Microbiologia da Água , Animais , Região dos Apalaches/epidemiologia , Proteínas da Membrana Bacteriana Externa/genética , RNA Polimerases Dirigidas por DNA/genética , Rim/microbiologia , Leptospira/classificação , Leptospira/genética , Leptospirose/epidemiologia , Leptospirose/microbiologia , Lipoproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real
7.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(11): 1130-1135, 2019 Nov 06.
Artigo em Chinês | MEDLINE | ID: mdl-31683400

RESUMO

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Ixodidae/microbiologia , Rickettsia/classificação , Rickettsia/isolamento & purificação , Rickettsiose do Grupo da Febre Maculosa/diagnóstico , Carrapatos , Animais , China , Feminino , Ixodidae/classificação , Ixodidae/crescimento & desenvolvimento , Masculino , Reação em Cadeia da Polimerase , Rickettsia/genética , Rios , Análise de Sequência
8.
Emerg Microbes Infect ; 8(1): 1711-1720, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31769735

RESUMO

Among the factors associated with the resurgence of whooping cough, special emphasis has been given to pathogen adaptation after the introduction of the acellular vaccine (ACV). To assess the impact of the vaccine transition strategy from whole-cell vaccine (WCV) to ACV on population dynamics of Bordetella pertussis in Barcelona (Spain), we studied 339 isolates collected from 1986 to 2015 by PFGE and multi-locus variable-number tandem repeat analysis (MLVA). Additionally, allelic variants for the pertussis toxin and its promoter, pertactin, type 3 fimbriae and fimbrial serotyping were assessed to determine its antigenic drift. A shift was observed in the B. pertussis population as well as in its antigenic profile concurrently with the introduction of ACV in Barcelona. Four out of the five most prevalent PFGE profiles were replaced by new profiles following the ACV introduction. MLVA type 27 was the dominant genotype, and its frequency increased from 25% to 79.3% after WCV replacement. Antigen typing demonstrated the emergence of prn2, ptxP3, fim3-2 and a shift from the fimbriae 3 to the fimbriae 2 serotypes after the ACV introduction. Our findings support the presence of population and antigenic dynamic changes in B. pertussis likely driven by the introduction of ACV.


Assuntos
Variação Antigênica , Vacinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Coqueluche/microbiologia , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Bordetella pertussis/genética , Genótipo , Humanos , Repetições Minissatélites , Dinâmica Populacional , Espanha , Fatores de Virulência de Bordetella/administração & dosagem , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologia , Coqueluche/prevenção & controle
9.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31636136

RESUMO

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/metabolismo , Fibrinogênio/metabolismo , Interações Hospedeiro-Patógeno , Streptococcus equi/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos de Bactérias/classificação , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/classificação , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Análise por Conglomerados , Variação Genética , Cavalos , Humanos , Fenótipo , Ligação Proteica , Homologia de Sequência , Fatores de Virulência/classificação , Fatores de Virulência/genética
10.
J Agric Food Chem ; 67(42): 11703-11709, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31578056

RESUMO

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Engenharia Metabólica , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/biossíntese
11.
Parasit Vectors ; 12(1): 497, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640755

RESUMO

BACKGROUND: Mass drug administration (MDA) with azithromycin is a cornerstone of the trachoma elimination strategy. Although the global prevalence of active trachoma has declined considerably, prevalence persists or even increases in some communities and districts. To increase understanding of MDA impact, we investigated the prevalence of active trachoma and ocular C. trachomatis prevalence, organism load, and circulating strains at baseline and one-year post-MDA in The Gambia and Senegal. METHODS: Pre- and one-year post-MDA, children aged 0-9 years were examined for clinical signs of trachoma in six Gambian and 12 Senegalese villages. Ocular swabs from each child's right conjunctiva were tested for evidence of ocular C. trachomatis infection and organism load (ompA copy number), and ompA and multi-locus sequence typing (MLST) was performed. RESULTS: A total of 1171 children were examined at baseline and follow-up in The Gambia. Active trachoma prevalence decreased from 23.9% to 17.7%, whereas ocular C. trachomatis prevalence increased from 3.0% to 3.8%. In Senegal, 1613 and 1771 children were examined at baseline and follow-up, respectively. Active trachoma prevalence decreased from 14.9% to 8.0%, whereas ocular C. trachomatis prevalence increased from 1.8% to 3.6%. Higher organism load was associated with having active trachoma and severe inflammation. Sequence typing demonstrated that all Senegalese samples were genovar A, whereas Gambian samples were a mix of genovars A and B. MLST provided evidence of clustering at village and household levels and demonstrated differences of strain variant frequencies in Senegal, indicative of an "outbreak". MLST, including partial ompA typing, provided greater discriminatory power than complete ompA typing. CONCLUSIONS: We found that one round of MDA led to an overall decline in active trachoma prevalence but no impact on ocular C. trachomatis infection, with heterogeneity observed between villages studied. This could not be explained by MDA coverage or number of different circulating strains pre- and post-MDA. The poor correlation between active trachoma and infection prevalence supports the need for further work on alternative indicators to clinical signs for diagnosing ocular C. trachomatis infection. MLST typing has potential molecular epidemiology utility, including better understanding of transmission dynamics, although relationship to whole-genome sequence variability requires further exploration.


Assuntos
Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Tracoma/epidemiologia , Tracoma/prevenção & controle , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Criança , Pré-Escolar , Chlamydia trachomatis/classificação , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/genética , Gâmbia/epidemiologia , Genótipo , Humanos , Lactente , Administração Massiva de Medicamentos , Tipagem de Sequências Multilocus , Filogenia , Testes Imediatos , Polimorfismo Genético , Prevalência , Senegal/epidemiologia , Tracoma/tratamento farmacológico , Sequenciamento Completo do Genoma
12.
PLoS Genet ; 15(10): e1008435, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613892

RESUMO

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Membrana Transportadoras/genética , Pectobacterium/genética , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/genética , Ferredoxinas/metabolismo , Genes Bacterianos/fisiologia , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica/fisiologia , Óperon/fisiologia , Pectobacterium/metabolismo , Peptídeo Hidrolases/metabolismo
13.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548319

RESUMO

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ehrlichia chaffeensis/imunologia , Ribonucleoproteínas/genética , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/genética , Autofagia/imunologia , Aderência Bacteriana/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Ehrlichia chaffeensis/genética , Técnicas de Inativação de Genes , Humanos , Imunidade Humoral/imunologia , NF-kappa B/genética , Células THP-1
14.
APMIS ; 127(12): 753-763, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31512768

RESUMO

Iron uptake system is expressed in early stages of Acinetobacter baumannii infections under iron-restricted conditions. This study is aimed at the evaluation of immuno-protectivity of BfnH in comparison with BauA in both mature and selected fragmental proteins. The study was designed in single and combined forms of antigens. BfnH is presented in 3472 strains of A. baumannii with more than 97% identity. The preliminary immune-informatics analysis of this protein indicated a region from the ß-barrel domain including exposed loops 2-5, with antigenic score comparable to that of BfnH. There was a significant rise in the specific IgG response in all test groups. The bacterial challenge with a lethal dose of A. baumannii demonstrated partial protection of whole proteins which coincides with a significant reduction in the bacterial population colonized in the main organs and an increase in the survival level. Passive immunization of the mice brought about 50% survival in the mice groups immunized with BfnH and with a combination of BfnH and BauA. The protectivity of siderophore receptors suggests their potential immunogenic role that could be considered as a component of multivalent subunit vaccine candidates against A. baumannii.


Assuntos
Infecções por Acinetobacter/prevenção & controle , Acinetobacter baumannii/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Receptores de Superfície Celular/imunologia , Infecções por Acinetobacter/imunologia , Infecções por Acinetobacter/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Epitopos de Linfócito B , Feminino , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
15.
Top Companion Anim Med ; 36: 12-15, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31472723

RESUMO

Vector-borne rickettsioses represent emerging threats to public health worldwide. The aim of this work was the screening for the presence of Rickettsia spp. in the blood of dogs and cats from southern Portugal. A PCR product of the expected size was amplified from DNA extracts obtained from blood samples of 29 out of 225 (12.9%) cats and in 2 out of 375 (.5%) dogs using genus-specific primers targeting Rickettsia gltA. Rickettsia conorii israelensis was identified by phylogenetical analysis of partial ompB sequences, amplified from blood samples taken from both a cat and a dog. The obtained results reinforce the idea that domestic animals may act as sentinels for the presence of vector-borne Rickettsia spp. in a given geographical area. In addition, rickettsioses should be included in the differential diagnosis of canine and feline vector-borne diseases.


Assuntos
Doenças do Gato/microbiologia , Doenças do Cão/microbiologia , Infecções por Rickettsia/veterinária , Rickettsia conorii/isolamento & purificação , Animais , Proteínas da Membrana Bacteriana Externa/genética , Gatos , Citrato (si)-Sintase/genética , DNA Bacteriano/sangue , Cães , Reação em Cadeia da Polimerase/veterinária , Portugal/epidemiologia , Infecções por Rickettsia/epidemiologia , Rickettsia conorii/genética , Análise de Sequência de DNA
16.
Parasitol Res ; 118(11): 3185-3189, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473856

RESUMO

A total of 482 bats representing 32 species and two families were captured in the Amazon forests of the Amapá state in northern Brazil. Nineteen Artibeus planirostris bats (3.9 %) were infested with 160 ticks, all identified as Ornithodoros hasei. Three pools of larvae were screened for rickettsial DNA via polymerase chain reaction (PCR) targeting three rickettsial genes: gltA, ompA and htrA. Only one of them yielded an amplicons of the expected size for all three molecular assays. Comparisons of the obtained sequences including a phylogenetic analysis confirmed the occurrence of "Candidatus Rickettsia wissemanii" in Brazil.


Assuntos
Quirópteros/microbiologia , Quirópteros/parasitologia , Ornithodoros/microbiologia , Infecções por Rickettsia/epidemiologia , Rickettsia/isolamento & purificação , Animais , Proteínas da Membrana Bacteriana Externa/genética , Brasil/epidemiologia , DNA Bacteriano/genética , Proteínas de Choque Térmico/genética , Ixodidae/microbiologia , Larva/microbiologia , Proteínas Periplásmicas/genética , Filogenia , Reação em Cadeia da Polimerase , Rickettsia/genética , Serina Endopeptidases/genética
17.
J Med Microbiol ; 68(10): 1540-1543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31483245

RESUMO

Four group A streptococcus (GAS) bacteraemia occurred in a small burn unit within 2 weeks. The GAS patient isolates, characterized as emm89, shared the same PFGE pulsotype with two other strains isolated 2 months later. The outbreak investigation revealed that a nurse was the most likely source of GAS transmission, as she was confirmed to carry the same outbreak strain in her throat and had direct and regular contact with the six outbreak patients in the unit. The outbreak was controlled after the nurse had undergone eradication treatment. This report highlights the emergence of the emm89 clone and its capacity to elicit invasive GAS outbreaks.


Assuntos
Unidades de Queimados/estatística & dados numéricos , Infecção Hospitalar/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/isolamento & purificação , Adulto , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecção Hospitalar/epidemiologia , Surtos de Doenças , Feminino , Humanos , Pessoa de Meia-Idade , Epidemiologia Molecular , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Tunísia , Adulto Jovem
18.
Vet Microbiol ; 235: 188-194, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383301

RESUMO

Outer membrane vesicles (OMVs) are produced and secreted virtually by every known Gram-negative bacterium. Despite their non-live nature, they share antigenic characteristics with the bacteria they originate from. This, together with their relative ease of purification, casts the OMVs as a very promising and flexible tool in both human and veterinary vaccinology. The aim of the current work was to get an insight into the antigenic pattern of OMVs from the pig pathogen Actinobacillus pleuropneumoniae in the context of vaccine development. Accordingly, we designed a protocol combining 2D Western Blotting and mass spectrometric identification to robustly characterize the antigenic protein pattern of the vesicles. Our analysis revealed that A. pleuropneumoniae OMVs carry several immunoreactive virulence factors. Some of these proteins, LpoA, OsmY and MIDG2331_02184, have never previously been documented as antigenic in A. pleuropneumoniae or other pathogenic bacteria. Additionally, we showed that despite their relative abundance, proteins such as FrpB and DegQ do not contribute to the antigenic profile of A. pleuropneumoniae OMVs.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Actinobacillus pleuropneumoniae/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Espectrometria de Massas , Mutação , Proteômica , Suínos , Fatores de Virulência/imunologia
19.
Indian J Med Microbiol ; 37(1): 50-53, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424010

RESUMO

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October-February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Orientia tsutsugamushi/genética , Tifo por Ácaros/diagnóstico , Feminino , Humanos , Masculino , Orientia tsutsugamushi/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Tifo por Ácaros/microbiologia , Tifo por Ácaros/patologia , Sensibilidade e Especificidade
20.
Indian J Med Microbiol ; 37(1): 113-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424021

RESUMO

Scrub typhus is one of the leading causes of acute febrile illness in India. This study aimed to determine the best diagnostic tool for the identification of scrub typhus and study the possible association between diagnostics and clinical characteristics. Patients with fever of ≤15 days admitted to the hospital satisfying the case definition of 47 kDa quantitative polymerase chain reaction (qPCR) positivity OR scrub typhus IgM ELISA positivity along with the presence of eschar OR Scrub typhus IgM ELISA positivity along with defervescence of fever within 72 h of initiation of specific therapy were recruited. Of the 116 patients satisfying the case definition, 47 kDa qPCR was positive in 43 (37%) patients, whereas IgM ELISA was positive in 104 (90%) patients and eschar was seen in 59 (51%) patients. The median duration of fever was 7.5 days (interquartile range 6-10 days). Multiorgan dysfunction syndrome (MODS) was described in 44 (37.9%) patients. Two patients (1.8%) succumbed to the illness. Presence of eschar and IgM ELISA positivity were detected in 106 (91%) cases. Scrub typhus, even with MODS, has low mortality because of immediate institution of specific therapy due to physician awareness. The presence of eschar and IgM ELISA positivity can be used to detect a majority of cases of scrub typhus.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Ensaio de Imunoadsorção Enzimática , Imunoglobulina M/sangue , Orientia tsutsugamushi/imunologia , Tifo por Ácaros/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Azitromicina , Criança , Pré-Escolar , Testes Diagnósticos de Rotina , Doxiciclina/uso terapêutico , Feminino , Humanos , Imunoglobulina M/imunologia , Lactente , Masculino , Pessoa de Meia-Idade , Orientia tsutsugamushi/isolamento & purificação , Reação em Cadeia da Polimerase , Tifo por Ácaros/tratamento farmacológico , Tifo por Ácaros/microbiologia , Tifo por Ácaros/patologia , Adulto Jovem
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