Distribution of non-LEE-encoded type 3 secretion system dependent effectors in enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important human gastroenteritis agents. The prevalence of six non-LEE genes encoding type 3 translocated effectors was investigated. The nleC, cif and nleB genes were more prevalent in typical than in atypical EPEC, although a higher diversity of genes combinations was observed in atypical EPEC.

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Construction of an ethanologenic Escherichia coli strain expressing beta-glucosidase / 生物工程学报

Constructing ethanologenic strains with cellulose activity is important to achieve consolidated bioprocessing of lignocellulose for ethanol production. In this study, we integrated the pyruvate decarboxylase gene pdc and alcohol dehydrogenase gene adhB from Zymomonas mobilis ZM4 into Escherichia coli JM109 by Red recombination method to generatea recombinant strain E. coli P81 that could produce ethanol from glucose. Abeta-glucosidase gene bglB from Bacillus polymyxa 1.794 was cloned into the recombinant E. coli P81 and beta-glucosidase was expressed to give a new recombinant strain E. coli P81 (pUC19-bglB) with dual functions of cellobiose degradation and ethanol production. The extracellular beta-glucosidaseactivity was 84.78 mU/mL broth and the extracellular cellobiase activity of E. coli P81 (pUC19-bglB) was 32.32 mU/mL broth. E. coli P81 (pUC19-bglB) fermented cellobiose to ethanol with a yield of 55.8% of the theoretical value, and when glucose and cellobiose were co-fermented, the ethanol yield reached 46.5% of thetheoretical value. The construction of consolidated bioprocessing strain opens the possibility to convert cellobiose to ethanol in a single bioprocess.

Study on the functions of potential new genes of Yersinia pestis type three secretion system / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).</p><p><b>METHODS</b>Mutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.</p><p><b>RESULTS</b>When grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.</p><p><b>CONCLUSION</b>YpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.</p>

The putative propeptide of MycP1 in mycobacterial type VII secretion system does not inhibit protease activity but improves protein stability

Mycosin-1 protease (MycP1) is a serine protease anchored to the inner membrane of Mycobacterium tuberculosis, and is essential in virulence factor secretion through the ESX-1 type VII secretion system (T7SS). Bacterial physiology studies demonstrated that MycP1 plays a dual role in the regulation of ESX-1 secretion and virulence, primarily through cleavage of its secretion substrate EspB. MycP1 contains a putative N-terminal inhibitory propeptide and a catalytic triad of Asp-His-Ser, classic hallmarks of a subtilase family serine protease. The MycP1 propeptide was previously reported to be initially inactive and activated after prolonged incubation. In this study, we have determined crystal structures of MycP1 with (MycP1²⁴⁻⁴²²) and without (MycP1⁶³⁻⁴²²) the propeptide, and conducted EspB cleavage assays using the two proteins. Very high structural similarity was observed in the two crystal structures. Interestingly, protease assays demonstrated positive EspB cleavage for both proteins, indicating that the putative propeptide does not inhibit protease activity. Molecular dynamic simulations showed higher rigidity in regions guarding the entrance to the catalytic site in MycP1²⁴⁻⁴²² than in MycP1⁶³⁻⁴²², suggesting that the putative propeptide might contribute to the conformational stability of the active site cleft and surrounding regions.

O Papel da imunoglobulina a secretora no mecanismo de defesa da mucosa bucal / The role of secretory immunoglobulin a in the defense mechanism of the oral mucosa

Introdução: As superficies mucosas mantem contato direto com o meio externo, sendo expostas a uma grande variedade de antigenos. Essas superficies possuem relativa vulnerabilidade e a secreção de anticorpos pelo sistema imune de mucosa constitui sua principal defesa, mantendo os antigenos desconhecidos e/ou potencialmente patogenicos fora do organismo. Entre esses anticorpos, o mais abundante ‚ uma IgA polimerica, denominada IgA secretora, que ‚ produzida por plasmócitos associados as mucosas e glandulas exocrinas. A sIgA possui uma estrutura capaz de resistir adegradação proteolitica de enzimas bacterianas e digestivas, o que a torna ideal para proteção do trato gastrointestinal. Na mucosa bucal a sIgA representa a mais importante barreira imunol¢gica, impedindo a aderencia e a penetração de microorganismos, sendo utilizada como parametro do status imunologico dessa mucosa. Objetivo: Realizar uma revisão de literatura a respeito da imunoglobulina A secretora liberada na saliva, enfocando sua formação, secreção e as principais funções imunológicas na proteção da mucosa bucal. Conclusão: A imunoglobulina A secretora possui ótimas caracteristicas para a proteção da mucosa bucal, atribuidas a sua estrutura e ação especifica e inespecifica. Os resultados de estudos clinico/laboratoriais associados a sua grande quantidade salivar, sugerem uma participação relevante da sIgA nos processos etiológicos e fisiopatológicos de lesões na mucosa bucal, evidenciando a necessidade de uma maior atenção as funções dessa imunoglobulina em estudos posteriores.

Introduction: Mucosa surfaces are in direct contact with the external environment, being exposed to a great variety of antigens. These surfaces have relative vulnerability and the secretion of antibodies by the mucosal immune system is the main defense mechanism, maintaining unknown and/or potentially pathogenic antigens out of the organism. Among these antibodies, the most abundant is a polymeric IgA, denominated secretory immunoglobulin A (sIgA), which is produced by plasmocytes associated with mucosas and exocrine glandules. A sIgA has a structure capable of resisting to the proteolytic degradation of bacterial and digestive enzymes, which makes it ideal for protecting the gastrointestinal tract. In the oral mucosa, sIgA represents the most important immunological barrier, impeding the adherence and penetration of microorganisms and being used as a parameter of immunological status of the mucosa. Objective: This paper presents a literature review on the sIgA released in the saliva, focusing on its formation, secretion and the main immunological functions on the protection of the oral mucosa. Conclusion: sIgA has excellent characteristics for protecting the oral mucosa, which are attributed to its structure and specific and unspecific actions. The results of clinical/laboratorial studies associated with its great amount in the saliva suggest a relevant participation of sIgA in the etiologic and physiopathological processes of oral mucosa injuries, demonstrating the necessity of a greater attention to the functions of this immunoglobulin in further studies.

Clinical significance of virulence-related genes of type III secretion system of Pseudomonas aeruginosa / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).</p><p><b>METHODS</b>One hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.</p><p><b>RESULTS</b>Among the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.</p><p><b>CONCLUSIONS</b>The TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.</p>