Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Unsupervised deep learning for identifying the O 6-carboxymethyl guanine by nanopore sequencing / 生物医学工程学杂志

O 6-carboxymethyl guanine(O 6-CMG) is a highly mutagenic alkylation product of DNA that causes gastrointestinal cancer in organisms. Existing studies used mutant Mycobacterium smegmatis porin A (MspA) nanopore assisted by Phi29 DNA polymerase to localize it. Recently, machine learning technology has been widely used in the analysis of nanopore sequencing data. But the machine learning always need a large number of data labels that have brought extra work burden to researchers, which greatly affects its practicability. Accordingly, this paper proposes a nano-Unsupervised-Deep-Learning method (nano-UDL) based on an unsupervised clustering algorithm to identify methylation events in nanopore data automatically. Specially, nano-UDL first uses the deep AutoEncoder to extract features from the nanopore dataset and then applies the MeanShift clustering algorithm to classify data. Besides, nano-UDL can extract the optimal features for clustering by joint optimizing the clustering loss and reconstruction loss. Experimental results demonstrate that nano-UDL has relatively accurate recognition accuracy on the O 6-CMG dataset and can accurately identify all sequence segments containing O 6-CMG. In order to further verify the robustness of nano-UDL, hyperparameter sensitivity verification and ablation experiments were carried out in this paper. Using machine learning to analyze nanopore data can effectively reduce the additional cost of manual data analysis, which is significant for many biological studies, including genome sequencing.

Vigilancia epidemiológica de Acinetobacter baumannii multirresistente a nivel hospitalario / Epidemiological surveillance of multidrug-resistant Acinetobacter baumannii at hospital level / Vigilância epidemiológica do Acinetobacter baumannii multirresistente em nível hospitalar

La Acinetobacter baumannii se considera como un agente microbiano de gran importancia clínica debido a la resistencia que ha adquirido a los antimicrobianos, esto trae como consecuencias complicaciones al referir una terapia antibiótica, prolongando la estancia en la hospitalización de los pacientes infectados con esta bacteria, causando un alto grado de mortalidad por su elevada capacidad de proliferación en las diferentes áreas hospitalarias. OBJETIVO. Describir la epidemiología de los brotes causados por Acinetobacter baumannii en Latinoamérica, así como también los mecanismos de Resistencia de este patógeno causando inconvenientes al momento de emplear los diferentes tratamientos terapéuticos. MATERIAL Y METODOS. Es una revisión sistemática bajo la declaración PRISMA de las investigaciones relacionadas al tema. Para la búsqueda de información se emplearon fuentes de datos provenientes de: Scielo, Redalyc, Google académico, se encontraron 43 artículos de los cuales solo 25 fueron válidos para la investigación. RESULTADOS. La resistencia que presenta la Acinetobacter baumannii a los antibióticos se incrementó con el pasar de los años. Este incremento de la resistencia se debe a diversos factores entre los cuales se destacan el desarrollo de ß-lactamasas dirigidas contra los betalactámicos, ya sea de amplio espectro o carbapenemasas; variaciones en las proteínas que forman parte de la membrana externa bacteriana, en las bombas de expulsión, perdida de porinas, modificaciones del lugar (blanco o diana) de acción de los antibióticos y variaciones en la expresión de proteínas fijadoras de penicilina. Esta variabilidad depende en parte de la capacidad de la bacteria para adquirir genes de resistencia, a menudo a través de una transferencia horizontal de genes CONCLUSIONES. La Acinetobacter baumannii se caracteriza por tener múltiples mecanismos de resistencia a antibióticos, lo que ha aumentado las consecuencias nocivas de este potencial patógeno y representa un desafío importante para los pacientes y el personal de salud.

Acinetobacter baumannii is considered as a bacterium of great clinical importance due to the resistance it has acquired to antimicrobials, which has triggered complications when referring an antibiotic therapy, prolonging the stay in the hospital of patients infected with this bacterium, causing a high degree of mortality due to its high proliferation capacity between different hospital areas. OBJETIVE. To describe the most relevant aspects about the epidemiology of the outbreaks caused by Acinetobacter baumannii in Latin America, as well as the different resistance mechanisms that this pathogen has acquired thus causing inconveniences when using the different therapeutic treatments. MATERIALS AND METHODS. A systematic review was carried out, under the PRISMA declaration, for the search of the information were used databases such as: Scielo, Redalyc, Google academic, 43 articles were found of which only 25 were valid for research. RESULTS. The resistance of Acitetobacter baumannii to different antibiotic groups has increased over the years. This increase in resistance is due to several factors among which stand out: ß-lactamases directed against beta-lactams, either broad spectrum or carbapenemases; variations in proteins of the outer membrane, ejection pumps, loss of porins, modifications of the place (target) of action of the antibiotics and alterations in the expression of penicillin-fixing proteins. This ability depends in part on the ability of this bacterium to acquire resistance genes, often through horizontal gene transfer. CONCLUSIONS. A. baumannii has developed multiple antibiotic resistance mechanisms, which increase the harmful consequences of this potential pathogen and represents a major challenge for patients and health personnel.

Acinetobacter baumannii é considerada como uma bactéria de grande importância clínica devido à resistência que adquiriu aos antimicrobianos, o que desencadeou complicações no momento de referir uma terapia antibiótica, prolongando a estadia na hospitalização dos pacientes infectados com esta bactéria, causando um elevado grau de mortalidade devido à sua elevada capacidade de proliferação entre diferentes áreas hospitalares. OBJECTIVO. Descrever os aspectos mais relevantes sobre a epidemiologia dos surtos causados por Acinetobacter baumannii a nível da América Latina, bem como os diferentes mecanismos de resistência que adquiriu este patógeno causando com isto inconvenientes o momento de empregar os diferentes tratamentos terapéuticos. MATERIAL E METODOS. Realizou-se uma revisão sistemática, sob a declaração PRISMA, para a busca da informação empregaram-se bases de dados como: Scielo, Redalyc, Google acadêmico, foram encontrados 43 artigos dos quais apenas 25 foram válidos para a pesquisa. RESULTADOS. A resistência que apresenta Acitetobacter baumannii aos diferentes grupos de antibioticos aumentou com o passar dos anos. Este aumento da resistência deve-se a diversos factores, entre os quais se destacam: ß-lactamases dirigidos contra os beta-lactâmicos, quer de largo espectro quer carbapenemases; variações nas proteínas da membrana externa, as bombas de expulsão, perda de porinas, modificações do local (branco) de acção dos antibioticos e alterações na expressão das proteínas fixadoras de penicilina. Esta capacidade depende em parte da capacidade desta bactéria para adquirir genes de resistência, muitas vezes através da transferência horizontal de genes. CONCLUSOES. Acinetobacter baumannii desenvolveu múltiplos mecanismos de resistência aos antibióticos, o que aumentou as consequências nocivas deste potencial patogênico e representa um desafio importante para os pacientes e o pessoal de saúde.

Polymorphism of P66 in / 生物医学与环境科学（英文）

Objective@#To study the polymorphism in P66 and its human B-cell epitopes of @*Methods@#Polymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese @*Results@#Results showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in @*Conclusion@#In P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of

Caracterización molecular y fenotípica de aislamientos clínicos de Salmonella Typhimurium variante monofásica (1, 4, [5],12: i: -) recuperados en Colombia / Molecular and phenotypic characterization of Salmonella Typhimurium monophasic variant (1, 4, [5],12: i: -) from Colombian clinical isolates

Resumen: Introducción. La variante monofásica (1,4,[5],12:i:-) de Salmonella Typhimurium ocupa los primeros lugares en los programas de vigilancia de Salmonella a nivel mundial. En Colombia, Salmonella enterica variante monofásica alcanza el cuarto lugar en cuanto a los aislamientos clínicos recuperados por medio de la vigilancia por laboratorio del Grupo de Microbiología del Instituto Nacional de Salud, pero se desconoce si dichos aislamientos están relacionados con la variante monofásica de Typhimurium que circula a nivel global, y con sus características genéticas y fenotípicas. Objetivo. Caracterizar los aislamientos de Salmonella monofásica recuperados en Colombia entre el 2015 y el 2018 por el Grupo de Microbiología del Instituto Nacional de Salud. Materiales y métodos. Se analizaron 286 aislamientos clínicos de Salmonella enterica variante monofásica mediante PCR o secuenciación del genoma completo (Whole Genome Sequencing, WGS) para confirmar si correspondían a Salmonella Typhimurium variante monofásica, en tanto que, en 54 aislamientos, se determinó la estructura genética del operón que codifica la segunda fase flagelar y, en 23, se evaluó la motilidad, el crecimiento y la expresión de las proteínas de membrana externa. Resultados. El 61 % (n=174) de los aislamientos de Salmonella monofásica correspondió a Salmonella Typhimurium serovar monofásico. El 64,8 % (n=35/54) se relacionó con el clon europeo-español y, el 13 % (n=7/54), con el estadounidense. En dos aislamientos de orina se encontró una diferencia significativa en la motilidad y el crecimiento, así como ausencia de la porina OmpD en medio mínimo M9. Conclusiones. En el periodo de estudio, circuló en Colombia la variante monofásica de Salmonella Typhimurium relacionada con el clon europeo-español, y se registró ausencia total del operón fljAB. Los resultados evidenciaron cambios fenotípicos en los aislamientos provenientes de muestras de orina que sugieren adaptación en procesos invasivos.

Abstract: Introduction. The Salmonella Typhimurium monophasic variant (1,4,[5],12:i:-) is currently the most commonly detected variant in Salmonella surveillance programs worldwide. In Colombia, the Salmonella enterica monophasic variant is the fourth most common clinical isolate recovered through the laboratory surveillance of the Grupo de Microbiología from the Instituto Nacional de Salud; however, it is unknown whether these isolates are closely related to the monophasic Typhimurium variant, which circulates globally, and their genetic and phenotypic characteristics have not been reported. Objective. To characterize monophasic Salmonella enterica isolates identified in Colombia from 2015 to 2018 by the Instituto Nacional de Salud. Materials and methods. Two hundred eighty-six clinical isolates of the monophasic Salmonella enterica variant were analyzed by PCR or whole-genome sequencing to confirm whether they corresponded to the Salmonella Typhimurium monophasic variant while the genetic structure of the operon encoding the second flagellar phase was determined in 54 isolates. Motility, growth, and expression of the outer membrane proteins were evaluated in 23 isolates. Results. During the study period in Colombia, 61% (n=174) of Salmonella monophasic isolates belonged to Salmonella Typhimurium serovar monophasic (1,4,[5],12:i-). Of these, 64.8% (n=35/54) were related to the European/Spanish clone and 13% (n=7/54) to the U.S. clone. Two isolates recovered from urine samples showed differences in motility, growth, and the absence of the OmpD porin in M9 minimal medium. Conclusions. Most of the monophasic Salmonella Typhimurium variants that have circulated in Colombia since 2015 lacked the second phase of operon fljAB, which is related to the European/Spanish clone. The results evidenced phenotypic changes in urine samples suggesting bacterial adaptation in the case of these invasive samples.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Mecanismos de resistência antimicrobiana em pseudomonasaeruginosa / Mechanisms responsible for antimicrobial resistance in pseudomonas aeruginosa

Pseudomonas aeruginosa é uma bactéria de grande importância para indivíduos imunocomprometidos. No Brasil, ela é um dos principais agentes em infecções hospitalares e pode provocar diversos tipos de processos clínicos. Atualmente, um dos maiores desafios em infecções provocadas por P. aeruginosa é a resistência apresentada diante de inúmeros antimicrobianos. Além da resistência intrínseca de P.aeruginosa, essa bactéria facilmente desenvolve mecanismos de resistência adicionais, através de mutações e da aquisição de elementos genéticos móveis, por exemplo. Dessa forma, P. aeruginosa é considerada um patógeno multirresistente, o que limita as alternativas terapêuticas capazes de combatê-lo. Portanto, compreender osmecanismos que levam a essa resistência é de extrema importância para enfrentar as infecções por P. aeruginosa.

Pseudomonas aeruginosa is a bacterium of great importance forimmunocompromised individuals. In Brazil, it is one of the leadingcauses of hospital infections and can cause many types of infections.Currently, one of the biggest challenges in infections caused by P.aeruginosa is the resistance presented against numerousantimicrobials. In addition to the intrinsic resistance of P. aeruginosa,inherent in the species, this bacterium easily acquire additionalmechanisms of resistance via mutation and acquisition of mobilegenetic elements, for example. Accordingly, P. aeruginosa isconsidered a multidrug-resistant pathogen, which limits thetherapeutic alternatives able to fight it. Therefore, understanding themechanisms that lead to this resistance is of utmost importance totackle infections by P. aeruginosa.

[ effects of cell phone electromagnetic fields on the behavior of single ompf nanochannel forming protein: a practical approach]

Widespread of telecommunication systems in recent years, have raised the concerns on the possible danger of cell phone radiations on human body. Thus, the study of the electromagnetic fields on proteins, particularly the membrane nano channel forming proteins is of great importance. These proteins are responsible for keeping certain physic-chemical condition within cells and managing cell communication. Here, the effects of cell phones radiation on the activity of a single nanopore ion channel forming protein, OmpF, have been studied biophysically. Planar lipid bilayers were made based on Montal and Muller technique, and the activity of single OmpF channel reconstituted by electrical shock was recorded and analyzed by means of voltage-clamp technique at 20[degree]C. The planar lipid bilayers were formed from the monolayers made on a 60 micro m diameter aperture in the 20 micro m thick Teflon film that separated two [cis and trans] compartments of the glass chamber. In this practical approach we were able to analyze characteristics of an individual channel at different chemical and physical experimental conditions. The voltage clamp was used to measure the channel's conductance, voltage sensitivity, gating patterns in time scales as low as microseconds in real time. Our results showed that exposure of single voltage dependent channel, OmpF, to EMF of cell phone at high-frequency has a significant influence on the voltage sensitivity, gating properties and substate numbers of the single channel but has no effect on single-channel conductance. Regarding to the relaxation time, the channel also recovers in the millisecond time range when the field is removed. We observed an increase in the voltage sensitivity of the OmpF single channel while it had no effect on the single-channel conductance, which is remained to be further elucidated.

Busca por alvos de regulação pelo segundo mensageiro c-diGMP em Pseudomonas aeruginosa / Search for c-di-GMP regulation targets in Pseudomonas aeruginosa

Recentemente, o bis-(3',5')-di-guanosina monofosfato cíclico (c-di-GMP) surgiu como uma importante molécula sinalizadora nas bactérias. Essa molécula foi identificada como uma das responsáveis pelo controle do comportamento bacteriano e está relacionada com a patogenicidade e a adaptação de diversas bactérias, coordenando a expressão de genes envolvidos com virulência, motilidade e formação de biofilme. O mecanismo pelo qual c-diGMP atua vem sendo motivo de estudo de vários grupos de pesquisa nos últimos anos. Já foi demonstrado o papel dessa molécula em diferentes etapas do controle da expressão gênica. Acredita-se que a manipulação dos níveis de c-di-GMP pode ser uma nova abordagem terapêutica contra bactérias patogênicas. Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que atua como um patógeno oportunista, causando infecções em pacientes imunocomprometidos, sendo o maior causador de infecções crônicas em pacientes portadores de fibrose cística. O genoma de P. aeruginosa PA14 apresenta vários genes que codificam proteínas envolvidas no metabolismo e/ou ligação de c-di-GMP, o que pode indicar um amplo papel regulatório deste nucleotídeo nessa bactéria. Uma associação infundada entre níveis elevados de c-di-GMP e a resistência aos antibióticos é geralmente assumida, já que altos níveis de c-di-GMP levam à formação de biofilme, que é comprovadamente um modo de crescimento mais resistente. Nesse trabalho, utilizando uma abordagem proteômica, mostramos que Pseudomonas aeruginosa PA14 regula a expressão de cinco porinas em resposta a variações nos níveis de c-di-GMP, independentemente dos níveis de mRNA. Uma dessas porinas, OprD, é responsável pela entrada do antibiótico ß-lactâmico imipenem na célula e é menos abundante em condições de alto c-di-GMP. Também demonstramos que linhagens com altos níveis de c-di-GMP apresentam uma vantagem competitiva de crescimento em relação a linhagens com níveis mais baixo de c-di-GMP quando crescidas em meio contendo imipenem. Em contraste, observamos que células planctônicas com elevados níveis c-di-GMP são mais sensíveis a tobramicina. Em conjunto, estes resultados mostram que c-di-GMP pode regular a resistência a antibióticos em sentidos opostos, e independentemente do crescimento em biofilme

Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this dinucleotide emerged as an important intracellular signal molecule controlling bacterial behavior. Diverse molecular mechanisms have been described as targets for c-di-GMP, but several questions remain to be addressed. An association between high c-di-GMP levels and antibiotic resistance is largely assumed, since high c-di-GMP upregulates biofilm formation and the biofilm mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Pseudomonas aeruginosa is a versatile gamma-proteobacterium that behaves as an opportunistic pathogen to a broad range of hosts. The ability of P. aeruginosa to form biofilms contributes to its virulence and adaptation to different environments. The P. aeruginosa PA14 genome presents several genes encoding proteins involved in metabolism or binding to c-di-GMP, which may indicate a wide regulatory role of this nucleotide in this bacterium. Here, using a proteomic approach, we show that Pseudomonas aeruginosa PA14 regulates the amount of five porins in response to c-di-GMP levels, irrespective of their mRNA levels. One of these porins is OprD, decreased in high c-di-GMP conditions, which is responsible for the uptake of the ß-lactam antibiotic imipenem. We also demonstrate that this difference leads strains with high c-di-GMP to be more resistant to imipenem even when growing as planktonic cells, giving them a competitive advantage over cells with low c-di-GMP. Contrastingly, we found that planktonic cells with high c-di-GMP levels are more sensitive to aminoglycosides antibiotics. Together, these findings show that c-di-GMP levels can regulate the antibiotic resistance to different drugs in opposite ways and irrespective of a biofilm mode of growth

Carbapenem and cefoxitin resistance of Klebsiella pneumoniae strains associated with porin OmpK36 loss and DHA-1 β-lactamase production

Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.

Purificação e caracterização da VDAC de mitocôndrias corticais aviares: identificação de modificações pós-traducionais nas porinas neuronais murinas e aviares / Purification and characterization of avian cortical mitochondrial VDAC: identification of post-translational modifications of rat and avian neuronal porins

A VDAC é uma porina presente na MME cuja função é crucial no metabolismo energético, sobrevivência e morte celular. A caracterização da VDAC torna-se importante para a compreensão das inter-relações da mitocôndria com os diferentes componentes citosólicos, tais como a HK. A ligação HK-VDAC favorece a utilização do ATP intramitocondrial em células neuronais, a HK cerebral pode interagir de formas diferentes com a VDAC, o que resulta em diferentes sítios de ligação (sítios A e B). Os variados papéis metabólicos das isoformas da VDAC podem ser explicados pela presença de alterações pós-traducionais. No presente trabalho purificamos a VDAC1 mitocondrial neuronal proveniente de cérebro aviar. Paralelamente, comprovamos que a presença de múltiplas formas das VDACs 1 e 2 em cérebros murino e aviar, seja devida à presença de modificações pós-traducionais, nomeadamente a fosforilação. A proteína isolada apresentou peso molecular de 30KDa. Quando submetida à eletroforese e posteriormente à coloração para a identificação de fosfoproteínas, a mesma mostrou-se desfosforilada. O conhecimento da presença, ou ausência de fosforilação das VDACs, reside na importância de estabelecer-se as bases moleculares ligadas à existência de sítios A e B nas mitocôndrias neuronais.

VDAC (voltage-dependent anion channel) is a pore forming protein from outer mitochondrial membrane. It has key functions on energetic metabolism, and cell death and survival. VDAC characterization is important for understanding mitochondrial interactions with cytosolic proteins, such as hexokinase (HK). HK-VDAC interaction supports preferential access to intramitochondrial ATP in neural cells. Brain HK interacts in different ways with VDAC. It results in two HK binding sites (A and B). VDAC isoforms differential metabolic roles may be explained by the presence of post-translational modifications. In this study we purified avian neuronal mitochondrial VDAC1. At same time we showed that VDACs 1 and 2 pI heterogeneity in rat and avian brains is due to phosphorylation. Purified VDAC had a molecular weight of 30 KDa. The purified VDAC submitted to phosphorylated protein staining on gel, was dephosphorylated. The knowledge of presence or absence of VDAC phosphorylation is important for understanding the molecular nature basis of A and B HK binding sites in brain mitochondria.

Construction and functional studies of uropathogenic E. coli strains with ompT gene knockout / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of ompT gene in uropathogenic E. coli (UPEC) CFT073 strain in urinary tract infection (UTI).</p><p><b>METHODS</b>An ompT deletion mutant (COTD) was generated by λ Red recombineering in the UPEC CFT073 strain, which was characterized by PCR and sequencing. C57B/L6 mouse models of acute UTI with the mutant and wild-type strains were established to compare the colonization abilities of the two strains in the bladder. The adhesion of CFT073 mutant to human unthelial 5637 cells was also investigated in vitro.</p><p><b>RESULTS</b>PCR and DNA sequencing confirmed the loss of ompT gene in the mutant COTD. The in vitro adhesion rate of the mutant strain COTD to 5637 cells was (6.7±2.2)%, significantly lower than that of (8.3±1.9)% of the wild-type strain (P<0.05). In the murine models of acute UTI, the mutant strain showed a mean colonization number of about (17±8)×10⁴ cfu, which was significantly lower than that of (7∓2)×10⁵ cfu of the wide-type CFT073 strain (P<0.05).</p><p><b>CONCLUSION</b>OmpT gene can be involved in the colonization of UPEC in the bladder tissue and plays an important role in the pathogenesis of UPEC-induced UTI.</p>

Resistencia a carbapenemes en aislamientos de pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos / Carbapenem resistance in pseudomonas aeruginosa isolates: an example of interaction between different mechanisms

Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.

Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.

Pseudomonas aeruginosa multirresistente: um problema endêmico no Brasil / Multidrug-resistant Pseudomonas aeruginosa: an endemic problem in Brazil

Relatos mundiais têm documentado a problemática da endemicidade de isolados clínicos de Pseudomonas aeruginosa multirresistente (MDR) aliada a elevados índices de morbidade/mortalidade. No Brasil, surtos de infecção ocasionados por P. aeruginosa têm sido relacionados com uma disseminação clonal da espécie. Atualmente, as opções terapêuticas para o tratamento das infecções causadas por esse microrganismo são limitadas, muitas vezes restringindo-se ao uso de carbapenêmicos (p. ex., imipenem [IPM]). Assim, a resistência ao IPM é uma questão de saúde pública, uma vez que esse antibiótico é empregado como último recurso no tratamento de infecções de origem hospitalar, causadas por bactérias Gram-negativas multirresistentes. No Brasil, os principais mecanismos relacionados com fenótipos multirresistentes de P. aeruginosa são produção de metalobetalactamase (MBL) do tipo SPM-1, presença de metilase 16S rRNA RmtD, perda de porina OprD e superexpressão de bombas de efluxo, o que pode explicar os altos índices de resistência a carbapenêmicos e aminoglicosídeos. A emergência de cepas com essas características é preocupante, tendo em vista a escassez de terapias efetivas no tratamento de infecções por esse patógeno. Finalmente, com base em relatos nacionais, publicados por diferentes grupos de pesquisa, podemos deduzir que a convergência de múltiplos mecanismos de resistência em P. aeruginosa tem sido um evento favorável para a seleção de diferentes clones endêmicos multirresistentes disseminados no Brasil.

Global reports have documented the endemicity of multidrug-resistant (MDR) Pseudomonas aeruginosa associated with high levels of morbidity/mortality. In Brazil, outbreaks of MDR P. aeruginosa have been related to clonal dissemination. Currently, therapeutic options for the treatment of these infections are restricted to carbapenemic antibiotics (i.e., imipenem [IPM]). Thus, carbapenem resistance is a public health issue, since carbapenems are considered the last resort to nosocomial infections caused by MDR Gram-negative bacteria. In Brazil, the main mechanisms associated with MDR P. aeruginosa phenotypes are metallo-betalactamase (MBL) production (SPM-1 enzyme), presence of 16S rRNA methylase RmtD, loss of OprD porin, and overexpression of efflux pumps, which may explain the high level of carbapenem and aminoglycoside resistance. Accordingly, the emergence and dissemination of MDR strains is worrisome. Finally, based on national reports published by different groups of investigators, it is deduced that the convergence of multiple mechanisms of P. aeruginosa resistance has played a major role in the selection of endemic MDR clones widespread in Brazil.

Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB.</p><p><b>METHODS</b>A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli.</p><p><b>CONCLUSION</b>The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.</p>

Porinas e suas ações imunomoduladoras dependentes de TLR2 / Porins and their immunomodulatory effects triggered by TLR2

Os micro-organismos podem infectar seu hospedeiro por diferentes vias, sendo a principal o trato respiratório. O reconhecimento pela mucosa dessas vias pode desencadear inibição da proliferação e bloqueio da entrada microbiana, assim como estimular resposta direcionada a memória imunológica para prevenir posteriores infecções. Alguns micro-organismo, como as bactérias Neisseria meningitidis e Neisseria lactamica, são capazes de modular a resposta imune de mucosa diretamente, ou por meio das células epiteliais respiratórias. Este trabalho propôs, então, a avaliação das porinas B provenientes destas bactérias como moduladoras da produção de IL-8 nas linhagens BEAS-2B e Detroit 562. Também foi avaliada a dependência deste estímulo ao receptor TLR2. Ambas as porinas se ligaram a TLR2 e por este receptor estimularam a produção de IL-8. O perfil de produção foi dependente da expressão de TLR2 pelas células. A porina lactâmica induziu menos IL-8 por regular negativamente a expressão de TLR2, mas sua afinidade pelo receptor se mostrou maior que a da porina meningocócica. As porinas são então moduladoras das células de mucosa, fato que somado a atividade adjuvante destas proteínas por via parenteral estimulou a avaliação destas como adjuvantes de mucosa. O modelo escolhido para a avaliação foi o de inoculação intranasal de camundongos, utilizando como antígeno o lipopolissacarídio pouco imunogênico de Franciscella tularensis atenuada (Ft-LPS). A análise foi baseada no título de anticorpos IgG e IgM séricos. A porina meningocócica se mostrou a mais imunogênica, mas por ser originária de patógeno acarreta maior risco biológico em sua produção. Para viabilizar a porina meningocócica como adjuvante, a mesma foi substituída por porina homóloga produzida de modo recombinante em Escherichia coli não patogênica. A porina recombinante foi avaliada pelo mesmo sistema in vivo e comparada a adjuvantes experimentais de ação conhecida (rCTB, QS-21 e ODN 1826). A porina apresentou...

Microorganisms can invade the host through many routes, specially the respiratory tract. The respiratory mucosa is responsible for recognition, inhibition, proliferation and entry blockade of microorganisms, besides incitation of immunological memory to prevent further infections. Some microorganisms, such as Neisseria meningitidis and Neisseria lactamica, can modulate the mucosa immune response directly or through stimulation of respiratory epithelial cells. The present work proposed the evaluation of porin B proteins, derived from these microorganisms, as modulators of IL-8 production on respiratory epithelial cell strains BEAS-2B and Detroit 562. TLR2 receptor dependency for the modulation was also evaluated. Both porins bounded to TLR2 and through this receptor were able to stimulate IL-8 production, whereas this profile was correlated with TLR2 expression. Lactamica porin (Nlac PorB) induced less IL-8 and TLR2 expression, also for a shorter period of time. The effect caused by Nlac PorB was attributed to TLR2 down regulated expression, since its binding affinity to the receptor is greater than meningococcal porin (Nmen PorB). Porins were therefore able to immune modulate mucosal cells, fact that allied with their parenteral adjuvant activity incited evaluation of porins as potential mucosal adjuvants. The model chosen for the evaluation was intranasal immunization of mice, using as the antigen a low immunogenic lipopolysaccharide extracted from attenuated Franciscella tularensis (Ft-LPS). The evaluation was based on IgG and IgM serum titers. After the immunization scheme, Nmen PorB induced higher IgG and IgM titers than Nlac PorB. Although Nmen PorB was more efficient, it comes from a pathogen. To overcome the risk of its production, it was replaced by recombinant porin (rPorB) produced by Escherichia coli. rPorB was evaluated by the same model and compared with well known experimental adjuvants (rCTB, QS-21 e ODN 1826). rPoB had the highest IgM and IgG...

Immunogenicity of multi-epitopes gene of major outer membrane protein of Chlamydia trachomatis / 中华预防医学杂志

<p><b>OBJECTIVE</b>To construct a recombinant eukaryotic expression plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene, and evaluate the Ct MOMP-specific humoral and cellular immune response induced by pcDNA3.1-Ct MOMP168 in BALB/c mice.</p><p><b>METHODS</b>Recombinant plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene was constructed. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1-Ct MOMP168 or pcDNA3.1 or PBS (n = 12, 100 microg/time per mouse), and the same immunization schedule was repeated for the third time at 2 week intervals. The titers of anti-Ct MOMP antibody and its antibody subtypes in sera, the cytotoxicity of Ct MOMP-specific cytotoxic T lymphocyte (CTL) in spleen, and the level of cytokine (IFN-gamma, IL-4, IL-10)-producing CD3(+) T cells in spleen were detected by ELISA, LDH release assays and intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS), respectively.</p><p><b>RESULTS</b>The recombinant plasmid pcDNA3.1-Ct MOMP168 was able to induce Ct-specific antibody response (A(490) = 0.973 +/- 0.136; serum titer was 1:1000) as compared with pcDNA3.1 (A(490) = 0.180 +/- 0.025) and PBS (A(490) = 0.110 +/- 0.015), and the major antibody subtype was IgG2a with statistical significance (F = 106.884, P < 0.05). When the ratio of effector cells and target cells reached to 50:1, the activity of cytotoxic T-lymphocyte in pcDNA3.1-Ct MOMP168 immunized mice (41.71% +/- 8.34%) was significantly higher (F = 22.315, P < 0.05) than that in pcDNA3.1 immunized mice (18.40% +/- 3.45%) and PBS immunized mice (14.50% +/- 2.42%). The levels of CD3(+) IFN-gamma(+) T cells in pcDNA3.1-Ct MOMP168 immunized mice (1.15% +/- 0.16%) were significantly higher (F = 99.638, P < 0.05) than that in pcDNA3.1 immunized mice (0.12% +/- 0.08%) and PBS immunized mice (0.09% +/- 0.03%), while the significant difference in the levels of IL-4(+) CD3(+) T cells and IL-10(+) CD3(+) T cells was not observed (F = 0.886 and 1.112, P > 0.05) between pcDNA3.1-Ct MOMP168 immunized mice (0.13% +/- 0.08% and 0.14% +/- 0.08%) and pcDNA3.1 (0.07% +/- 0.05% and 0.13% +/- 0.06%) or PBS immunized mice (0.08% +/- 0.04% and 0.07% +/- 0.04%).</p><p><b>CONCLUSION</b>In BALB/c mice, the recombinant plasmid pcDNA3.1-Ct MOMP168 might induce not only the generation of Ct-specific antibody, but also the high level of Ct MOMP-specific CD3(+) IFN-gamma(+) T cells.</p>

Prevalencia de infección cervical por Chlamydia trachomatis en mujeres de la Región Metropolitana / Prevalence of cervical infection by Chlamydia trachomatis among Chilean women living in the Metropolitan Región

Background: Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) worídwide. In women, chlamydia infections are 75 percent asymptomatic and can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy. Infants exposed to the microorganism at birth also have a high risk to develop conjunctivitis and pneumonía. Aim: To determine the prevalence of C trachomatis in women in the Metropolitan área of Santiago (Chile). Patients and methods: Cervical specimens were collected from 403 women attending three gynecological outpatient settings from Apríl 2003 to June 2005. These included one public hospital (n =100), a prívate medical center (n =268), and a clinic for adolescents (n =35). Mean ages ofeach group of patients were 35.6±8,2, 33.4±8.1 and 16.9±4.2 years, respectively. The diagnosis of C trachomatis was performed by the amplification byPCRofa 517-base pair segment of the cryptic plasmid on specimens extracted by a commercial procedure. Positive specimens were conñrmed by nested PCRs targeting the ompl gene. The presence of vaginal infections and its association with C trachomatis was investigated in a subset of 223 women ofthe prívate center. Residís: C trachomatis was detected in the cervix of 19 out of 403 women, resulting in a prevalence of 4.7 percent. The distribution of positive cases among different age groups was not significantly different. Women presenting with bacterial vaginosis had a significantly higher prevalence of C trachomatis infection (p <0.01). Conclusions: This study found a high prevalence of C trachomatis among gynecologic patients that should prompt preventive strategies.