Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat

The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences ofblaCTX-M and plasmid-mediated ampCand qnrgenes were investigated in Escherichia colifrom 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. TheblaCTX-M-15, blaCTX-M-2 andblaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates). Twenty-one biotypes (46 isolates) harboured blaCMY-2.Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 orblaCTX-M-8. The qnrB and/orqnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL) and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description ofblaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 andblaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Erkrankungen durch verotoxinbildende Escherichia coli beim Menschen [Verotoxin-producing Escherichia coli in human disease]

Les colibacilles producteurs de vérotoxines sont impliqués dans la pathogénie de syndromes diarrhéiques et dans celle de certains syndromes hémolytiques et urémiques. Le syndrome diarrhéique est caractérisé par l'apparition soudaine de douleurs abdominales sévères à type de crampes, suivies d'une diarrhée aqueuse qui, ensuite, devient sanglante. Les diarrhées peuvent être accompagnées de vomissements et d'une fièvre modérée. La période d'incubation varie entre 3 et 9 jours. Le syndrome hémolytique et urémique, la première cause d'insuffisance rénale aiguë du nourrisson et de l'enfant, est caractérisé par une triade typique: anémie hémolytique microangiopathique avec thrombocytopénie et insuffisance rénale glomérulonéphritique aiguë. La cause du syndrome hémolytique et urémique avec diarrhées prodromique a été attribuée aux vérotoxines. Les toxines produites par les colibacilles seraient à l'origine de lésions vasculaires endothéliales prédominant au niveau rénal et induisant le syndrome hémolytique et urémique.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares ( CORBIA et al., 2000; MELO, 2009 ) . A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC nº 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia

O presente trabalho monográfico elaborado como parte dos requisitos para obtenção do grau de licenciatura em Análises Clínicas e Saúde Pública, teve como objectivo proceder à contagem de Staphylococcus coagulase positivo e Escherichia coli nas amostras de queijo fresco da ilha do Fogo comercializado no mercado municipal da cidade da Praia. O queijo é um derivado do leite muito apreciado devido ao seu valor nutritivo como também pelo seu sabor, que consegue atender aos mais diferentes paladares. Mas, muitas vezes as condições de processamento, armazenamento e comercialização poderão comprometer as suas características organolépticas, ou torná-lo impróprio para o consumo humano através de contaminação por microrganismos causadores das toxinfecções alimentares (CORBIA et al., 2000; MELO, 2009). A presença desses microrganismos no queijo poderá ter como fonte de contaminação a utilização do leite cru, utensílios contaminados utilizados durante o processo de fabrico, condições higiénico-sanitária precárias. Para a avaliação da qualidade microbiológica do queijo fresco artesanal comercializado no mercado da cidade da Praia, foram analisadas 40 amostras de queijos provenientes da ilha do Fogo que são comercializados neste estabelecimento. A técnica utilizada para a contagem das bactérias Staphylococcus coagulase positiva foi a de sementeira a superfície e para Escherichia coli fez-se pelo método de incorporação. Dos resultados obtidos para a contagem de Staphylococcus coagulase positiva 87,5% das amostras analisadas estavam contaminadas e para Escherichia coli 82,5% das amostras estavam também contaminadas, o que de acordo com a Resolução - RDC no 12, de 2 de Janeiro de 2001 da ANVISA, estão fora do limite estabelecido, e estando assim insatisfatórias para o consumo humano. Concluiu-se que os queijos frescos da ilha do Fogo comercializados no mercado municipal da cidade da Praia não são de boa qualidade para o consumo humano. Os produtores do queijo e as vendedeiras precisam de formações sobre as boas práticas de higiene e de fabrico a fim de obtermos um produto de qualidade e isento de microrganismos que são prejudiciais à saúde do consumidor.

Enterohemorraginen Escherichia coli (EHEC) ja sen osoittaminen PCR-menetelmällä

Summary: Enterohemorrhagic Escherichia coli (EHEC) and its detection with a PCR method

Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 35 units of Ler. DNA wraps around Ler particles that contain more than 1516 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.