Recombinant PilS: Cloning, Expression and Biochemical Characterization of a Pil-Fimbriae Subunit.

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

Distribution of Major Pilin Subunit Genes Among Atypical Enteropathogenic Escherichia coli and Influence of Growth Media on Expression of the ecp Operon.

Atypical enteropathogenic Escherichia coli (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coli pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94-100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1-8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coli common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37°C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens.

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes.

Low-molecular mass comparative proteome of four atypical enteropathogenic Escherichia coli isolates showing different adherence patterns.

Atypical enteropathogenic Escherichia coli (aEPEC) are heterogeneous in terms of serotypes, adherence patterns and the presence of non-locus of enterocyte effacement virulence factors. In this study, the low-molecular mass proteomes of four representative aEPEC, comprising three different adhesion phenotypes (localized-like, aggregative and diffuse) and one non-adherent isolate, were analyzed and compared by 2D gel electrophoresis and LC-MS/MS. By mass spectrometry, a total of 59 proteins were identified according to their annotated function, with most of them being involved in metabolism, protection, and transport; some of them still classified as hypothetical proteins. Thus, in this comparative proteomic analysis of low-molecular mass extracted proteins from different aEPEC isolates, the proteins identified are mainly involved in key metabolic pathways. Also, the majority of the hypothetical and filamentous proteins identified in the isolates studied are products of genes originally identified in the genome of enterohemorrhagic E. coli.