Base Pairing Interaction between 5'- and 3'-UTRs Controls icaR mRNA Translation in Staphylococcus aureus.

The presence of regulatory sequences in the 3' untranslated region (3'-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3'-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3'-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3'-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3'-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3'-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3'-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3'-UTR with the 5'-UTR of the same mRNA

Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy—and also to its side effects, which include folate deficiency and gastrointestinal upset

Protein-mediated biofilm development in Staphylococcus aureus

Póster presentado en el Joint Annual Seminar on the Projects of the 1st and 2nd calls of the ERA-NET PathoGenoMics, celebrado en Costa Adeje (Tenerife) el 18 y 19 de enero de 2010.Despite the general agreement that the exopolysaccharides plays a key role in biofilm development, increasing number of evidences indicate the existence of proteinaceous- dependent biofilm matrix. The existence of biofilm matrixes of different composition suggests that each matrix might confer different properties to the embedded bacteria. Because the proteomic analysis of the exopolysaccharide matrix of Salmonella enteritidis and Staphylococcus aureus revealed the presence of very few proteins, whose presence is not specific for biofilm matrix, we focused our e fforts to investigate protein-mediated biofilm development in S. aureus . The ultimate goal will be to understand the influence that biofilm matrix nature (polysaccharidic or proteinaceuos) has on embedded bacteria. For that, we first analyzed the protein-mediated biofilm matrix developed by some S. aureus strains in the absence of ArlRS two-component system. To identify the protein component responsible for the PIA/PNAG-independent biofilm development in arlRS mutants, fixed bacterial cells were digested with trypsin to yield a peptide mixture that was fractionated and analyzed by 2DnLC coupled to electrospray ionization and mass spec trometry. The results of the study revealed that overexpression of Protein A induced in tercellular aggregation and biofilm development. Exogenous addition of purified protein A or supernatants containing secreted protein A to growth media induced biofilm development, indica ting that, at least, protein A can promote biofilm development without being covalently anchored to the cell wall. On the other hand, we investigated a clinical S. aureus MRSA strain able to switch between proteinaceous and polysaccharidic biofilm matrix depending on the environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth- glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to the activation of a LexA-dependent SOS response or FnBP overexpression from a mu lticopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Interestingly, scanning electron microscopy revealed that cells growing in these protein-mediated biofilms formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG -dependent biofilm were embedded in an abundant extracellular material. Finally, we compared the contribution of protein-mediated biofilm with respect to exopolysaccharide-mediated biofilm to colonization of subcutaneously implanted catheters. Unexpectedly, the results revealed that both Protein A and FnBP mutants displayed a significantly lower capacity to develop biofilm on implanted catheters than their corresponding isogenic PIA/PNAG-deficient mutants. These results strongly suggest that protein-mediated biofilms might play a more relevant role than exopolysaccharide-mediated biofilm in the colonization of the implanted devices.Peer Reviewe

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist

O tempo como dimensão de pesquisa sobre uma política de diversidade e relações de trabalho

Neste estudo nosso objetivo é apreender o processo de absorção dos deficientes físicos pelas empresas, considerando múltiplos olhares (público interno e externo), e se estes se modificaram ao longo dos anos. Assim, conduzimos uma pesquisa numa empresa aérea que alocou essas pessoas em posições de contato direto com o público. Num primeiro momento, constatamos que a empresa atendeu aos anseios dos seus clientes e empregados. Os empregados não deficientes viam na contratação dos deficientes a possibilidade de benefícios para a organização (melhoria de sua imagem pública) e para si mesmos (repasse de trabalho para os deficientes). Já para os empregados portadores de deficiência, tal situação era aceitável, pois viabilizava sua integração ao grupo. Em 2011, voltamos a campo e entrevistamos representantes das mesmas categorias (passageiros, empregados - deficientes e não - e os gerentes de RH e marketing) para avaliar o impacto da dimensão "tempo". As entrevistas foram transcritas e submetidas à análise do discurso, a qual nos remeteu aos seguintes marcos teóricos: ética, tempo como variável subjetiva e gestão da diversidade da força de trabalho nas organizações. O campo revelou que a naturalização da presença dos deficientes ocorreu de tal forma que, para os passageiros deixou de ser um diferencial da empresa e, para os empregados, algo relevante. A empresa, por sua vez, insiste em buscar visibilidade social e midiática por meio da espetacularização da deficiência. Já os deficientes reclamam que não conseguem fazer carreira, pois encontram o mesmo "teto de vidro" denunciado por outras minorias

The vaccine potential of Bordetella pertussis biofilm-derived membrane proteins

Pertussis is an infectious respiratory disease of humans caused by the gram-negative pathogen Bordetella pertussis. The use of acellular pertussis vaccines (aPs) which induce immunity of relative short duration and the emergence of vaccine-adapted strains are thought to have contributed to the recent resurgence of pertussis in industrialized countries despite high vaccination coverage. Current pertussis vaccines consist of antigens derived from planktonic bacterial cultures. However, recent studies have shown that biofilm formation represents an important aspect of B. pertussis infection, and antigens expressed during this stage may therefore be potential targets for vaccination. Here we provide evidence that vaccination of mice with B. pertussis biofilm-derived membrane proteins protects against infection. Subsequent proteomic analysis of the protein content of biofilm and planktonic cultures yielded 11 proteins which were ≥three-fold more abundant in biofilms, of which Bordetella intermediate protein A (BipA) was the most abundant, surface-exposed protein. As proof of concept, mice were vaccinated with recombinantly produced BipA. Immunization significantly reduced colonization of the lungs and antibodies to BipA were found to efficiently opsonize bacteria. Finally, we confirmed that bipA is expressed during respiratory tract infection of mice, and that anti-BipA antibodies are present in the serum of convalescent whooping cough patients. Together, these data suggest that biofilm proteins and in particular BipA may be of interest for inclusion into future pertussis vaccines