Gustavo Pelicioli Riboldi

Structural studies of the Enterococcus faecalis SufU [Fe-S] cluster protein

BackgroundIron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein.ResultsBLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed.ConclusionThe data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the G ram p ositive r egion (GPR), suggested as a possible candidate for interaction with other factors and/or regulators.

Antimicrobial resistance profile of Enterococcus spp isolated from food in Southern Brazil

Fifty-six Enterococcus spp. strains were isolated from foods in Southern Brazil, confirmed by PCR and classified as Enterococcus faecalis (27), Enterococcus faecium (23) and Enterococcus spp (6). Antimicrobial susceptibility tests showed resistance phenotypes to a range of antibiotics widely administrated in humans such as gentamycin, streptomycin, ampicillin and vancomycin.

Antimicrobial resistance and investigation of the molecular epidemiology of Listeria monocytogenes in dairy products

INTRODUCTION Listeria monocytogenes is a ubiquitous microorganism in nature and is responsible for listeriosis, an infectious disease caused by consumption of contaminated food. METHODS Molecular characterization was performed on 19 strains of Listeria monocytogenes (serovars 1/2a, 1/2b, 4b and 4c), isolated from dairy products in Rio Grande do Sul, Brazil. The molecular techniques applied were random amplification of polymorphic DNA and restriction enzyme analysis. In addition to the molecular analysis, the antimicrobial resistance profile was determined. RESULTS The strains studied showed a low degree of diversity. In relation to the antimicrobial resistance profile of those microorganisms from the samples analyzed, all of them were susceptible to the antimicrobials tested. CONCLUSIONS The molecular techniques that were used presented good discriminatory power for the strains studied. Furthermore, all of the samples that were analyzed were susceptible to the antimicrobials tested.

Phenotypic and genotypic heterogeneity of Enterococcus species isolated from food in Southern Brazil

Enterococcus is an important group of lactic acid bacteria (LAB), which inhabits the gastrointestinal tract of humans and animals. These microorganisms can also be found in large groups of foods where they can play a beneficial role during food maturation processes or, conversely, can be used as a food contamination indicator. These microorganisms have an additional importance in various aspects of clinical microbiology. The aim of this study was to investigate and determine the phenotypic and genetic diversity in 55 enterococci isolated from different food sources. Phenotypic characteristics based upon substrate hydrolysis differences were used to identify different Enterococcus species. Analysis of the resultant data divided these species into eight Enterococcus phenotype groups. E. faecalis was the food isolate species with the greatest phenotypic variability. Fifty‐five previously isolated Enterococcus strains were re‐confirmed as belonging to this genus by PCR techniques. Randomly amplified polymorphic DNA (RAPD‐PCR) was used to study the genetic variability using M13 primers and the resultant DNA fragments produced a database of different fingerprints. After statistical analyses of the RAPD‐PCR profiles, 42 patterns were obtained and 6 different Enterococcus RAPD clusters (ERC) were identified. Genetic diversity was highest in ERC I, which grouped together approximately 40% of the E. faecium and E. faecalis isolates obtained from dairy products. Samples isolated from meat and vegetables offered the greatest genotype variability. Results of the present study suggest the presence of both phenotypic and genotypic variability within enterococci strains isolated from diverse sources of food common to Southern Brazil. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Biogenesis of [Fe–S] cluster in Firmicutes: an unexploited field of investigation

Iron–sulfur clusters (ISC) ([Fe–S]) are evolutionarily ancient and ubiquitous inorganic prosthetic groups present in almost all living organisms, whose biosynthetic assembly is dependent on complex protein machineries. [Fe–S] clusters are involved in biologically important processes, ranging from electron transfer catalysis to transcriptional regulatory roles. Three different systems involved in [Fe–S] cluster assembly have already been characterized in Proteobacteria, namely, the nitrogen fixation system, the ISC system and the sulfur assimilation system. Although they are well described in various microorganisms, these machineries are poorly characterized in members of the Firmicutes phylum, to which several groups of pathogenic bacteria belong. Recently, several research groups have made efforts to elucidate the biogenesis of [Fe–S] clusters at the molecular level in Firmicutes, and many important characteristics have been described. Considering the pivotal role of [Fe–S] clusters in a number of biological processes, the review presented here focuses on the description of the biosynthetic machineries for [Fe–S] cluster biogenesis in prokaryotes, followed by a discussion on recent results observed for Firmicutes [Fe–S] cluster assembly.

Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation

The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

beta-lactamase production Haemophilus spp. and resistance to ampicillin in a general hospital in Porto Alegre city, RS, Brazil (2001-2005)

In a four-year period (July/2001-June/2005), 410 Haemophilus spp. isolates were studied. Those were isolated from sputum at Hospital Nossa Senhora da Conceição (NSC) in Porto Alegre city (RS). beta-lactamase enzyme was detected in 113 (27.6%) of isolates through chromogenic cephalosporin method. Fifty-eight (51.3%) of them showed sensibility to ampicillin through disc-diffusion method using Haemophilus Test Medium (HTM) by NCCLS criteria. In 297 (72.4%) isolates beta-lactamase was not detected by chromogenic cephalosporin method. Five (1.7%) of them were resistant and 1 (0.3%) intermediate to ampicillin using disc-diffusion method. The authors emphasized the importance of Haemophilus spp. resistance to ampicillin research in clinical laboratories routine and the use of more than one method for this analysis was proposed, due to different resistance mechanisms in Haemophilus spp.