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.

In Vitro Alkaline pH Resistance of Enterococcus faecalis

Enterococcus faecalis is a bacterial species often found in root canals with failed endodontic treatment. Alkaline pastes are widely used in Endodontics because of their biocompatibility and antimicrobial activity, but this microorganism can resist alkalinity. The purpose of this study was to evaluate in vitro the alkaline pH resistance of E. faecalis for different periods up to 14 days. Samples were obtained from the oral cavity of 150 patients from the Endodontic clinic. The pH of the experimental tubes (n=84) was first adjusted with 6M NaOH to pH values of 9.5, 10.5, 11.5 and 12.5 (21 tubes per pH). Twenty clinical isolates and the ATCC 29212 strain were tested. The 5 positive controls and experimental tubes of each pH were inoculated with 10 µL of bacterial suspension and incubated at 36 °C for 24, 48 and 72 h, 7 and 14 days. For each period, the turbidity of the medium was visually compared with a 0.5 McFarland standard. The presence of the microorganism was confirmed by seeding on M-Enterococcus agar. Four tubes containing BHI broth adjusted to the tested pHs were incubated for 14 days to verify if pH changes occurred. The pH of inoculated BHI broth was also measured on day 14 to determine if the microorganism acidified the medium. The growth of all E. faecalis strains occurred at pH 9.5 to 11.5 in all periods. Although turbidity was not observed at pH 12.5, there was growth of 13 and 2 strains at 24 and 48 h, respectively, on M-Enterococcus agar. No tube showed growth at pH 12.5 after 72 h. It was concluded that E. faecalis can survive in highly alkaline pH, and some clinical isolates require 72 h at pH 12.5 to be killed.

Enterococcus faecalis é uma espécie bacteriana frequentemente encontrada em canais radiculares com insucesso do tratamento endodôntico. Pastes alcalinas são amplamente utilizada em Endodontia por causa de sua biocompatibilidade e atividade antimicrobiana, porém esse microrganismo pode ser resistente a alcalinidade. Este estudo avaliou in vitro a resistência do E. faecalis ao pH alcalino por diferentes períodos até 14 dias. Amostras foram obtidas da cavidade oral de 150 pacientes da Clínica de Endodontia. O pH dos tubes experimentais (n=84) foram inicialmente ajustados com NaOH 6M a valores de pH 9.5, 10.5, 11.5 e 12.5 (21 tubes per pH). Vinte isolados clínicos e a cepa ATCC 29212 foram testados. Os 5 controles positivos e os tubos experimentais de cada pH foram inoculados com 10 µL de suspensão bacteriana e incubados a 36 °C por 24, 48 e 72 h, 7 e 14 dias. Para cada período, turvação do meio foi compara visualmente com padrão 0.5 da escala de McFarland. A presença de microorganism foi confirmada por semeadura no meio ágar M-Enterococcus. Quatro tubos contendo caldo BHI ajustado aos pHs testados foram incubados por 14 dias para verificar a ocorrência de alterações de pH. O pH do caldo BHI inoculado também foi medido no 14° dia para determinar se o microrganismo acidificou o meio. O crescimento de todas as cepas de E. faecalis ocorreu com pH entre 9.5 e 11.5 em todos os períodos. Embora não tenha sido observada turvação do meio no pH 12.5, houve crescimento de 13 e 2 cepas às 24 e 48 h, respectivamente, no meio ágar M-Enterococcus. Nenhum tube apresentou crescimento bacteriano no pH 12.5 após 72 h. Concluiu-se que o E. faecalis pode sobreviver em pH altamente alcalino, que alguns isolados clínicos requerem 72 h em pH 12.5 para serem eliminados.

Propeptide processing and proteolytic activity of proenzymes of the Staphylococcal and Enterococcal GluV8-family protease.

Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser-1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn-1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family proenzymes with shortened propeptides intrinsically possess proteolytic activity and are involved in the propeptide shortening that facilitates the final hetero-catalytic maturation.