RESUMO
Campylobacter jejuni represents one of the major causes of bacterial enteritis caused by food in humans. There are still mechanisms to be deciphered to better understand better its physiology and pathogenesis. Study of gene expression levels by RT-qPCR could be used, but to be accurate and reproducible, a good internal control has to be chosen. The aim of this study was to identify a highly stable housekeeping gene in Campylobacter jejuni that could constitute a good internal control to study gene expression variations between different growth phases or stress conditions. Expression levels of six different housekeeping genes (gyrA, ilvC, rpoA, slyD, thiC and rrs) were measured by RT-qPCR under different conditions (exponential phase, stationary phase, cold shock, cold shock+oxidative stress, oxidative stress). The rpoA gene was chosen as the best internal control. In a previous study, 9 proteins were identified as involved in oxidative stress response, among which 3 virulence factors. Expression levels of genes coding for these proteins was evaluated by RT-qPCR using rpoA as an internal control. The results obtained were concordant with what had been observed at the proteomic level, validating the methods used and confirming the hypothesis of a potential link between oxidative stress and virulence factors expression.
Assuntos
Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estresse Fisiológico/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Girase/genética , DNA Girase/metabolismo , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Estabilidade de RNA , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normasRESUMO
Campylobacter jejuni is a microaerophilic pathogen but is able to survive oxidative stress conditions during its transmission to the human host. Strains of different origins (reference, poultry, or human clinical) were tested for survival under oxidative stress conditions. C. jejuni strains were grown in Mueller Hinton broth to obtain late exponential-phase cultures. Then they were exposed to 2 different stresses: (1) cultures were either plated on Columbia agar plates and exposed to atmospheric oxygen or (2) paraquat (a chemical oxidizing agent) was added to liquid cultures to reach a 500-microM concentration. Both of these experimental conditions were realized at 3 different temperatures: 4 degrees C, 25 degrees C, and 42 degrees C. Results obtained with paraquat and atmospheric oxygen were similar. Surprisingly, C. jejuni was found to be very sensitive to oxidative stress at 42 degrees C, which is its optimal growth temperature, whereas it was more resistant at 4 degrees C. A strain effect was observed, but no relationship was found between the origin of the strains and level of resistance. High temperature (42 degrees C) combined with oxidative stress allowed a rapid decrease in the C. jejuni population, whereas low temperature considerably decreased the effect of oxidative stress.