Koolman Leonard, Whyte Paul, Burgess Catherine, Bolton Declan
Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland; Institute of Food and Health & School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Institute of Food and Health & School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
Int J Food Microbiol. 2016 Mar 2;220:33-8. doi: 10.1016/j.ijfoodmicro.2016.01.002. Epub 2016 Jan 7.
Studies were undertaken to investigate the effect of oxidative stress conditions (exposure to hydrogen peroxide, H2O2) on [1] the expression of 14 Campylobacter jejuni virulence-associated genes associated with motility and/or invasion (flaA, flaB, flhA, flhB, ciaB, iamA), adhesion (cadF), cytotoxin production (cdtA, cdtB, cdtC) as well as some of the regulators of these genes (rpoN, fliA, luxS, cj1000), in 10 C. jejuni strains (5 poultry and 5 human) and [2] the ability of these cells to adhere to and invade Caco-2 cells. Using 16S rRNA as the reference gene (preliminary research demonstrated that this gene was stably expressed), the expression of the 14 virulence associated genes was investigated under normal and oxidative stress conditions using reverse transcription PCR. A Caco-2 cell tissue culture assay was used to examine adhesion and invasion. The response to oxidative stress was strain-dependent. Two strains showed significant (p<0.05) up or down regulation in 7 of the 14 genes tested, while only 1-2 genes were affected in the remaining strains. Expression of cadF was significantly (p<0.05) changed in all strains, cdt B in 4 strains and cj1000 in 3 strains. Expression of the remaining genes was either unaffected or significantly altered in 1-2 strains. NCTC 11168 completely lost the ability to adhere to and invade Caco-2 cells. One other strain also demonstrated reduced adherence while two others were unable to invade Caco-2 cells after exposure to oxidative stress conditions. In contrast strain 7, a poultry isolate, showed increased invasion. It was concluded that oxidative stress affects expression of C. jejuni virulence genes in a strain-dependent manner, CadF may have a secondary survival function and the cdtB gene may have a different promoter than cdtA and cdtC.
开展了多项研究，以调查氧化应激条件（暴露于过氧化氢，H2O2）对[1]10株空肠弯曲菌（5株来自家禽，5株来自人类）中14个与运动性和/或侵袭相关的空肠弯曲菌毒力相关基因（flaA、flaB、flhA、flhB、ciaB、iamA）、黏附相关基因（cadF）、细胞毒素产生相关基因（cdtA、cdtB、cdtC）以及这些基因的一些调节因子（rpoN、fliA、luxS、cj1000）表达的影响，以及[2]这些细胞黏附并侵袭Caco-2细胞的能力。以16S rRNA作为参照基因（初步研究表明该基因表达稳定），采用逆转录PCR在正常和氧化应激条件下研究14个毒力相关基因的表达。使用Caco-2细胞组织培养试验检测黏附和侵袭情况。对氧化应激的反应具有菌株依赖性。在检测的14个基因中，有2株在7个基因上表现出显著（p<0.05）上调或下调，而其余菌株仅1 - 2个基因受到影响。所有菌株中cadF的表达均有显著（p<0.05）变化，4株中cdt B的表达有变化，3株中cj1000的表达有变化。其余基因的表达在1 - 2株中要么未受影响，要么有显著改变。NCTC 11168完全丧失了黏附并侵袭Caco-2细胞的能力。另一株在暴露于氧化应激条件后也表现出黏附能力降低，还有两株无法侵袭Caco-2细胞。相比之下，家禽分离株7型表现出侵袭能力增强。得出的结论是，氧化应激以菌株依赖性方式影响空肠弯曲菌毒力基因的表达，CadF可能具有次要的生存功能，并且cdtB基因可能具有与cdtA和cdtC不同的启动子。
