Investigating the intracellular growth, cytotoxicity and apoptotic effects of solar irradiated Campylobacter jejuni in a murine macrophage cell line (RAW 264.7)

Campylobacter jejuni is a leading cause of gastroenteritis worldwide. Solar disinfected (SODIS) water can reduce diarrhoeal incidences in communities where potable water is inaccessible. SODIS water has been associated with the reduction of diarrhoeal incidences amongst SODIS users. Thus solar attenuation of bacteria may have a significant impact on immune cells such as macrophages. When solar irradiated cells die the release antigenic materials such lipopolysaccharides and intracellular contents into the water. The solar irradiated bacterial may have an impact on the cytotoxicity and cell death of immune cells such as macrophages. This study assessed the effects of solar irradiation on the viability, metabolic activity and three virulence characteristics (invasion, cytotoxicity, and apoptosis) of C. jejuni on RAW 264.7 cells. Samples of C. jejuni were suspended in water and treated in the following ways (i) heat and chemically attenuated (1% formalin at 60°C for one hour), (ii)) exposure to solar ultraviolet radiation (SUVR) for 0, 4 and 8 and, (iii) non-exposure to SUVR. The metabolic activity was assayed spectrophotometrically using Alamar Blue. The C. jejuni samples were used to infect macrophage RAW264.7 cells and its intracellular growth was assessed by using the gentamicin protection assay. Cytotoxicity was assessed by using the Lactate Dehydrogenase Assay (LDH). Apoptosis of the treated macrophages was analysed by Flow Cytometry. The results showed that all the C. jejuni were not culturable. However, the non-solar irradiated Campylobacter jejuni retained its metabolic activity (40.2%±1%) whereas no metabolic activity was observed in the heat and chemically attenuated and solar irradiated bacteria. Intracellular growth of the bacteria in the RAW264.7 cells was not detected in all the treated samples. The non-irradiated C. jejuni showed higher cytotoxic and apoptotic effects on macrophages than the heat attenuated and solar irradiated samples. In conclusion, solar irradiation of C. jejuni eliminates its metabolic activity and also reduces its ability to induce cytotoxicity and apoptosis in the macrophages.

空肠弯曲菌（Campylobacter jejuni）是全球范围内引发肠胃炎的首要致病菌。太阳能消毒（SODIS，Solar Disinfected）用水可在难以获取饮用水的社区降低腹泻发病率，既往研究已证实SODIS用水与使用者腹泻发生率降低存在关联。因此，细菌的太阳能灭活作用可能对巨噬细胞等免疫细胞产生显著影响。当经太阳能照射的细菌死亡后，其会将脂多糖（lipopolysaccharides）及胞内内容物等抗原物质释放至水体中；经太阳能照射的细菌还可能对巨噬细胞等免疫细胞的细胞毒性与细胞死亡产生影响。本研究评估了太阳能照射对空肠弯曲菌（C. jejuni）在RAW 264.7细胞上的存活率、代谢活性，以及三项毒力特征（侵袭力、细胞毒性与细胞凋亡）的影响。实验将空肠弯曲菌样品悬浮于水中，采用以下三种方式处理：(i) 热与化学联合灭活（于60℃下用1%福尔马林处理1小时）；(ii) 暴露于太阳紫外线辐射（SUVR，solar ultraviolet radiation），处理时长分别为0、4和8；(iii) 未暴露于太阳紫外线辐射。采用阿尔玛蓝（Alamar Blue）分光光度法检测样品的代谢活性。用上述处理后的空肠弯曲菌样品感染巨噬细胞RAW264.7细胞，通过庆大霉素保护试验评估其胞内增殖情况。采用乳酸脱氢酶检测法（LDH，Lactate Dehydrogenase Assay）评估细胞毒性。通过流式细胞术分析经处理的巨噬细胞的凋亡情况。结果显示，所有空肠弯曲菌样品均无法培养。然而，未经过太阳能照射的空肠弯曲菌仍保留了40.2%±1%的代谢活性，而经热与化学联合灭活及太阳能照射的细菌则未检测到代谢活性。所有处理组样品中均未检测到细菌在RAW 264.7细胞内的增殖。未照射组的空肠弯曲菌对巨噬细胞的细胞毒性与凋亡诱导效应均强于热灭活组与太阳能照射组。综上，对空肠弯曲菌进行太阳能照射可消除其代谢活性，同时削弱其诱导巨噬细胞产生细胞毒性与凋亡的能力。