Kuley Runa, Smith Hilde E, Frangoulidis Dimitrios, Smits Mari A, Jan Roest Hendrik I, Bossers Alex
Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands; Host Microbe Interactomics, Wageningen University, Wageningen, The Netherlands.
Department of Infection Biology, Central Veterinary Institute part of Wageningen UR, Lelystad, The Netherlands.
PLoS One. 2015 Mar 20;10(3):e0121661. doi: 10.1371/journal.pone.0121661. eCollection 2015.
Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.
Q热由专性细胞内细菌伯氏考克斯体引起。该细菌的体外生长通常局限于有活力的真核宿主细胞，这给分子研究带来了实验限制，比如主要毒力因子的鉴定和表征。致病性研究可能受益于最近开发的一种用于伯氏考克斯体的细胞外生长培养基。然而，研究由两种根本不同的培养系统传代的菌株的毒力表型的一致性至关重要。在本研究中，我们评估了伯氏考克斯体在两种培养系统中的活力以及该细菌的脂多糖（LPS）编码区域，将其作为伯氏考克斯体感染潜力的间接但关键参数。基于单叠氮化丙锭（PMA）处理的实时PCR用于对有活力的伯氏考克斯体进行计数，这通过荧光感染灶形成单位计数测定法得到验证。此外，对在两种培养系统中传代的伯氏考克斯体分离的RNA进行了LPS相关基因表达检测。发现所有目前已知的LPS相关基因在两种培养系统的早期传代中均有表达，表明主要存在伯氏考克斯体的I相形式。最后，我们使用有免疫能力的小鼠来提供直接证据，即不同伯氏考克斯体菌株对于无菌和基于细胞的传代方法而言，其相对毒力基本相同。
