Pei Jianwu, Kahl-McDonagh Melissa, Ficht Thomas A
Department of Veterinary Pathobiology, Texas A&M University and Texas Agricultural Experiment Station College Station, TX, USA.
Front Cell Infect Microbiol. 2014 Mar 5;4:23. doi: 10.3389/fcimb.2014.00023. eCollection 2014.
It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.
长期以来人们一直观察到，光滑型布鲁氏菌可解离为对巨噬细胞具有细胞毒性的粗糙型突变体。然而，布鲁氏菌解离和细胞毒性的体内生物学意义和/或机制细节仍不完整。在本报告中，利用表现出不同程度细胞毒性的布鲁氏菌菌株开发了一种噬斑测定法。每天使用相差显微镜观察感染的单层细胞中噬斑的形成，同时使用免疫荧光测定法（IFA）监测布鲁氏菌的摄取和复制。在感染后4 - 5天（p.i.），用细胞毒性布鲁氏菌16MΔmanBA以0.1的感染复数（MOI）可检测到可见噬斑。IFA染色表明，噬斑由带有正在复制的布鲁氏菌的巨噬细胞组成。在以0.1的MOI感染非细胞毒性的16MΔmanBAΔvirB2的单层细胞中未检测到可见噬斑。然而，IFA染色确实揭示了在感染16MΔmanBAΔvirB2的单层细胞中有少量带有正在复制的布鲁氏菌的巨噬细胞小群体（病灶）。在感染粗糙型布鲁氏菌的巨噬细胞单层中观察到的病灶大小与液体培养中测得的细胞毒性直接相关，这表明细胞毒性对于布鲁氏菌的逸出和传播至关重要。在感染16M的单层细胞中观察到了小病灶和大病灶。双重抗体染色显示，在感染16M的单层细胞的大病灶而非小病灶中有自发的粗糙型突变体。此外，在源自16M感染的大病灶中观察到了噬斑形成。最后，向培养基中添加庆大霉素可抑制噬斑形成，这表明细胞间传播仅在细菌从细胞中释放后才发生。综上所述，这些结果表明布鲁氏菌诱导的细胞毒性对于布鲁氏菌的逸出和传播至关重要。
