Brucella dissociation is essential for macrophage egress and bacterial dissemination.
Brucella dissociation is essential for macrophage egress and bacterial dissemination.
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DOI：
10.3389/fcimb.2014.00023
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发表时间：
2014
影响因子：
5.7
通讯作者：
Ficht TA
中科院分区：
文献类型：
--
作者：
Pei J;Kahl-McDonagh M;Ficht TA
长期以来观察到光滑布鲁氏菌可以解离成对巨噬细胞具有细胞毒性的粗糙突变体。然而，布鲁氏菌解离和细胞毒性的体内生物学意义和/或机制细节仍然不完整。在本报告中，使用表现出不同程度细胞毒性的布鲁氏菌菌株开发了空斑试验。每天使用相差显微镜观察感染的单层菌斑形成，同时使用免疫荧光试验（IFA）监测布鲁氏菌的摄取和复制。在感染后4-5天（p.i.）与细胞毒性布鲁氏菌16 M ΔmanBA以0.1的MOI进行。IFA染色显示噬斑由巨噬细胞和复制的布鲁氏菌组成。在MOI为0.1的非细胞毒性16 M ΔmanBAΔ virB 2感染的单层细胞中未检出可见空斑。然而，IFA染色确实显示了在16 M ΔmanBAΔ virB 2感染的单层中具有复制布鲁氏菌的小群巨噬细胞（病灶）。在感染粗糙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，表明细胞毒性对布鲁氏菌的外出和传播至关重要。在16 M感染的单层中，观察到小和大病灶。双抗体染色显示自发粗糙突变体内的大，但不是在16 M感染的单层的小病灶。此外，在源自16 M感染的大病灶中观察到斑块形成。最后，在培养基中加入庆大霉素可抑制噬菌斑的形成，这表明只有在微生物从细胞中释放出来后才发生细胞间的扩散。综上所述，这些结果表明，布鲁氏菌诱导的细胞毒性是布鲁氏菌外出和传播的关键。
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.
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影响因子：
3.7
作者：
Chen F;He Y
通讯作者：
He Y
DOI：
10.3181/0904-rm-124
发表时间：
2009-12
期刊：
Experimental biology and medicine (Maywood, N.J.)
影响因子：
--
作者：
Covert J;Mathison AJ;Eskra L;Banai M;Splitter G
通讯作者：
Splitter G
影响因子：
3.4
作者：
Comerci, DJ;Martínez-Lorenzo, MJ;Ugalde, RA
通讯作者：
Ugalde, RA
DOI：
10.1084/jem.20030088
发表时间：
2003-08-18
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Celli J;de Chastellier C;Franchini DM;Pizarro-Cerda J;Moreno E;Gorvel JP
通讯作者：
Gorvel JP
影响因子：
3.1
作者：
Eskra, L;Mathison, A;Splitter, G
通讯作者：
Splitter, G