The Shigella flexneri Type 3 Secretion System Is Required for Tyrosine Kinase-Dependent Protrusion Resolution, and Vacuole Escape during Bacterial Dissemination

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.

福氏志贺菌（Shigella flexneri）是一种人类致病菌，可主动侵入肠上皮细胞并逃离初级液泡，以进入胞质溶胶区域。当胞质内的运动细菌接触细胞皮层时，其会通过形成膜突起在细胞间扩散，这些突起会在邻近细胞内形成次级液泡。本研究以感染了2a血清型菌株2457T的HT-29肠道细胞为模型，探究了Ⅲ型分泌系统（Type 3 Secretion System，T3SS）在福氏志贺菌播散过程中的作用。我们构建了一株T3SS针状结构核心组分MxiG表达缺陷的2457T菌株。正如预期，ΔmxiG菌株侵袭HT-29细胞的能力严重受损；而在阿拉伯糖诱导表达系统下表达mxiG的ΔmxiG/pmxiG菌株则恢复了完整的感染性。在该实验体系中，侵袭步骤完成后撤除诱导剂的ΔmxiG/pmxiG（Ara withdrawal）菌株，可在HT-29细胞胞质内正常进行肌动蛋白介导的运动，但细菌在突起中停留至液泡形成的时长显著增加，且无法成熟为液泡的突起数量也有所增多。相应地，该菌株无法在膜突起中触发酪氨酸磷酸化——这一信号事件是突起成熟为次级液泡所必需的。最后，该菌株无法从已形成的次级液泡中逃逸，这与此前在非肠道细胞中的研究结果一致。综上，Ⅲ型分泌系统在福氏志贺菌肠道细胞播散过程中发挥多重作用，包括依赖酪氨酸激酶信号通路将膜突起成熟为次级液泡，以及介导细菌从已形成的次级液泡中逃逸。