Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using an in vitro keratinocyte infection model, we show that a subpopulation of E. faecalis becomes internalized via macropinocytosis into single membrane-bound compartments, where they can survive and replicate. These intracellular E. faecalis can persist in late endosomes up to 72 hours after infection in the absence of colocalization with the lysosomal protease cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in a marked reduction in Rab7 expression, a small GTPase required for endosome-lysosome fusion. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection.

中文翻译：

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伤口感染期间粪肠球菌在体外和体内的上皮细胞内持续存在和复制

粪肠球菌是一种常见的伤口机会性病原体，其感染与生物膜形成、持久性和对治疗的抵抗有关。我们之前已经表明粪肠球菌伤口感染持续至少 7 天。在这里我们报告说，感染后长达 5 天，伤口部位的免疫和非免疫细胞中都存在存活的粪肠球菌，这提高了细胞内持久性导致慢性粪肠球菌感染的可能性。使用体外角质形成细胞感染模型，我们发现粪肠球菌亚群通过巨胞饮作用被内化到单个膜结合区室中，在那里它们可以存活和复制。在没有与溶酶体蛋白酶组织蛋白酶 D 共定位或与溶酶体明显融合的情况下，这些细胞内粪肠球菌可以在感染后的晚期内体中持续存在长达 72 小时，这表明粪肠球菌阻止了内体成熟。事实上，细胞内粪肠球菌感染导致 Rab7 表达显着降低，Rab7 是一种内体-溶酶体融合所需的小 GTP 酶。最后，我们证明细胞内粪肠球菌来自受感染的角质形成细胞的细菌在重新感染新的角质形成细胞方面明显更有效。总之，这些数据表明，面对强大的免疫反应，粪肠球菌的细胞内增殖可能有助于其持续存在，为随后的再感染提供准备好的细菌库。