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Spatial Segregation of Virulence Gene Expression during Acute Enteric Infection with Salmonella enterica serovar Typhimurium

机译：肠道沙门氏菌血清鼠伤寒沙门氏菌急性肠感染过程中毒力基因表达的空间分离

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To establish a replicative niche during its infectious cycle between the intestinal lumen and tissue, the enteric pathogen Salmonella enterica serovar Typhimurium requires numerous virulence genes, including genes for two type III secretion systems (T3SS) and their cognate effectors. To better understand the host-pathogen relationship, including early infection dynamics and induction kinetics of the bacterial virulence program in the context of a natural host, we monitored the subcellular localization and temporal expression of T3SS-1 and T3SS-2 using fluorescent single-cell reporters in a bovine, ligated ileal loop model of infection. We observed that the majority of bacteria at 2?h postinfection are flagellated, express T3SS-1 but not T3SS-2, and are associated with the epithelium or with extruding enterocytes. In epithelial cells, S. Typhimurium cells were surrounded by intact vacuolar membranes or present within membrane-compromised vacuoles that typically contained numerous vesicular structures. By 8?h postinfection, T3SS-2-expressing bacteria were detected in the lamina propria and in the underlying mucosa, while T3SS-1-expressing bacteria were in the lumen. Our work identifies for the first time the temporal and spatial regulation of T3SS-1 and -2 expression during an enteric infection in a natural host and provides further support for the concept of cytosolic S. Typhimurium in extruding epithelium as a mechanism for reseeding the lumen. >IMPORTANCE The pathogenic bacterium Salmonella enterica serovar Typhimurium invades and persists within host cells using distinct sets of virulence genes. Genes from Salmonella pathogenicity island 1 (SPI-1) are used to initiate contact and facilitate uptake into nonphagocytic host cells, while genes within SPI-2 allow the pathogen to colonize host cells. While many studies have identified bacterial virulence determinants in animal models of infection, very few have focused on virulence gene expression at the single-cell level during an in vivo infection. To better understand when and where bacterial virulence factors are expressed during an acute enteric infection of a natural host, we infected bovine jejunal-ileal loops with S. Typhimurium cells harboring fluorescent transcriptional reporters for SPI-1 and -2 (PinvF and PssaG, respectively). After a prescribed time of infection, tissue and luminal fluid were collected and analyzed by microscopy. During early infection (≤2?h), bacteria within both intact and compromised membrane-bound vacuoles were observed within the epithelium, with the majority expressing SPI-1. As the infection progressed, S. Typhimurium displayed differential expression of the SPI-1 and SPI-2 regulons, with the majority of tissue-associated bacteria expressing SPI-2 and the majority of lumen-associated bacteria expressing SPI-1. This underscores the finding that Salmonella virulence gene expression changes as the pathogen transitions from one anatomical location to the next.
机译：为了在肠腔和组织之间的感染周期中建立复制性生态位，肠病原体<肠>沙门氏菌血清型鼠伤寒需要大量毒力基因，包括两个III型分泌系统（T3SS）的基因及其同源效应子。为了更好地了解宿主-病原体之间的关系，包括自然宿主中细菌毒力程序的早期感染动力学和诱导动力学，我们使用荧光单细胞监测了T3SS-1和T3SS-2的亚细胞定位和时间表达记者在牛结扎回肠感染模型中进行了研究。我们观察到，感染后2小时的大多数细菌都是带鞭毛的，表达T3SS-1但不表达T3SS-2，并且与上皮细胞或挤出的肠上皮细胞有关。在上皮细胞中， S 。鼠伤寒细胞被完整的液泡膜包围或存在于膜受损的液泡中，液泡通常包含许多囊泡结构。感染后8小时，在固有层和下层粘膜中检测到表达T3SS-2的细菌，而在管腔中检测到T3SS-1的细菌。我们的工作首次确定了天然宿主肠道感染期间T3SS-1和-2表达的时空调节，并为细胞溶质 S 提供了进一步的支持。鼠伤寒菌在挤压上皮中作为再种管腔的一种机制。 >重要病原菌 Salmonella enterica 鼠伤寒沙门氏菌利用不同的毒力基因侵入并在宿主细胞中持续存在。沙门氏菌致病岛1（SPI-1）中的基因用于启动接触并促进非吞噬宿主细胞的摄取，而SPI-2中的基因则使病原体能够定居在宿主细胞中。尽管许多研究已经确定了感染动物模型中的细菌毒力决定因素，但很少有研究集中在体内感染过程中单细胞水平的毒力基因表达。为了更好地了解自然宿主急性肠感染期间何时以及在何处表达细菌毒力因子，我们用 S 感染了牛空肠回肠loop。携带SPI-1和-2（分别为P invF 和P ssaG ）的荧光转录报告基因的鼠伤寒细胞。在规定的感染时间后，收集组织和腔液并通过显微镜进行分析。在早期感染（≤2？h）期间，在上皮内观察到完整和受损的膜结合液泡内的细菌，其中大多数表达SPI-1。随着感染的进展， S 。鼠伤寒表现出SPI-1和SPI-2调节子的差异表达，大多数组织相关细菌表达SPI-2，大多数管腔相关细菌表达SPI-1。这强调了沙门氏菌毒力基因表达随病原体从一个解剖位置过渡到另一个解剖位置而改变的发现。

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