Spatial Segregation of Virulence Gene Expression during Acute Enteric Infection with Salmonella enterica serovar Typhimurium
Authors: Laughlin Richard C. , Knodler Leigh A. , Barhoumi Roula , Payne H. Ross , Wu Jing , Gomez Gabriel , Pugh Roberta , Lawhon Sara D. , Bäumler Andreas J. , Steele-Mortimer Olivia , Adams L. Garry 
Published: February 2014

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 (P
invF
and P
ssaG
, 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.