Genome Expression Analysis of Nonproliferating Intracellular Salmonella enterica Serovar Typhimurium Unravels an Acid pH-Dependent PhoP-PhoQ Response Essential for Dormancy

Abstract

Genome-wide expression analyses have provided clues on how Salmonella proliferates inside cultured macrophages and epithelial cells. However, in vivo studies show that Salmonella does not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined. In vitro infection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurring in vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in which S. enterica serovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model that S. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ϳ2% (98 genes) of the S. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate that S. Typhimurium restrains intracellular growth in vivo and support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.

Intracellular Salmonella induces aggrephagy of host endomembranes in persistent infections

“…The S. enterica serovar Typhimurium strain SV5015 used in this study, described in the fibroblast infection model, 18 is a His C derivative of the mouse virulent strain SL1344. 61 Derivative SV5015 strains were generated bearing plasmids pC.IGdsRed33 and pFPVmcherry 38 for constitutive production of fluorescent proteins DsRed and mCherry, respectively.…”

“…15 S. Typhimurium displays similar limited growth capacity in cultured fibroblasts 16,17 and in vivo inside nonphagocytic stromal cells located in the intestinal lamina propria. 18 Conversely, S. Typhimurium proliferates actively following entry into cultured epithelial cells. 19 Most reports using cultured epithelial cell lines describe net increases in intracellular bacterial loads up to 30-to 40-fold.…”

Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

“…These findings suggest that crosstalk between SPI-1 and SPI-2 could also occur in intracellular bacteria, although this aspect remains to be demonstrated. More recent studies have shown that intracellular bacteria express SPI-1 genes and protein effectors translocated by the TTSS encoded by this island (25)(26)(27)(28)(29). Representative examples are the SPI-1 effector proteins SopB and SipA.…”

“…Thus, most of these bacteria remain in a nongrowing state, reaching an average progeny of 3 to 4 cells per infected fibroblast at late infection times (36). In vivo, the PhoP-PhoQ system was reported to attenuate growth of intracellular bacteria located in nonphagocytic stromal cells present in the lamina propria of the mouse intestine (29). Recently, bacteria located inside phagocytes present in mouse organs have been shown to reach high numbers in the absence of SPI-2 (37).…”

Single-cell analysis: Understanding infected cell heterogeneity

“…11 Low numbers of Salmonella have also been found in nonphagocytic stromal cells of the intestinal lamina propria that are probably fibroblasts. 12 In this cell type Salmonella actively restrain their proliferation. Carrier state infections are commonly observed in human and domestic fowl following an acute phase.…”