Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids

“…These variations between serovars in early interactions with the host may direct the timing and magnitude of some aspects of the innate immune response, based on relative proportions of luminal to intracellular bacteria. Additionally, intracellular STM can be found in either the vacuole or the cytosol, and a recent study identified specific bacterial adaptations required for these two different intracellular lifestyles [33]. In the HIOs, we observed that some inflammatory pathways were decreased early during ST infection when compared to NTS.…”

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium

“…Previous studies have shown that a small subpopulation of hyper-replicating STM resides within the cytosol of epithelial cells and serves as a reservoir for dissemination. These motile bacteria retain both SPI-1 and flagella expression, but present a low expression of SPI-2 genes, and therefore are primed for invasion [65,87,88], and fuels high luminal density that facilitates fecal shedding [89]. This expression profile is somewhat similar to the identified intracellular profile of SPA.…”

“…Recently, a similar RNA-Seq approach was applied to determine the transcriptome of STM populations that are either free-living in the cytosol or residing within the SCV of HeLa cells 8 h p.i. [65]. Earlier studies have implemented a proteomic approach to determine the expression of STM proteins within HeLa cells at 6 [66] and 18 h [30] p.i.…”

Human variation impacting MCOLN2 restricts Salmonella Typhi replication by magnesium deprivation

“…Three potentially restricting conditions in the SCV lead to more PhoP activity: PhoP/Q is repressed by high Mg 2+ (18) and activated by cationic antimicrobial peptides (CAMPs) (19) or acidification (20,21). Since MCOLN2 is a divalent cation channel, PhoP/Q was most likely responding to reduced Mg 2+ concentrations, which along with Zn 2+ , are limited in SCVs (22,23). Indeed, the PhoP-activated Mg 2+ importers mgtA and mgtB were both upregulated more in bacteria inside wild-type THP-1s (Fig.…”

“…If knocking out MCOLN2 increases Mg 2+ availability, we theorized that these transporters would be necessary to uptake that extra Mg 2+ and therefore essential for the enhanced replication inside MCOLN2 -/- host cells. To test this, we generated a double knockout (Δ mgtA Δ mgtB ), which lacks the high affinity Mg 2+ importers used in low-Mg 2+ environments, like the ≤10μM concentration in the SCV ( 22 ). The double importer mutant is killed, instead of replicating, inside THP-1s ( Fig.…”