(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



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

['Tushun R. Powers', 'Paul G. Allen School For Global Health', 'College Of Veterinary Medicine', 'Washington State University', 'Pullman', 'Washington', 'United States Of America', 'Amanda L. Haeberle', 'Alexander V. Predeus', 'Institute Of Infection']

Date: 2021-09

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.

Intracellular pathogens reside either within a membrane-bound vacuole or are free-living in the cytosol and their virulence programs are tailored towards survival within a particular intracellular compartment. Some bacterial pathogens (such as Salmonella enterica) can successfully colonize both intracellular niches, but how they do so is unclear. Here we have exploited the parallel intracellular lifestyles of S. enterica in epithelial cells to identify the niche-specific bacterial expression profiles and environmental cues encountered by S. enterica. We have also discovered bacterial genes that are required for colonization of the cytosol, but not the vacuole. Our results advance our understanding of pathogen-adaptation to alternative replication niches and highlight an emerging concept in the field of bacteria-host cell interactions.

Funding: This work was supported by a Wellcome Trust Senior Investigator award (Grant 106914/Z/15/Z; https://wellcome.org ) to JCDH; NIH NIAID ( https://www.niaid.nih.gov/ ) T32 training grant AI007025 to TRP; start-up funds provided by the Paul G. Allen School for Global Health and intramural grants from WSU College of Veterinary Medicine (Mr. and Mrs. Delbert Caldwell Endowment and USDA NIFA Animal Health and Disease Funds) to LAK. LAK holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund ( https://www.bwfund.org/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Previous studies have largely investigated the infectious cycle of S. Typhimurium in epithelial cells by population-based analyses, which do not account for the heterogeneous population of intracellular bacteria. Only by determining the distinct responses of intracellular Salmonella to the cytosolic and the vacuolar niche can the infection biology of this important pathogen be accurately defined. Whilst the distinct milieus encountered within a vacuole versus the cytosol provide site-specific cues for Salmonella gene induction, little is known about what these cues might be, or which bacterial genes drive replication/survival in the cytosol. Using a combination of RNA-seq-based transcriptomics and single-cell microscopic analysis, here we describe niche-specific environments encountered by S. Typhimurium in epithelial cells and identify S. Typhimurium genes that are required for bacterial proliferation in the cytosolic compartment.

A specialized form of autophagy, called xenophagy, protects the mammalian cytosol by targeting intracellular pathogens to autophagosomes for their eventual degradation in lysosomes (reviewed in [ 22 ]). In fact, many of the components of the autophagic machinery have been identified using S. Typhimurium as a model pathogen. In the first report of autophagic recognition, Brumell and colleagues showed that a sub-population of internalized S. Typhimurium damage their nascent vacuole in epithelial cells in a T3SS1-dependent manner and these bacteria are decorated with the autophagy marker, microtubule-associated light chain-3 (LC3) [ 23 ]. A recently identified type III effector, SopF, limits the decoration of SCVs with LC3 [ 24 , 25 ]. Damage exposes host glycans restricted to the vacuole lumen to the cytosol, which are then recognized by β-galactoside-binding lectins, specifically galectin-3 (GAL3), GAL8 and GAL9 [ 26 ]. Galectin binding acts as an “eat me” signal that initiates a cascade of receptor binding, phagophore formation and tethering to the bacterium, culminating in autophagosome formation [ 26 , 27 ]. However, autophagic control of Salmonella is temporal and incomplete, at least in epithelial cells [ 12 , 23 ]. Furthermore, S. Typhimurium can also use autophagy to promote replication in the cytosol of epithelial cells [ 28 ] and repair damaged SCVs in mouse embryonic fibroblasts [ 29 ]. Autophagy therefore serves both a pro- and anti-bacterial role in S. enterica infections.

S. enterica resides within a membrane-bound vacuole within epithelial cells, fibroblasts, macrophages and dendritic cells (reviewed in [ 11 ]). However, the intracellular lifestyle of S. enterica differs between cell types, with a proportion of the total bacterial population living freely in the cytosol of epithelial cells, a phenomenon described for Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. Typhi infections in vitro and/or in vivo [ 12 – 17 ]. The eventual outcome of epithelial cells harboring cytosolic bacteria is their expulsion from the monolayer into the lumen of the gut and gall bladder [ 12 , 16 – 19 ], serving as a mechanism for bacterial spreading within and between hosts. Notably, the cytosol of macrophages is not permissive for S. Typhimurium replication [ 20 , 21 ], possibly due to nutrient limitation or enhanced host cell innate immune defenses such as autophagy and/or inflammasomes [ 21 ]. Considering that the site of intracellular replication is cell-type restricted, we propose that S. enterica is an “opportunistic” cytosolic pathogen.

Of the foodborne bacterial, protozoal and viral diseases, non-typhoidal Salmonella enterica (NTS) cause the largest burden of illness and death worldwide [ 4 ]. Infection can cause either a self-limiting gastroenteritis or a life-threatening, invasive disease (invasive non-typhoidal salmonellosis) in immunocompromised individuals, which is particularly a public health problem in sub-Saharan Africa and south-east Asia [ 5 ]. Upon ingestion of contaminated food or water, S. enterica enters epithelial cells lining the gut and resides within a membrane-bound compartment derived from the endocytic pathway [ 6 , 7 ], the Salmonella-containing vacuole (SCV). Entry into non-phagocytic cells is largely governed by a type III secretion system, T3SS1, that is encoded by Salmonella pathogenicity island (SPI) 1. The T3SS acts as a molecular syringe to inject bacterial effector proteins across the eukaryotic cell plasma membrane to modulate host signaling networks that induce rearrangement of the actin cytoskeleton, leading to the formation of plasma membrane “ruffles” and engulfment of bacteria (reviewed in [ 8 ]). Establishment and maintenance of the SCV is dependent upon effectors being translocated across the vacuolar membrane by T3SS1 and another T3SS, T3SS2, which is specifically induced by environmental cues sensed by bacteria within the SCV lumen [ 9 , 10 ]. T3SS2 is also important for survival within phagocytic cells, which S. enterica encounters in the lamina propria during an enteric infection or in the mesenteric lymph nodes, reticuloendothelial tissues (liver and spleen) and circulating blood during invasive disease.

There are two major niches in which intracellular bacteria survive and proliferate after internalization into host cells, confined within a membrane-bound vacuole or free-living within the cytosol. Different pathogenic mechanisms are required to occupy these diverse environments. While bacterial pathogens have been historically categorized as being either vacuolar or cytosolic, it has recently been realized that some bacteria can occupy both niches, often in a cell-type specific manner. Examples are Salmonella enterica, Mycobacterium tuberculosis and Listeria monocytogenes [ 1 – 3 ]. What is unclear is how bacteria that are adapted to survive within one intracellular niche can successfully colonize a distinct cellular compartment.

Results

Modulation of autophagy affects bacterial proliferation in the epithelial cell cytosol We hypothesized that the modulation of autophagy in epithelial cells would selectively perturb the cytosolic, and not vacuolar, proliferation of S. Typhimurium. Autophagy levels can be manipulated by pharmacological or genetic means (reviewed in [30]). To test our hypothesis, we used nutrient starvation to upregulate autophagy and the class III phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin (WTM), to inhibit autophagy (Fig 1A). To enumerate cytosolic bacteria after autophagy activation/inhibition, we used the digitonin permeabilization assay [12,13] to label the bacteria accessible to cytosol-delivered anti-S. Typhimurium lipopolysaccharide (LPS) antibodies at the early stages of infection (15 min– 3 h p.i., Fig 1B). Wild-type bacteria were constitutively expressing mCherry from a plasmid, pFPV-mCherry. In untreated cells (basal levels of autophagy), 6.8% of bacteria were accessible to the cytosol as early as 15 min p.i. This proportion increased to ~20% by 45 min p.i. and remained at a steady-state thereafter (Fig 1B). Treatment with Earle’s balanced salt solution (EBSS), i.e. starvation-induced autophagy, reduced the fraction of bacteria that were accessible to the cytosol at 45 min, 90 min and 180 min p.i. (Fig 1B), consistent with enhanced autophagic capture of bacteria in, or repair of, damaged vacuoles. In contrast, inhibition of autophagy (WTM) increased the proportion of cytosolic bacteria at all timepoints examined (Fig 1B), in agreement with previous findings [14,31]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. EBSS and wortmannin inversely affect the cytosolic population. (A) Schematic of the experimental design employed to differentially modulate host cell autophagy. (B) Epithelial cells seeded on glass coverslips were pretreated with EBSS or 100 nM WTM as depicted in (A) and infected with S. Typhimurium wild-type bacteria harboring pFPV-mCherry for plasmid-borne, constitutive expression of mCherry. The proportion of bacteria accessible to anti-Salmonella LPS antibodies delivered to the mammalian cell cytosol was determined by digitonin permeabilization assay and fluorescence microscopy. n≥3 independent experiments. (C) Epithelial cells seeded on coverslips were pretreated as in (A) and infected with mCherry-S. Typhimurium (wild-type glmS::Ptrc-mCherryST::FRT bacteria constitutively expressing a chromosomal copy of mCherry) harboring the fluorescent reporter plasmid, PuhpT-gfpova (pNF101). At the indicated times, cells were fixed and the proportion of infected cells containing cytosolic bacteria (GFP-positive) was scored by fluorescence microscopy. The mean from each experiment is represented as a large dot (n = 3). (D) Cells seeded on coverslips were pretreated as in (A) and infected with wild-type bacteria harboring pFPV-mCherry. The number of bacteria in each infected cell was scored by fluorescence microscopy. Cells with ≥100 bacteria contain cytosolic S. Typhimurium. Each small dot represents one infected cell. n = 2 (1 h) or 3 (8 h) experiments. (E) Epithelial cells were infected with wild-type bacteria and the number of intracellular bacteria at 1 h and 8 h p.i. was determined by gentamicin protection assay. Fold-replication is CFUs at 8 h/1 h. n = 3 independent experiments. The mean from each experiment is represented as a large dot (n = 3). For all panels, control = untreated cells; EBSS = Earle’s balanced salt solution treatment; WTM = wortmannin treatment. Asterisks indicate data significantly different from control (ANOVA with Dunnett’s post-hoc test, p<0.05). https://doi.org/10.1371/journal.ppat.1009280.g001 To verify cytosolic localization, we used a transcriptional reporter plasmid, pNF101, that only expresses gfp-ova in the sub-population of S. Typhimurium that are in damaged vacuoles and/or free in the cytosol. Epithelial cells were infected with mCherry-S. Typhimurium (S. Typhimurium wild-type bacteria constitutively expressing mCherry on the chromosome) harboring pNF101. In untreated cells, 35% and 13% of infected cells harbored GFP-positive (cytosolic) bacteria at 2 h and 8 h p.i., respectively (Fig 1C). EBSS treatment lowered this proportion at both timepoints to 23% and 8%, respectively. Conversely, WTM treatment increased the level of infected cells with cytosolic bacteria at both 2 h and 8 h p.i. (79% and 48%). To quantify the niche-specific effects of EBSS and WTM treatments on bacterial replication, epithelial cells were infected with S. Typhimurium harboring pFPV-mCherry and the number of bacteria per cell was scored. Cytosolic S. Typhimurium that evade autophagic clearance in epithelial cells replicate at a much faster rate than vacuolar bacteria [12,13,32], eventually occupying the entire cytosolic space (≥100 bacteria/cell). By contrast, vacuolar bacteria display low to moderate replication (2–40 bacteria/cell). Comparing the three treatment conditions, the incidence of infected cells with ≥100 bacteria at 8 h p.i. (8.2±1.6% in control, n = 3 experiments) was reduced by EBSS treatment (1.2±2.0%) and promoted by WTM treatment (16±5.4%) (Fig 1D). A similar trend was seen with the chloroquine (CHQ) resistance assay, which quantifies the proportion of cytosolic bacteria in the total population—51±8.7%, 28±14% and 65±10% (n = 4 experiments) of intracellular bacteria were cytosolic at 8 h p.i. in untreated, EBSS-treated and WTM-treated cells, respectively. Consequently, total bacterial proliferation was restricted and promoted by EBSS and WTM treatment, respectively, compared to untreated cells (Fig 1E). Importantly, neither bacterial invasion at 1 h p.i. nor vacuolar replication at 8 h p.i. was overtly affected (Fig 1D). Collectively, these data confirm that modulation of autophagy selectively affects the proliferation of S. Typhimurium in the cytosol of epithelial cells.

Extraneous genes that are induced in the epithelial cytosol Several osmotically-sensitive genes that are up-regulated when S. Typhimurium is exposed to NaCl shock [34] were identified as candidate cytosol-induced genes, namely cysP (cysPUWA operon), soxS and proV (proVWX operon) (S1 Dataset). We qualitatively and quantitatively confirmed the cytosol-specific expression of cysP and soxS using transcriptional reporters (S8 Fig). However, the proV-gfpmut3 transcriptional fusion was not up-regulated in the cytosol (S4 Fig). The cysPUWA operon encodes a sulfate/thiosulfate permease and periplasmic binding protein [81]. SoxS is a member of the AraC/XylS family of transcriptional regulators that is required for bacterial resistance to oxidative stress [82,83]. Six additional S. Typhimurium genes were identified as being up-regulated in the cytosol according to the transcriptomics data, namely uhpT, sfbA (sfbABC operon), fruB (fruBKA operon), grxA, mtr and trpE (trpEDCBA operon) (S1 Dataset). All six genes are constitutively expressed in infection-relevant broth conditions [34] and sfbA, mtr, trpE and fruB are also up-regulated upon S. Typhimurium infection of macrophages ([43]; S1 Dataset). Infection of epithelial cells with mCherry-S. Typhimurium harboring PuhpT-gfpmut3, PsfbA-gfpmut3, PgrxA-gfpmt3 and PfruB-gfpmut3 confirmed the RNA-seq-based prediction of their induction in the cytosol (S8 Fig). However, mtr and trpE were not induced in the cytosol (S4 Fig). UhpT is a hexose phosphate transporter whose induction has been reported previously for cytosolic S. Typhimurium [36]. fruBKA, known as the fructose operon, encodes for three enzymes involved in fructose uptake [84]. GrxA, or glutaredoxin 1 (Grx1), is a redox enzyme that detoxifies oxidizing agents such as reactive oxygen species (ROS), thereby defending against oxidative stress [85]. The sfbABC operon is predicted to encode a periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC [86].

[END]

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009280

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/