Formation Of Salmonella-containing Vacuole Research Articles

Salmonella Typhimurium induces genome-wide expression and phosphorylation changes that modulate immune response, intracellular survival and vesicle transport in infected neutrophils

Salmonella Typhimurium is a food-borne pathogen that causes salmonellosis. When in contact with the host, neutrophils are rapidly recruited to act as first line of defense. To better understand the pathogenesis of this infection, we used an in vitro model of neutrophil infection to perform dual RNA-sequencing (both host and pathogen). In addition, and given that many pathogens interfere with kinase‐mediated phosphorylation in host signaling, we performed a phosphoproteomic analysis. The immune response was overall diminished in infected neutrophils, mainly JAK/STAT and toll-like receptor signaling pathways. We found decreased expression of proinflammatory cytokine receptor genes and predicted downregulation of the mitogen-activated protein (MAPK) signaling pathway. Also, Salmonella infection inhibited interferons I and II signaling pathways by upregulation of SOCS3 and subsequent downregulation of STAT1 and STAT2. Additionally, phosphorylation of PSMC2 and PSMC4, proteasome regulatory proteins, was decreased in infected neutrophils. Cell viability and survival was increased by p53 signaling, cell cycle arrest and NFkB-proteasome pathways activation. Combined analysis of RNA-seq and phosphoproteomics also revealed inhibited vesicle transport mechanisms mediated by dynein/dynactin and exocyst complexes, involved in ER-to-Golgi transport and centripetal movement of lysosomes and endosomes. Among the overexpressed virulence genes from Salmonella we found potential effectors responsible of these dysregulations, such as spiC, sopD2, sifA or pipB2, all of them involved in intracellular replication. Our results suggest that Salmonella induces (through overexpression of virulence factors) transcriptional and phosphorylation changes that increases neutrophil survival and shuts down immune response to minimize host response, and impairing intracellular vesicle transport likely to keep nutrients for replication and Salmonella-containing vacuole formation and maintenance.

Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle.

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while many S. enterica serovar Typhimurium (STM) strains have a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies single cell analyses approaches, which are flow cytometry of Salmonella harboring dual fluorescent protein reporters, effector translocation, and correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyostelium discoideum.

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.

Tracking Pathogen Infections by Time-Resolved Chemical Proteomics.

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time-resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host-cell proteins at different time points initiated by UV for label-free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella-containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.

Salmonella-containing vacuole development in avian cells and characteristic of cigR in Salmonella enterica serovar Pullorum replication within macrophages

Salmonella enterica serovar Pullorum (S. Pullorum) is one of the host-restricted serotypes causing systemic infection in poultry. Survival in macrophages is one of the mechanisms underlying the persistent infection of S. Pullorum in hosts. Formation of Salmonella-containing vacuole (SCV) is essential for bacteria to be concealed in macrophages. In this study, confocal microscopy was applied to detect the SCV development by S. Pullorum in the chicken hepatocellular carcinoma cell line LMH and the macrophage cell line HD-11, respectively. The results showed that macrophages were more appropriate for the SCV maturation during S. Pullorum infection compared to epithelial cells. We evaluated the role of the CigR effector protein in the formation of SCVs. CigR is a membrane-binding protein, which is one of the type III secretion system 2 (T3SS2) effectors encoded within Salmonella pathogenicity island 3 (SPI3). The deletion of cigR in S. Typhimurium and S. Pullorum enhanced bacterial virulence to both mice and chickens. To analyze the influence of CigR on the SCV development during S. Pullorum infection, this study compared the formation of SCVs by using S. Pullorum C79-13 and the cigR deleted strain C79-13ΔcigR. Compared to the wild type strain, the loss of cigR gene in strain C79-13ΔcigR caused a four-fold increase in the recruitment ability of the SCV marker protein Rab7 in infected macrophages after 30 min, 5 h, and 16 h post-infection. In addition, infection and proliferation of strain C79-13ΔcigR in the avian macrophage cell line HD-11 was higher than that of the wild type strain. Our findings suggest that CigR is an anti-virulence effector inhibiting replication of S. Pullorum and SCV development in macrophages.

The entry of Salmonella in a distinct tight compartment revealed at high temporal and ultrastructural resolution.

Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.

The Critical Role of Membrane Cholesterol in Salmonella-Induced Autophagy in Intestinal Epithelial Cells

It was previously observed that plasma membrane cholesterol plays a critical role in the Salmonella-induced phosphatidylinositol 3-kinase-dependent (PI3K)-dependent anti-inflammatory response in intestinal epithelial cells (IECs). The PI3K/Akt pathway is associated with autophagy which has emerged as a critical mechanism of host defense against several intracellular bacterial pathogens. Plasma membrane contributes directly to the formation of early Atg16L1-positive autophagosome precursors. Therefore, this study aimed to investigate the role of plasma membrane cholesterol on the Salmonella-induced autophagy in IECs. By using methyl-beta-cyclodextrin (MBCD), it was demonstrated that disruption of membrane cholesterol by MBCD enhanced NOD2 and Atg16L1 proteins expression in membrane, and autophagic LC3II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells, which was counteracted by Atg16L1 siRNA. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) siRNA enhanced the Salmonella-induced activation of Akt in Caco-2 cells. However, inhibitors of Akt or extracellular signal-regulated kinases (ERK) had no significant effect on Salmonella-induced autophagy Beclin 1 or LC3 proteins expression. In conclusion, our study suggests that cholesterol accumulation in the plasma membrane at the entry site of Salmonella results in the formation of Salmonella-containing vacuole (SCV) and decreased autophagy. Our results offer mechanistic insights on the critical role of membrane cholesterol in the pathogenesis of Salmonella infection in intestinal epithelial cells and the therapeutic potential of its antagonists.

Involvement of TIP60 acetyltransferase in intracellular Salmonellareplication

BackgroundSalmonella enterica is a facultative intracellular pathogen that replicates within a membrane-bound compartment termed Salmonella containing vacuole (SCV). The biogenesis of SCV requires Salmonella type III protein secretion/translocation system and their effector proteins which are translocated into host cells to exploit the vesicle trafficking pathways. SseF is one of these effectors required for SCV formation and Intracellular Salmonella replication through unknown mechanisms.ResultsIn an attempt to identify host proteins that interact with SseF, we conduct a yeast two-hybrid screening of human cell cDNA library using SseF as the bait. We identified that TIP60, an acetyltransferase, interacts with SseF. We showed that the TIP60 acetylation activity was increased in the presence of SseF, and TIP60 was upregulated upon Salmonella infection. In addition, TIP60 is required for efficient intracellular Salmonella replication in macrophages.ConclusionTaken together, our data suggest that Salmonella may use SseF to exploit the host TIP60 acetyltransferase activity to promote efficient Salmonella replication inside host cells.