An advanced intestinal tissue model reveals host and pathogen strategies during Salmonella infection
Ontology highlight
ABSTRACT: Here, we introduce an intestinal tissue model to study human enteric infections. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. We use Dual RNA-seq to chart the communication amongst several different cell types at the intestinal barrier and the pathogen. The results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelial compartment. Our approach promises to reveal more human-specific infection strategies employed by Salmonella and other pathogens.
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 Homo sapiens
Project description:Salmonella is a human and animal pathogen causing gastro-enteric diseases worldwide. The key feature of Salmonella infection is its entry into intestinal epithelial cells within a Salmonella-Containing Vacuole (SCV). This original compartment is distinct from empty macropinosomes formed around the infection site. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. On the opposite, Salmonella induces the formation of elongated tubules leading to SCV membrane and volume loss. Later, the SCV can mature into a vacuolar pathogen niche, or be ruptured releasing Salmonella in the host cytosol where the bacteria hyper-replicates. Here, we describe how size control of the early SCV is the main contributor to its stability and consequently determines the Salmonella intracellular niche and growth. We identify the SNAREs required for increasing the SCV size through fusions. We show that this fusion promotes the maintenance of the SCV integrity and the establishment of a vacuolar niche
Project description:The intestinal epithelium is the first line of defence against invasive enteric pathogens. Removal of infected cells by exfoliation prevents mucosal translocation and systemic infection in the adult host, but is less commonly observed in the neonatal small intestine. Instead, here we describe non-professional efferocytosis of Salmonella-infected enterocytes by neighbouring intestinal epithelial cells in the neonatal intestine. Intestinal epithelial stem cell organoid co-cultures of neonatal and adult cell monolayers with damaged enterocytes replicated this observation, confirmed the age-dependent ability of intestinal epithelial cells for efferocytosis and identified the critical involvement of the 'eat-me' signals and adaptors phosphatidylserine and C1q as well as the 'eat-me' receptors integrin-v (CD51) and CD36 in cellular uptake. Consistent with this, massive epithelial cell membrane protrusions and CD36 accumulation at the contact site with apoptotic cells were observed in the infected neonatal host in vivo. Efferocytosis of infected small intestinal enterocytes by neighbouring epithelial cells may represent a previously unrecognised mechanism of neonatal antimicrobial host defense to maintain barrier integrity.
Project description:lysozyme-untreated Salmonella spent media and lysozyme-treated salmonella supernatant have different effects on intestinal epithelial permeability. To analyze the content and the different between the two supernatants, we did the proteomics to identify the proteins.
Project description:The enteric pathogen Salmonella enterica serovar Typhimurium infects humans and animals. Invasion of intestinal epithelial cells is an important step in gut colonization. Both the Salmonella invasion-associated Type III Secretion System 1 (T3SS1) and motility are required for efficient invasion. The master regulator HilD induces expression of T3SS1 and flagella genes, thus coordinating expression of invasion and motility. Here we show that HilD also induces smooth swimming and that this is important for invasion of epithelial cells, in vitro and in vivo. HilD-induced bacteria express the chemotaxis receptor McpC, which suppresses tumbling and increases smooth swimming, even in the absence of exogenous ligands. mcpC is repressed by H-NS binding, which can be displaced by HilD. This is the first time that bacteria have been shown to use the smooth swimming phenotype in order to optimize invasion.
Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonella’s close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique.
Project description:The purpose of this experiment was to identify intestinal epithelial responses to various strains of Salmonella enterica. Human intestinal organoids were infected with three serovars of Salmonella; Typhimurium, Enteritidis and Typhi, as well as type 3 secretion system -1 and -2 mutants in Typhimurium in order to identify host responses that were similar and unique to each serovar, and responses that were dependent on these secretion systems.
Project description:The immune response against pathogens involves multiple cell state transitions and complex gene expression changes. Here we established an in vivo single-cell nascent RNA labeling sequencing method (scnasRNA-seq) and applied it to survey time-resolved RNA dynamics during immune response to acute enteric infection with Salmonella. We showed that detection of nascent RNA synthesis reflects more realistic information on cell activation and gene transcription than total RNA level. Interplay of nascent RNA synthesis and RNA degradation together modulate dynamics of total RNA. We found that bone marrow macrophages are first primed at very early stage upon Salmonella infection. In contrast, the innate immune response of macrophages in intestine is limited. Notably, intestinal CD8+ T cells and plasma cells are rapidly and specifically activated at early stage post infection. Intestinal late enterocytes quickly express MHC-I molecules and present Salmonella antigen to CD8+ T cells for their activation, serving as antigen presenting cells for initiation of adaptive immunity. Our findings unveil novel RNA control strategies of immune cells and dynamic time course of immune response activation upon Salmonella infection, challenging the doctrine boundary between innate immunity and adaptive immunity against bacterial infection.
Project description:The immune response against pathogens involves multiple cell state transitions and complex gene expression changes. Here we established an in vivo single-cell nascent RNA labeling sequencing method (scnasRNA-seq) and applied it to survey time-resolved RNA dynamics during immune response to acute enteric infection with Salmonella. We showed that detection of nascent RNA synthesis reflects more realistic information on cell activation and gene transcription than total RNA level. Interplay of nascent RNA synthesis and RNA degradation together modulate dynamics of total RNA. We found that bone marrow macrophages are first primed at very early stage upon Salmonella infection. In contrast, the innate immune response of macrophages in intestine is limited. Notably, intestinal CD8+ T cells and plasma cells are rapidly and specifically activated at early stage post infection. Intestinal late enterocytes quickly express MHC-I molecules and present Salmonella antigen to CD8+ T cells for their activation, serving as antigen presenting cells for initiation of adaptive immunity. Our findings unveil novel RNA control strategies of immune cells and dynamic time course of immune response activation upon Salmonella infection, challenging the doctrine boundary between innate immunity and adaptive immunity against bacterial infection.
Project description:The purpose of this experiment was to identify the contribution of PMNs in intestinal epithelial responses to Salmonella infection.