Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT ...(microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000
cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.
Colorectal cancer (CRC) is a significant public health problem accounting for about 10% of all new cancer cases globally. Though genetic and epigenetic factors influence CRC, the gut microbiota acts ...as a significant component of the disease's etiology. Further research is still needed to clarify the specific roles and identify more bacteria related to CRC development. This review aims to provide an overview of the "driver-passenger" model of CRC. The colonization and active invasion of the "driver(s)" bacteria cause damages allowing other commensals, known as "passengers," or their by-products, i.e., metabolites, to pass through the epithelium . This review will not only focus on the species of bacteria implicated in this model but also on their biological functions implicated in the occurrence of CRC, such as forming biofilms, mucus, penetration and production of enterotoxins and genotoxins.
Tightly regulated immune responses must occur in the intestine to avoid unwanted inflammation, which may cause chronic sequela leading to diseases such as colorectal cancer. Toll-like receptors play ...an important role in preventing aberrant immune responses in the intestine by sensing endogenous commensal microbiota and delivering important regulatory signals to the tissue. However, the role that specific innate receptors may play in the development of chronic inflammation and their impact on the composition of the colonic microbiota is not well understood. Using a model of inflammation-induced colorectal cancer, we found that
species are lost more quickly in wild-type (WT) mice than TLR6-deficient mice resulting in overall differences in bacterial composition. Despite the longer retention of
, the TLR6-deficient mice presented with more tumors and a worse overall outcome. Restoration of the lost
species suppressed inflammation, reduced tumor number, and prevented change in the abundance of Proteobacteria only when given to WT mice, indicating the effect of these
are TLR6 dependent. We found that the TLR6-dependent effects of
could be dissociated from one another via the involvement of IL10, which was necessary to dampen the inflammatory microenvironment, but had no effect on bacterial composition. Altogether, these data suggest that innate immune signals can shape the composition of the microbiota under chronic inflammatory conditions, bias the cytokine milieu of the tissue microenvironment, and influence the response to microbiota-associated therapies.
The balance between regulatory and inflammatory immune responses is critical to maintain intestinal homeostasis. Furthermore, the nature of the inflammatory response needs to be tailored to the ...tissue to provide proper protective immunity while preserving host integrity. TLR2 (Toll-like receptor 2) is a unique TLR in that it has been shown to promote regulatory and inflammatory T cell responses. Using Yersinia enterocolitica, we show that oral infection promotes TH17 immunity, whereas systemic infection promotes TH1 immunity. Furthermore, induction of TH17 immunity during oral infection is dependent on TLR1 and results from the combinatorial effect of TLR2/TLR1-induced IL-6 and IL-23 and the presence of TGF-β in the intestinal environment. Interestingly, TLR2/TLR1 was not involved in TH1 immune responses during systemic infection, whereas the TLR2/TLR6 receptor complex induced IL-10+ regulatory T cell responses during both systemic and oral infections. Our results reveal that the route of infection is central in determining which pathways provide protective immunity. Furthermore, they also demonstrate that TLR2 has dual immune functions in the gut and identify TLR1 as a critical innate receptor for protective intestinal TH17 immunity.
The plague is caused by the bacterium Yersinia pestis. Plague bacteria are thought to inject effector Yop proteins into host cells via the type III pathway. The identity of the host cells targeted ...for injection during plague infection is unknown. We found, using Yop {szligbeta}-lactamase hybrids and fluorescent staining of live cells from plague-infected animals, that Y. pestis selected immune cells for injection. In vivo, dendritic cells, macrophages, and neutrophils were injected most frequently, whereas B and T lymphocytes were rarely selected. Thus, it appears that Y. pestis disables these cell populations to annihilate host immune responses during plague.
There is cross-talk between the intestinal epithelium and the microbiota that functions to maintain a tightly regulated microenvironment and prevent chronic inflammation. This communication is partly ...mediated through the recognition of bacterial proteins by host-encoded innate receptors, such as TLRs. However, studies examining the role of TLR signaling on colonic homeostasis have given variable and conflicting results. Despite its critical role in mediating immunity during enteric infection of the small intestine, TLR1-mediated recognition of microbiota-derived ligands and their influence on colonic homeostasis has not been well studied. In this study, we demonstrate that defective TLR1 recognition of the microbiome by epithelial cells results in disruption of crypt homeostasis specifically within the secretory cell compartment, including a defect in the mucus layer, ectopic Paneth cells in the colon, and an increase in the number of rapidly dividing cells at the base of the crypt. As a consequence of the perturbed epithelial barrier, we found an increase in mucosal-associated and translocated commensal bacteria and chronic low-grade inflammation characterized by an increase in lineage-negative Sca1
Thy1
innate lymphoid-like cells that exacerbate inflammation and worsen outcomes in a model of colonic injury and repair. Our findings demonstrate that sensing of the microbiota by TLR1 may provide key signals that regulate the colonic epithelium, thereby limiting inflammation through the prevention of bacterial attachment to the mucosa and exposure to the underlying immune system.
Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these ...microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.
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•Differences in microbiota found between patients with polyps and between polyp subtypes•NTBF is enriched in patients with polyps•NTBF from polyps is enriched for LPS biosynthesis genes, activates TLR4, and induces IL-12•Presence of LPS genes and elevated expression of LPS are found in polyp tissues
Kordahi and colleagues find that the common commensal bacteria, nontoxigenic B. fragilis (NTBF), is enriched in patients with precancerous colonic polyps. NTBF isolated from polyps is enriched in genes involved in LPS biosynthesis, which may allow for its increased ability to activate the immune system and cause inflammation.
The balance between regulatory and inflammatory immune responses is critical to maintain intestinal homeostasis. Furthermore, the nature of the inflammatory response needs to be tailored to the ...tissue to provide proper protective immunity while preserving host integrity. TLR2 (Toll-like receptor 2) is a unique TLR in that it has been shown to promote regulatory and inflammatory T cell responses. Using Yersinia enterocolitica, we show that oral infection promotes T(H)17 immunity, whereas systemic infection promotes T(H)1 immunity. Furthermore, induction of T(H)17 immunity during oral infection is dependent on TLR1 and results from the combinatorial effect of TLR2/TLR1-induced IL-6 and IL-23 and the presence of TGF-β in the intestinal environment. Interestingly, TLR2/TLR1 was not involved in T(H)1 immune responses during systemic infection, whereas the TLR2/TLR6 receptor complex induced IL-10(+) regulatory T cell responses during both systemic and oral infections. Our results reveal that the route of infection is central in determining which pathways provide protective immunity. Furthermore, they also demonstrate that TLR2 has dual immune functions in the gut and identify TLR1 as a critical innate receptor for protective intestinal T(H)17 immunity.
The intestine is a complex organ that must maintain tolerance to innocuous food antigens and commensal microbiota while being also able to mount inflammatory responses against invading pathogenic ...microorganisms. The ability to restrain tolerogenic responses while permitting inflammatory responses requires communication between commensal bacteria, intestinal epithelial cells and immune cells. Disruption or improper signaling between any of these factors may lead to uncontrolled inflammation and the development of inflammatory diseases. Toll-like receptors (TLR) recognize conserved molecular motifs of microorganisms and, not surprisingly, are important for maintaining tolerance to commensal microbiota, as well as inducing inflammation against pathogens. Perturbations in individual TLR signaling can lead to a number of different outcomes and illustrate a system of regulation within the intestine in which each TLR plays a largely non-redundant role in mucosal immunity. This review will discuss recent findings on the roles of individual TLRs and intestinal homeostasis.
Educating dendritic cells (DC) to become tolerogenic DC, which promote regulatory IL-10 immune responses, represents an effective immune evasion strategy for pathogens. Yersinia pestis virulence ...factor LcrV is reported to induce IL-10 production via interaction with Toll-like receptor (TLR) 2. However, TLR2-/- mice are not protected against subcutaneous plague infection. Using complementary in vitro and in vivo approaches and LcrV as a model, we show that TLR6 associates with TLR2 to induce tolerogenic DC and regulatory type-1 T cells selectively secreting IL-10. In contrast, TLR1 heterodimerizes with TLR2 to promote proinflammatory IL-12p40 cytokine, producing DC and inflammatory T cell differentiation. LcrV specifically hijacks the TLR2/6 pathway to stimulate IL-10 production, which blocks host protective inflammatory responses. These results explain why TLR2 can mediate both pro- and anti-inflammatory responses and identify TLR6 as a distinct receptor driving regulatory IL-10 responses.
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