The tumour-associated microbiota is an intrinsic component of the tumour microenvironment across human cancer types
. Intratumoral host-microbiota studies have so far largely relied on bulk tissue ...analysis
, which obscures the spatial distribution and localized effect of the microbiota within tumours. Here, by applying in situ spatial-profiling technologies
and single-cell RNA sequencing
to oral squamous cell carcinoma and colorectal cancer, we reveal spatial, cellular and molecular host-microbe interactions. We adapted 10x Visium spatial transcriptomics to determine the identity and in situ location of intratumoral microbial communities within patient tissues. Using GeoMx digital spatial profiling
, we show that bacterial communities populate microniches that are less vascularized, highly immuno‑suppressive and associated with malignant cells with lower levels of Ki-67 as compared to bacteria-negative tumour regions. We developed a single-cell RNA-sequencing method that we name INVADEseq (invasion-adhesion-directed expression sequencing) and, by applying this to patient tumours, identify cell-associated bacteria and the host cells with which they interact, as well as uncovering alterations in transcriptional pathways that are involved in inflammation, metastasis, cell dormancy and DNA repair. Through functional studies, we show that cancer cells that are infected with bacteria invade their surrounding environment as single cells and recruit myeloid cells to bacterial regions. Collectively, our data reveal that the distribution of the microbiota within a tumour is not random; instead, it is highly organized in microniches with immune and epithelial cell functions that promote cancer progression.
Type VI secretion systems (T6SSs) translocate effectors into target cells and are made of a contractile sheath and a tube docked onto a multi-protein transmembrane complex via a baseplate. Although ...some information is available about the mechanisms of tail contraction leading to effector delivery, the detailed architecture and function of the baseplate remain unknown. Here, we report the 3.7 Å resolution cryo-electron microscopy reconstruction of an enteroaggregative Escherichia coli baseplate subcomplex assembled from TssK, TssF and TssG. The structure reveals two TssK trimers interact with a locally pseudo-3-fold symmetrical complex comprising two copies of TssF and one copy of TssG. TssF and TssG are structurally related to each other and to components of the phage T4 baseplate and of the type IV secretion system, strengthening the evolutionary relationships among these macromolecular machines. These results, together with bacterial two-hybrid assays, provide a structural framework to understand the T6SS baseplate architecture.
Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule ...inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, including Escherichia coli, that are resistant to 5-FU. Further, CRC E. coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that ex vivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.
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•5-Fluorouracil (5-FU) inhibits growth of the oncomicrobe Fusobacterium nucleatum•CRC strains of Escherichia coli rapidly modify 5-FU into a nontoxic product•Bacterially modified 5-FU no longer inhibits cancer or F. nucleatum cell growth•50% of patient-derived ex vivo CRC microbial communities can reduce 5-FU levels
LaCourse et al. show that Fusobacterium nucleatum, a bacterium associated with colorectal cancer, is inhibited by the chemotherapeutic 5-fluorouracil, suggesting that 5-fluorouracil efficacy may in part be due to its antimicrobial activity. Specific intratumoral microbiota members modify 5-fluorouracil into a nontoxic form, rescuing Fusobacterium nucleatum and cancer epithelial cell growth.
The contribution of the microbiota to disease progression and treatment efficacy is often neglected when determining who is at the highest risk of developing gastrointestinal cancers or designing ...treatment strategies for patients. We reviewed the current literature on the effect of the human microbiota on cancer risk, prognosis, and treatment efficacy. We highlight emerging research that seeks to identify microbial signatures as biomarkers for various gastrointestinal cancers, and discuss how we could harness knowledge of the microbiome to detect, prevent, and treat these cancers. Finally, we outline further research needed in the field of gastrointestinal cancers and the microbiota, and describe the efforts required to increase the accuracy and reproducibility of data linking the microbiome to cancer.
Single-cell RNA sequencing (scRNAseq) technologies have been beneficial in revealing and describing cellular heterogeneity within mammalian tissues, including solid tumors. However, many of these ...techniques apply poly(A) selection of RNA, and thus have primarily focused on determining the gene signatures of eukaryotic cellular components of the tumor microenvironment. Microbiome analysis has revealed the presence of microbial ecosystems, including bacteria and fungi, within human tumor tissues from major cancer types. Imaging data have revealed that intratumoral bacteria may be located within epithelial and immune cell types. However, as bacterial RNA typically lacks a poly(A) tail, standard scRNAseq approaches have limited ability to capture this microbial component of the tumor microenvironment. To overcome this, we describe the invasion-adhesion-directed expression sequencing (INVADEseq) approach, whereby we adapt 10x Genomics 5' scRNAseq protocol by introducing a primer that targets a conserved region of the bacterial 16S ribosomal RNA gene in addition to the standard primer for eukaryotic poly(A) RNA selection. This 'add-on' approach enables the generation of eukaryotic and bacterial DNA libraries at eukaryotic single-cell level resolution, utilizing the 10x barcode to identify single cells with intracellular bacteria. The INVADEseq method takes 30 h to complete, including tissue processing, sequencing and computational analysis. As an output, INVADEseq has shown to be a reliable tool in human cancer cell lines and patient tumor specimens by detecting the proportion of human cells that harbor bacteria and the identities of human cells and intracellular bacteria, along with identifying host transcriptional programs that are modulated on the basis of associated bacteria.
Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals
, is enriched in human colorectal cancer (CRC) ...tumours
. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis
. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.
Bacteria in polymicrobial habitats contend with a persistent barrage of competitors, often under rapidly changing environmental conditions
. The direct antagonism of competitor cells is thus an ...important bacterial survival strategy
. Towards this end, many bacterial species employ an arsenal of antimicrobial effectors with multiple activities; however, the benefits conferred by the simultaneous deployment of diverse toxins are unknown. Here we show that the multiple effectors delivered to competitor bacteria by the type VI secretion system (T6SS) of Pseudomonas aeruginosa display conditional efficacy and act synergistically. One of these effectors, Tse4, is most active in high-salinity environments and synergizes with effectors that degrade the cell wall or inactivate intracellular electron carriers. We find Tse4 synergizes with these disparate mechanisms by forming pores that disrupt the ΔΨ component of the proton motive force. Our results provide evidence that the concomitant delivery of a cocktail of effectors serves as a bet-hedging strategy to promote bacterial competitiveness in the face of unpredictable and variable environmental conditions.
Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, ...whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that
possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in
that reveal three conserved and widespread interbacterial antagonism resistance clusters (
). We find that although
are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of
to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.
We report the complete genome sequence of Morganella morganii CTX51T, a strain isolated from the resected tumor of a patient with cecal colorectal adenocarcinoma of the cecum. The genome comprises a ...circular chromosome of 4.19 Mbp, with an overall GC content of 50.4% and one circular plasmid of 8.48 kbp.
We report the complete genome sequence of Clostridium cadaveris IFB3C5, a strain isolated from the resected tumor of a treatment naive colorectal cancer patient. This genome is comprised of a ...singular chromosome of approximately 3.63 Mbp in length, contains two plasmids, and has an overall mean GC content of 31.7%.
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