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Microbial interactions shape the composition and stability of the human gut microbiome. Yet, few studies have systematically investigated species-species growth interactions and the mechanisms behind these. Here we show that among 36 representative gut bacterial strains, when two species interact, the interactions are mostly inhibitory. To provide biological insight into specific interactions, we further investigate the basis of a positive interaction, showing that Clostridium perfringens promotes the growth of Mediterraneibacter gnavus through extracellular vesicles. Additionally, we identify Veillonella parvula as a species capable of modulating environmental pH, thereby enabling the growth of Parabacteroides merdae, a strain highly sensitive to acidic conditions. This pH-increasing effect is enhanced by guanine supplementation and persists in multi-species communities containing different pH-lowering strains from diverse bacterial phyla. Although V. parvula is commonly present in human gut microbiomes, it is generally found at low levels. Given the spatial organization of bacteria in the gut, the local pH modulation by V. parvula might support the growth of acid-sensitive strains. Overall, the comprehensive dataset and mechanistic insights presented here provide a starting point to predict microbiome composition by integrating growth interactions.

Colorectal cancer (CRC), the third-most prevalent and second deadliest cancer, requires new therapeutic strategies due to the significant side effects of current treatments. We investigated the anticancer properties of MakA, a cytotoxin from Vibrio cholerae, administered systemically in a mouse model, with a focus on its impact on the tumor microenvironment (TME) and immune cell infiltration. Our findings demonstrate that MakA administration is non-toxic and does not cause systemic tissue damage. It increases immune cell abundance in the TME, suppresses tumor growth, promotes cancer cell apoptosis, and enhances leukocyte recruitment and activation. Elevated neutrophil and macrophage densities were associated with increased production of pro-inflammatory mediators with anti-neoplastic properties. These findings highlight MakA’s potential as a targeted, less harmful CRC therapy by modulating the TME immune response.

Pore-forming toxins (PFTs) are recognized as major virulence factors produced by both Gram-positive and Gram-negative bacteria. While the effects of PFTs have been extensively investigated using mammalian cells as a model system, their interactions with the environmental host, Acanthamoeba castellanii remains less understood. This study employed high-throughput image screening (HTI), advanced microscopy, western blot analysis, and cytotoxicity assays to evaluate the impact of PFT-producing bacterial species on their virulence against A. castellanii. Our unbiased HTI data analysis reveals that the cyst induction of A. castellanii in response to various bacterial species does not correlate with the presence of PFT-producing bacteria. Moreover, A. castellanii demonstrates resistance to PFT-mediated cytotoxicity, in contrast to mammalian macrophages. Notably, Vibrio anguillarum and Ralstonia eutropha triggered a high frequency of cyst formation and cytotoxicity in infected A. castellanii. In summary, our findings reveal that A. castellanii exhibits a unique resistance to PFTs, unlike mammalian cells, suggesting its potential ecological role as a reservoir for diverse pathogenic species and its influence on their persistence and proliferation in the environment. (Figure presented.)

Urochordate Ciona spp. are ideal marine model organisms for studying embryogenesis and developmental and evolutionary biology. However, the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging. This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp. The destination vector sets contained two Gateway cassettes flanked by Minos sites, allowing the N- or C-terminal tagging of a protein of interest with various fluorescent markers. In addition, we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry, a fluorescent indicator for Cas9 expression at cellular resolution. We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs. Furthermore, we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues. The genetic tools developed in this study offer a robust method for gene expression, cell tracking, and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.

Recent studies reveal that Vibrio cholerae secretes virulence factors impacting host cell viability, though their effects on cancer cells remain unclear. However, the bacterial components and mechanisms influencing cancer cells remain largely unknown. This study investigated the effects of V. cholerae mutants lacking secreted proteins on carcinoma cells. We identified the hemagglutinin zinc-metalloprotease HapA as the main factor reducing cancer cell viability. HapA cleaves protease-activated receptors 1 and 2 on epithelial cancer cells at unique sites, unlike human proteases. This cleavage triggers an early and transient activation of the kinases MEK and ERK. Transient MEK and ERK activation initiates caspase 7, leading to apoptosis and reduced viability in epithelial cancer cells. Our findings underscore the significance of human protease-activated receptors as targets for bacterial protease HapA. Furthermore, we demonstrate that selective cleavage of PAR-1/2 by HapA adjusts MEK-ERK signalling dynamics, suggesting potential new avenues for the development of novel anticancer therapies. Understanding how pathogens like V. cholerae interact with cancer cells sheds light on potential mechanisms underlying cancer progression and suggests new therapeutic targets for cancer treatment.

This study explores the virulence mechanisms of Vibrio cholerae, with a particular emphasis on HapA, a zinc metalloprotease delivered via outer membrane vesicles (OMVs). The findings reveal that OMV-associated HapA disrupts the integrity of tight and adherens junctions in intestinal epithelial cell models more effectively than its purified counterpart, suggesting that association with OMVs substantially potentiates the pathogenic effects of HapA. The study further details the uptake of V. cholerae OMVs by epithelial cells, as well as their targeted degradation of key junctional proteins, including claudin, ZO-1, and ?-catenin. These results highlight the critical role of OMV-associated HapA in compromising epithelial barrier function. Additionally, the use of spheroids and intestinal organoids in our experiments provides deeper insight into bacterial pathogenesis, offering valuable information for the development of targeted therapeutic strategies.

The subject of this chapter is The protease of Vibrio cholerae (PrtV). The PrtV peptidase is a secreted, Asp-zincin metallo-endopeptidase best characterized from the bacterium Vibrio cholerae, the causative agent of cholera. PrtV degrades host fibronectin, fibrinogen, and plasminogen. PrtV is stabilized at low temperatures by binding Ca2+. PrtV protects the bacterium from predation, killing Caenorhabditis elegans, by allowing the bacterium to colonize the nematode intestine. PrtV also processes the bacterial cytolysin VCC, modulating its exotoxin activity including lysis of erythrocytes and intestinal epithelial cells.

The Shaker/Kv1 subfamily of voltage-gated potassium (K+) channels is essential for modulating membrane excitability. Their loss results in prolonged depolarization and excessive calcium influx. These channels have also been implicated in a variety of other cellular processes, but the underlying mechanisms remain poorly understood. Through comprehensive screening of K+ channel mutants in C. elegans, we discovered that shk-1 mutants are highly susceptible to bacterial pathogen infection and oxidative stress. This vulnerability is associated with reduced glycogen levels and substantial mitochondrial dysfunction, including decreased ATP production and dysregulated mitochondrial membrane potential under stress conditions. SHK-1 is predominantly expressed and functions in body wall muscle to maintain glycogen storage and mitochondrial homeostasis. RNA-sequencing data reveal that shk-1 mutants have decreased expression of a set of cation-transporting ATPases (CATP), which are crucial for maintaining electrochemical gradients. Intriguingly, overexpressing catp-3, but not other catp genes, restores the depolarization of mitochondrial membrane potential under stress and enhances stress tolerance in shk-1 mutants. This finding suggests that increased catp-3 levels may help restore electrochemical gradients disrupted by shk-1 deficiency, thereby rescuing the phenotypes observed in shk-1 mutants. Overall, our findings highlight a critical role for SHK-1 in maintaining stress tolerance by regulating glycogen storage, mitochondrial homeostasis, and gene expression. They also provide insights into how Shaker/Kv1 channels participate in a broad range of cellular processes.

Cytolysin A (ClyA) is a pore-forming protein from a strongly silenced gene in non-pathogenic Escherichia coli, including typical commensal isolates in the intestinal microbiome of healthy mammalian hosts. Upon overproduction, ClyA-expressing bacteria display a cytolytic phenotype. However, it remains unclear whether sublytic amounts of native ClyA play a role in commensal E. coli-host interactions in vivo. Here, we show that sublytic amounts of ClyA are released via outer membrane vesicles (OMVs) and affect host cells in a remarkable manner. OMVs isolated from ClyA+ E. coli were internalised into cultured colon cancer cells. The OMV-associated ClyA caused reduced levels of cancer-activating proteins such as H3K27me3, CXCR4, STAT3 and MDM2 via the EZH2/H3K27me3/microRNA 622/CXCR4 signalling axis. Our results demonstrate that sublytic amounts of ClyA in OMVs from non-pathogenic E. coli can influence the stability of the EZH2 protein, reducing its activity in epigenetic regulation, causing elevated level of the tumour suppressor protein p53.

Microbial ecosystems experience spatial and nutrient restrictions, leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here we demonstrate first time that oral symbiont Aggregatibacter aphrophilus possesses a T6SS and can eliminate its close relative oral pathobiont Aggregatibacter actinomycetemcomitans using its T6SS. These findings bring newer the anti-bacterial prospects of symbionts against cohabiting pathobionts while introducing presence of an active T6SS in the oral cavity.