Biology subjects

Watnick, P.

Publications and source records attributed to Watnick, P..

5 recordsLinked to original sources

bioRxiv2026

The tumor suppressor Tip60 inhibits TORC1 signaling in response to microbial acetate to promote autophagy and enterocyte differentiation

The intestinal microbiota is critical for maintenance of local and systemic immune and metabolic homeostasis in animals, but few molecular mechanisms of action have been delineated. Here, using a Drosophila model, we elucidate the role of the microbial fermentation product acetate in maintenance of the intestinal barrier and enterocyte maturation. Tip60 is a lysine acetyl transferase that modifies histone and non-histone targets. We previously showed that Tip60 activates innate immune signaling in enteroendocrine cells in response to microbe-derived acetate. Here we elucidate a distinct mechanism of action in enterocytes. mTOR is a serine-threonine kinase that regulates cell growth and autophagy based on nutrient availability as part of the TORC1 complex. We report that microbe-derived acetate represses enterocyte TORC1 signaling in a Tip60-dependent manner. This licenses autophagy, which is required to destroy commensal microbes phagocytosed by enterocytes, resulting in bacterial dissemination. Single cell sequencing shows accumulation of poorly differentiated enterocytes in Tip60 knockdown intestines. The microbiota, Tip60, and mTOR have been implicated in the development and progression of colorectal cancer. As accumulation of undifferentiated precursors is a harbinger of malignant transformation and metastasis, we propose our findings provide a mechanistic link between the microbiota, Tip60, and mTOR, epithelial innate immunity and oncogenesis.

immunology↗

bioRxiv2026

Actuation of CRP activating region 3 by acetylation modulates V. cholerae sugar utilization and virulence

The cyclic AMP receptor protein or CRP is a global regulator of bacterial metabolism that activates transcription of genes required for utilization of alternative carbon sources in response to the second messenger cAMP, which is synthesized in the setting of glucose scarcity. CRP activates transcription through contact with RNA polymerase at three sites termed activating regions (ARs) 1-3. AR3 was previously reported to be functional only when CRP K52 was mutated to a neutral residue and to be essential for transcription only in the absence of AR1 and AR2. Multiple proteomic studies have reported acetylation of CRP K52. This post-translational modification is predicted to activate AR3. To probe the role of K52 acetylation (K52QAc) and AR3 at the genome level, we used ChIP-seq and RNA-seq analysis to compare WT CRP with a CRP K52Q mutant that mimics CRP K52Ac. We report that CRP K52Q binds to hundreds of new sites on the chromosome, resulting in increased abundance of known as well as previously unknown transcripts. These transcripts increase uptake and metabolism of dietary sugars such as maltose and galactose, repress acetate consumption, and augment virulence gene expression. We attribute the repression of acetate consumption to a novel small RNA, CrbZ, which is positively regulated by CRP K52Q in LB broth and by WT CRP specifically in minimal medium containing maltose. This study highlights the role of post-translational modifications in molding the CRP regulon to optimize pathogen metabolism and virulence gene expression in the human intestine in response to nutritional cues. Significance statementAs a model in the field of bacterial transcription, the structure and function of the cAMP receptor protein (CRP), a global transcription regulator, has been exhaustively investigated. These studies have established three activating regions (ARs) where CRP contacts RNA polymerase, of which only two were thought to participate in transcription activation by native CRP. Here we provide evidence that post-translational acetylation of V. cholerae CRP lysine 52 actuates AR3, enabling occupancy of hundreds of novel CRP binding sites and the transcription of genes encoding novel small RNAs. These changes alter virulence gene expression, promote utilization of dietary carbon sources, and delay acetate uptake. We propose that acetylation of CRP K52 engages AR3, thus optimizing V. cholerae fitness in the human intestine.

microbiology↗

bioRxiv2025

Vibrio cholerae colonization of the arthropod intestine activates innate immune signaling in enteroendocrine cells via phosphorylation of the nuclear receptor ultraspiracle

The Gram-negative rod Vibrio cholerae causes profuse diarrhea in humans and is found in close association with both terrestrial and aquatic arthropods in the environment. We have previously shown that V. cholerae colonizes the arthropod intestine. Here we show that tryptophan produced by V. cholerae in the arthropod intestine is used by enterocytes to synthesize serotonin and signal to enteroendocrine cells (EECs) to activate the TNF-like immune deficiency pathway IMD. We define the EEC serotonin signaling pathway, which involves a subset of serotonin G protein-coupled receptors, Gq, phospholipase C, and protein kinase C. This pathway culminates in phosphorylation and potentiation of the nuclear receptor ultraspiracle, which binds the sex hormone ecdysone to activate IMD signaling. While IMD signaling increases antimicrobial peptide synthesis in all intestinal cell types, IMD signaling in EECs uniquely activates expression of the enteroendocrine peptide Tachykinin and the V. cholerae colonization factor Peritrophin-15a. We propose that, because V. cholerae secretes a metabolite that activates IMD signaling in EECs, it has evolved to exploit the arthropod intestinal innate immune response to maximize adhesion to the arthropod intestine.

microbiology↗

bioRxiv2025

A conserved, immune-regulated peritrophin promotes Vibrio cholerae colonization of the arthropod intestine

Vibrio cholerae is a human diarrheal pathogen and an estuarine organism that associates with both terrestrial and aquatic arthropods. Using the model terrestrial arthropod Drosophila melanogaster, we previously showed that V. cholerae forms a multi-layered bacterial structure called a biofilm in the arthropod intestine and activates the arthropod intestinal innate immune response. Here we show that activation of the immune response in enterocytes decreases V. cholerae colonization of the arthropod intestine, while activation of the immune response in enteroendocrine cells that express the enteroendocrine peptide tachykinin (Tk) promotes V. cholerae colonization. To uncover the basis of this observation, we measured the impact of TkRNAi on intestinal gene expression by RNA-seq analysis. In addition to increasing expression of antimicrobial peptides and lipases, Tk activated the expression of chitinases and chitin-binding proteins. These proteins interact with chitin fibrils in the peritrophic matrix (PM), a protective coating that overlies the arthropod intestinal epithelium. One of these Tk-activated PM components, the small, secreted chitin-binding protein Peritrophin 15a (Peri-15a), is essential for robust V. cholerae colonization of the gut. Homologs of Peri-15a are widespread in both terrestrial and aquatic organisms including marine non-biting midges, marine copepods, rotifers, and cyanobacteria. We propose that Peri-15a and its homologs, found in the intestines of diverse arthropods, either serves as a receptor or reveals a PM epitope that promotes V. cholerae attachment to the intestinal surface. Therefore, activation of the enteroendocrine cell intestinal innate immune response by V. cholerae may, in fact, represent a colonization strategy.

microbiology↗

bioRxiv2025

Carbon source, cell density, and the microbial community control inhibition of V. cholerae surface colonization by environmental nitrate

The intestinal diarrheal pathogen Vibrio cholerae colonizes the host terminal ileum, a microaerophilic, glucose-poor, nitrate-rich environment. In this environment, V. cholerae respires nitrate and increases transport and utilization of alternative carbon sources via the cAMP receptor protein (CRP), a transcription factor that is active during glucose scarcity. Here we show that V. cholerae nitrate respiration in aerated cultures is under control of CRP and, therefore, glucose availability. V. cholerae nitrate respiration results in extracellular accumulation of nitrite because V. cholerae does not possess the machinery for nitrite reduction. This nitrite inhibits V. cholerae biofilm formation via an as yet unelucidated mechanism that depends on the high cell density master regulator HapR. The genome of Paracoccus aminovorans, an intestinal microbe shown to enhance V. cholerae biofilm accumulation in the neonatal mouse gut and predispose household contacts to cholera, encodes enzymes that reduce nitrite to nitrogen gas. We report that, in nitrate-supplemented co-cultures, P. aminovorans metabolizes the nitrite generated by V. cholerae and, thereby, enhances V. cholerae surface accumulation. We propose that V. cholerae biofilm formation in the host intestine is limited by nitrite production but can be rescued by intestinal microbes such as P. aminovorans that have the capacity to metabolize nitrite. Such microbes increase V. cholerae colonization of the host ileum and predispose to infection. ImportanceV. cholerae colonizes the terminal ileum where both oxygen and nitrate are available as terminal electron acceptors. V. cholerae biofilm formation is inhibited by nitrate due to its conversion to nitrite during V. cholerae respiration. When co-cultured with a microbe that can further reduce nitrite, V. cholerae surface accumulation in the presence of nitrate is rescued. The contribution of biofilm formation to ileal colonization depends on the composition of the microbiota. We propose that the intestinal microbiota predisposes mammalian hosts to cholera by consuming the nitrite generated by V. cholerae in the terminal ileum. Differences in the intestinal abundance of nitrite-reducing microbes may partially explain the differential susceptibility of humans to cholera and the resistance of non-human mammalian models to intestinal colonization with V. cholerae.

microbiology↗