Linking microbial genes to plasma and stool metabolites uncovers host-microbial interactions underlying ulcerative colitis disease course.

Understanding the role of the microbiome in inflammatory diseases requires the identification of microbial effector molecules. We established an approach to link disease-associated microbes to microbial metabolites by integrating paired metagenomics, stool and plasma metabolomics, and culturomics. We identified host-microbial interactions correlated with disease activity, inflammation, and the clinical course of ulcerative colitis (UC) in the Predicting Response to Standardized Colitis Therapy (PROTECT) pediatric inception cohort. In severe disease, metabolite changes included increased dipeptides and tauro-conjugated bile acids (BAs) and decreased amino-acid-conjugated BAs in stool, whereas in plasma polyamines (N-acetylputrescine and N1-acetylspermidine) increased. Using patient samples and Veillonella parvula as a model, we uncovered nitrate- and lactate-dependent metabolic pathways, experimentally linking V. parvula expansion to immunomodulatory tryptophan metabolite production. Additionally, V. parvula metabolizes immunosuppressive thiopurine drugs through xdhA xanthine dehydrogenase, potentially impairing the therapeutic response. Our findings demonstrate that the microbiome contributes to disease-associated metabolite changes, underscoring the importance of these interactions in disease pathology and treatment.

Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Veillonella parvula in the intestine.

Colonization of the intestine by oral microbes has been linked to multiple diseases such as inflammatory bowel disease and colon cancer, yet mechanisms allowing expansion in this niche remain largely unknown. Veillonella parvula, an asaccharolytic, anaerobic, oral microbe that derives energy from organic acids, increases in abundance in the intestine of patients with inflammatory bowel disease. Here we show that nitrate, a signature metabolite of inflammation, allows V. parvula to transition from fermentation to anaerobic respiration. Nitrate respiration, through the narGHJI operon, boosted Veillonella growth on organic acids and also modulated its metabolic repertoire, allowing it to use amino acids and peptides as carbon sources. This metabolic shift was accompanied by changes in carbon metabolism and ATP production pathways. Nitrate respiration was fundamental for ectopic colonization in a mouse model of colitis, because a V. parvula narG deletion mutant colonized significantly less than a wild-type strain during inflammation. These results suggest that V. parvula harness conditions present during inflammation to colonize in the intestine.

Roles and regulation of Spx family transcription factors in Bacillus subtilis and related species.

Bacillus subtilis Spx is the prototype for a large family of redox-responsive transcription factors found in many bacteria, most notably those from the phylum Firmicutes. Unusually for a transcription factor, B. subtilis Spx protein modulates gene expression by binding as a monomer to the αCTD domain of RNA polymerase (RNAP), and only interacts with DNA during subsequent promoter engagement. B. subtilis Spx drives the expression of a large regulon in response to proteotoxic conditions, such as heat and disulfide stress, as well as cell wall stress. Here, we review the detailed mechanisms that control the expression, stability, and activity of Spx in response to a variety of stress conditions. We also summarize current knowledge regarding Spx homologs in other Firmicutes, the environmental conditions in which those homologs are activated, and their biological role.

Identification of Novel Spx Regulatory Pathways in Bacillus subtilis Uncovers a Close Relationship between the CtsR and Spx Regulons.

In Bacillus subtilis, the Spx transcription factor controls a large regulon in response to disulfide, heat, and cell wall stresses. The regulatory mechanisms that activate the Spx regulon are remarkably complex and involve changes in transcription, proteolysis, and posttranslational modifications. To identify genes involved in Spx regulation, we performed a transposon screen for mutations affecting expression of trxB, an Spx-dependent gene. Inactivation of ctsR, encoding the regulator of the Clp proteases, reduced trxB expression and lowered Spx levels. This effect required ClpP, but involved ClpC rather than the ClpX unfoldase. Moreover, cells lacking McsB, a dual function arginine kinase and ClpCP adaptor, largely reverted the ctsR phenotype and increased trxB expression. The role of McsB appears to involve its kinase activity, since loss of the YwlE phosphoarginine phosphatase also led to reduced trxB expression. Finally, we show that Spx is itself a regulator of the ctsR operon. Altogether, this work provides evidence for a role of CtsR regulon members ClpC, ClpP, and McsB in Spx regulation and identifies a new feedback pathway associated with Spx activity in B. subtilisIMPORTANCE In Bacillus subtilis, the Spx transcription factor is proteolytically unstable, and protein stabilization figures prominently in the induction of the Spx regulon in response to oxidative and cell envelope stresses. ClpXP is largely, but not entirely, responsible for Spx instability. Here, we identify ClpCP as the protease that degrades Spx under conditions that antagonize the ClpXP pathway. Spx itself contributes to activation of the ctsR operon, which encodes ClpC as well as the McsB arginine kinase and protease adaptor, thereby providing a negative feedback mechanism. Genetic studies reveal that dysregulation of the CtsR regulon or inactivation of the YwlE phosphoarginine phosphatase decreases Spx activity through mechanisms involving both protein degradation and posttranslational modification.

Stabilization of Bacillus subtilis Spx under cell wall stress requires the anti-adaptor protein YirB.

Spx is a global transcriptional regulator present in low-GC Gram-positive bacteria, including the model bacterium Bacillus subtilis and various human pathogens. In B. subtilis, activation of Spx occurs in response to disulfide stress. We recently reported, however, that induction of Spx also occurs in response to cell wall stress, and that the molecular events that result in its activation under both stress conditions are mechanistically different. Here, we demonstrate that, in addition to up-regulation of spx transcription through the alternative sigma factor σM, full and timely activation of Spx-regulated genes by cell wall stress requires Spx stabilization by the anti-adaptor protein YirB. YirB is itself transcriptionally induced under cell wall stress, but not disulfide stress, and this induction requires the CssRS two-component system, which responds to both secretion stress and cell wall antibiotics. The yirB gene is repressed by YuxN, a divergently transcribed TetR family repressor, and CssR~P acts as an anti-repressor. Collectively, our results identify a physiological role for the YirB anti-adaptor protein and show that induction of the Spx regulon under disulfide and cell wall stress occurs through largely independent pathways.

Induction of the Spx regulon by cell wall stress reveals novel regulatory mechanisms in Bacillus subtilis.

The transcription factor Spx is the master regulator of the disulfide stress response in Bacillus subtilis. Intriguingly, the activation of Spx by diamide relies entirely on posttranslational regulatory events in spite of the complex transcriptional control of the spx gene. Here, we show that cell wall stress, but not membrane stress, also results in induction of the Spx regulon. Remarkably, two major differences were found regarding the mechanism of induction of Spx under cell wall stress in comparison to disulfide stress. First, transcriptional induction of the spx gene from a σM -dependent promoter is required for accumulation of Spx in response to cell wall stress. Second, activation of the Spx regulon during cell wall stress is not accompanied by oxidation of the Spx disulfide switch. Finally, we demonstrate that cells lacking Spx have increased sensitivity toward antibiotics inhibiting both early and late steps in peptidoglycan synthesis, suggesting that the Spx regulon plays an important adaptive role in the cell wall stress response. This study expands the functional role of the Spx regulon and reveals novel regulatory mechanisms that result in induction of Spx in B. subtilis.