In Streptococcus mutans, ComX, an alternative sigma factor, drives the transcription of the “late-competence genes” required for genetic transformation. ComX activity is modulated by inputs from two signaling pathways, ComDE and ComRS, that respond to the competence stimulating peptide (CSP) and the SigX-inducing peptide (XIP), respectively. In particular, the comRS, encoding the ComR regulatory protein and the ComS precursor to XIP, functions as the proximal regulatory system for ComX activation. Here, we investigated the individual and combinatorial effects of CSP and XIP on genetic transformation and cell killing of S. mutans. Our transformation results confirm the recent reports by Mashburn-Warren et al. and Desai et al. that XIP functions optimally in a chemically defined medium (CDM), whereas its activity is inhibited when cells are grown in complex medium. Using tandem mass spectrometry (MS/MS) fragmentation, a drastic reduction in XIP levels in ComX-deficient cultures were observed, suggesting a ComX-mediated positive feedback mechanism for XIP synthesis. Our evaluation of cell viability in the presence of 10μM XIP resulted in the killing nearly 82% of the population. The killing activity was shown to be dependent on the presence of comR/S and comX. These results suggest a novel role for XIP as a compelling effector of cell death. This is the first report that demonstrates a role for XIP in cell killing.

Streptococcus mutans, a Gram-positive human commensal and pathogen, is commonly recognized as a primary causative agent in dental caries. Metabolic activity of this strain results in the creation of acids and secreted products are recognized as pathogenic factors and agents that promote immunomodulation by stimulating the release of pro-inflammatory cytokines. Products of secondary metabolic pathways of microorganisms from the human microbiome are increasingly investigated for their immunomodulatory functions. In this study, we sought to explore the metabolomic output of nonribosomal peptide pathways within the model S. mutans strain, S. mutans UA159, using a systems metabolomic approach to gain in-depth analysis on products created by this organism and probe these molecules for their immunomodulatory function. Comparative metabolomics and biosynthetic studies using wild-type and nonribosomal peptide deletion strains (within the mutanobactin biosynthetic locus), precursor feedings (fatty acid derivatives) led to the identification of 58 metabolites, 13 of which were structurally elucidated. In addition to these, an assembly line derailment product, mutanamide, was also identified and used to assess immunomodulatory properties of mutanobactins and actions relating to their previously reported functions describing hyphal inhibitory profiles in Candida albicans. The results of this study demonstrate both the complexity and the divergent roles of products stemming from this unique biosynthetic assembly line.

Lactobacillus plantarum strains are noted for their presence in the human gastrointestinal tract and are distinguished for their immunomodulatory actions and therapeutic applications. Despite the uncertainty in the underlining molecular mechanisms, recent evidence suggests that L. plantarum secretes immunomodulatory agents that alter immunological signaling cascades. Elaboration of these metabolic products from L. plantarum strain WCFS1 was demonstrated previously to correlate with the mid-log-stationary transition, perhaps consistent with secondary metabolite expression. Here, we present the metabolomic shifts revealed by principal component analysis that correspond to the mid-log-stationary transition of L. plantarum, and identify pyroglutamic (pyro) dipeptides within this transition as correlative with the immunomodulatory actions. Four of these (pyro-phenylalanine, pyro-leucine, pyro-isoleucine, pyro-tryptophan) were characterized and the two dominant members, pyro-phenylalanine and pyro-tryptophan, were directly interrogated for immunomodulatory activity through in vivo administration using C57BL/6 mice. Administration of these compounds resulted in decreased production of pro-inflammatory cytokine interferon (IFN)-gamma, which is of noted importance in gastrointestinal immune homeostasis.

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