Biology subjects

Ton-That, H.

Publications and source records attributed to Ton-That, H..

6 recordsLinked to original sources

bioRxiv2026

Higher-order metabolic interactions among oral commensals gate pathobiont expansion through succinate-driven pH modulation

Despite advances in defining microbiome shifts associated with inflammatory mucosal diseases, the ecological interactions that constrain or facilitate pathobiont expansion remain poorly understood. Using a six-species synthetic oral microbial community and factorial interaction screening, we identified a higher-order interaction governing the growth of the periodontal pathobiont Porphyromonas gingivalis. The core community member Veillonella parvula was the strongest positive determinant of P. gingivalis growth, but this effect was substantially reduced by the health-associated commensal Actinomyces oris. A. oris-mediated antagonism resulted from extracellular acidification and was enhanced under anaerobic conditions. Genetic dissection of A. oris fermentation revealed that lactate dehydrogenase (ldh1) contributes to anaerobic lactate production, but ldh1 deletion only partially relieved P. gingivalis inhibition. Instead, succinate, produced through a succinate dehydrogenase/fumarate reductase pathway (sdh2), emerged as the principal driver of acidification and P. gingivalis suppression. At high cell densities, however, V. parvula consumed both lactate and succinate, reversing acidification and restoring conditions permissive for P. gingivalis growth. To assess the relevance of these interactions in vivo, we analyzed subgingival microbiome from 1,355 adults, including 555 with detectable P. gingivalis. Among these individuals, P. gingivalis abundance was negatively associated with Actinomyces abundance after adjustment for periodontitis severity, whereas this association was attenuated at high V. parvula abundance, consistent with the interaction observed in vitro. Together, these findings identify succinate as a previously unrecognized mediator of interbacterial antagonism and reveal how higher-order metabolic interactions among oral commensals regulate pathobiont expansion through pH modulation, with protective effects shaped by oxygen availability and metabolic cross-feeding.

microbiology↗

bioRxiv2025

Inactivation of the Fusobacterium nucleatum Rnf complex reduces FadA-mediated amyloid formation and tumor development

The Gram-negative anaerobe Fusobacterium nucleatum is an oral oncobacterium that promotes colorectal cancer (CRC) development with the amyloid-forming cell surface adhesin FadA integral to CRC tumorigenesis. We describe here molecular genetic studies uncovering a novel mode of metabolic regulation of FadA-mediated tumor formation by a highly conserved respiratory enzyme known as the Rnf complex. First, we show that genetic disruption of Rnf, via rnfC deletion, significantly reduces the level of fadA transcript, accompanied by a near-complete abolishment of the precursor form of FadA (pFadA), reduced assembly of FadA at the mature cell pole, and severe defects in the osmotic stress-induced formation of FadA amyloids. We show further that the Rnf complex regulates three response regulators (CarR, ArlR, and S1), which modulate the expression of pFadA, without affecting fadA transcript. Consistent with our hypothesis that these response regulators control factors that process FadA, deletion of rnfC, carR, arlR, or s1 each impairs expression of the signal peptidase gene lepB, and FadA production is nearly abolished by CRISPR-induced depletion of lepB. Importantly, while rnfC deletion does not affect the ability of the mutant cells to adhere to CRC cells, rnfC deficiency significantly diminishes the fusobacterial invasion of CRC cells and formation of spheroid tumors in vitro. Evidently, the Rnf complex modulates the expression of the FadA adhesin and tumorigenesis through a gene regulatory network consisting of multiple response regulators, each controlling a signal peptidase that is critical for the post-translational processing of FadA and surface assembly of FadA amyloids. IMPORTANCEThe Rhodobacter nitrogen-fixation (Rnf) complex of Fusobacterium nucleatum plays an important role in the pathophysiology of this oral pathobiont, since genetic disruption of this conserved respiratory enzyme negatively impacts a wide range of metabolic pathways, as well as bacterial virulence in mice. Nonetheless, how Rnf deficiency weakens the virulence potential of F. nucleatum is not well understood. Here, we show that genetic disruption of the Rnf complex reduces surface assembly of adhesin FadA and FadA-mediated amyloid formation, via regulation of signal peptidase LepB by multiple response regulators. As FadA is critical in the carcinogenesis of colorectal cancer (CRC), the ability to invade CRC cells and promote spheroid tumor growth is strongly diminished in an Rnf-deficient mutant. Thus, this work uncovers a molecular linkage between the Rnf complex and LepB-regulated processing of FadA - likely via metabolic signaling - that maintains the virulence potential of this oncobacterium in various cellular niches.

microbiology↗

bioRxiv2024

Ethanolamine-induced assembly of microcompartments is required for Fusobacterium nucleatum virulence

Many bacteria metabolize ethanolamine as a nutrient source through cytoplasmic organelles named bacterial microcompartments (BMCs). Here we investigated the molecular assembly, regulation, and function of BMCs in Fusobacterium nucleatum - a Gram-negative oral pathobiont that is associated with adverse pregnancy outcomes. The F. nucleatum genome harbors a conserved ethanolamine utilization (eut) locus with 21 genes that encode several putative BMC shell proteins and a two-component signal transduction system (TCS), in addition to the enzymes for ethanolamine transport and catabolism. We show that the expression of most of these genes as well as BMC formation is highly increased in wild type fusobacteria when cultured in the presence of ethanolamine as a nutrient source. Deletion of the response regulator EutV eliminated this induction of eut mRNAs and BMCs, thus demonstrating that BMC formation is transcriptionally regulated by the TCS EutV-EutW in response to ethanolamine. Mass spectrometry of isolated BMCs unveiled the identity of the constituent proteins EutL, EutM1, EutM2, and EutN. Consistent with the role of these proteins in BMC assembly and metabolism, deletion of eutN, eutL/eutM1/eutM2, or eutL/eutM1/eutM2/eutN not only affected BMC formation, but also ethanolamine utilization, causing cell growth defects with ethanolamine as nutrient. BMCs also assembled in fusobacteria cultured with placental cells or the culture media, a process that is dependent on the BMC shell proteins. Significantly, we show that the eutN mutant is defective in inducing preterm birth in a mouse model. Together, these results establish that BMC-mediated metabolism of ethanolamine is critical for fusobacterial virulence. IMPORTANCEThe oral anaerobe Fusobacterium nucleatum can spread to distal internal organs, such as the colon and placenta, and thereby promote the development of colorectal cancer and induce preterm birth, respectively. Yet, how this opportunistic pathogen adapts to the various metabolically distinct host cellular niches remains poorly understood. We demonstrated here that this microbe assembles specialized metabolic organelles, termed bacterial microcompartments (BMCs), to utilize environmental ethanolamine (EA) as a key environmental nutrient source. The formation of F. nucleatum BMCs, containing BMC shell proteins EutLM1M2N, is controlled by a two-component system, EutV-EutW, responsive to EA. Significantly, this ability of F. nucleatum to form BMCs in response to EA is crucial for its pathogenicity evidenced by the fact that the genetic disruption of BMC formation reduces fusobacterial virulence in a mouse model of preterm birth.

microbiology↗

bioRxiv2023

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-[A] resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

microbiology↗

bioRxiv2023

An ancient respiratory enzyme metabolically modulates Fusobacterium nucleatum virulence

A prominent oral commensal and opportunistic pathogen, Fusobacterium nucleatum can traverse to extra-oral sites such as placenta and colon, promoting adverse pregnancy outcomes and colorectal cancer, respectively. How this anaerobe sustains many metabolically changing environments enabling its virulence potential remains unclear. Informed by our genome-wide transposon mutagenesis, we report here that the highly conserved Rnf complex, encoded by the rnfCDGEAB gene cluster, is key to fusobacterial metabolic adaptation and virulence. Genetic disruption of the Rnf complex via non-polar, in-frame deletion of rnfC ({Delta}rnfC) abrogates polymicrobial interaction (or coaggregation) associated with adhesin RadD and biofilm formation. The defect in coaggregation is not due to reduced cell surface of RadD, but rather an increased level of extracellular lysine, which binds RadD and inhibits coaggregation. Indeed, removal of extracellular lysine via washing {Delta}rnfC cells restores coaggregation, while addition of lysine inhibits this process. These phenotypes mirror that of a mutant ({Delta}kamA) that fails to metabolize extracellular lysine. Strikingly, the {Delta}rnfC mutant is defective in ATP production, cell growth, cell morphology, and expression of the enzyme MegL that produces hydrogen sulfide from cysteine. Targeted metabolic profiling demonstrated that catabolism of many amino acids, including histidine and lysine, is altered in {Delta}rnfC cells, thereby reducing production of ATP and metabolites including H2S and butyrate. Most importantly, we show that the {Delta}rnfC mutant is severely attenuated in a mouse model of preterm birth. The indispensable function of Rnf complex in fusobacterial pathogenesis via modulation of bacterial metabolism makes it an attractive target for developing therapeutic intervention. IMPORTANCEAlthough viewed as an oral commensal, the Gram-negative F. nucleatum is an opportunistic pathogen that can spread to extra-oral sites such as placenta and colon, promoting adverse pregnancy outcomes and colorectal cancer, respectively. How this anaerobe sustains various metabolically changing environments enabling its virulence potential remains unclear. We demonstrate here that the highly conserved Rnf complex is key to fusobacterial metabolic adaptation and virulence. Genetic disruption of this Rnf complex causes global defects in polymicrobial interaction, biofilm formation, cell growth and morphology, H2S production, and ATP synthesis. Targeted metabolomic profiling demonstrates that the loss of this respiratory enzyme significantly diminishes catabolism of numerous amino acids, which negatively impacts fusobacterial virulence as tested in a preterm birth model in mice.

microbiology↗

bioRxiv2023

The Basal and Major Pilins in the Corynebacterium diphtheriae SpaA Pilus Adopt Similar Structures that Competitively React with the Pilin Polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme-substrate reaction intermediate.

biochemistry↗