Transport and Metabolism of Citrate by Streptococcus mutans

Streptococcus mutans, a normal inhabitant of dental plaque, is considered a primary etiological agent of dental caries. Two virulence determinants of S. mutans are its acidogenicity and aciduricity (the ability to produce acid and the ability to survive and grow at low pH, respectively). Citric acid is ubiquitous in nature; it is a component of fruit juices, bones, and teeth. In lactic acid bacteria citrate transport has been linked to increased survival in acidic conditions. We identified putative citrate transport and metabolism genes in S. mutans, which led us to investigate citrate transport and metabolism. Our goals in this study were to determine the mechanisms of citrate transport and metabolism in S. mutans and to examine whether citrate modulates S. mutans aciduricity. Radiolabeled citrate was used during citrate transport to identify citrate metal ion cofactors, and thin-layer chromatography was used to identify metabolic end products of citrate metabolism. S. mutans was grown in medium MM4 with different citrate concentrations and pH values, and the effects on the growth rate and cell survival were monitored. Intracellular citrate inhibited the growth of the bacteria, especially at low pH. The most effective cofactor for citrate uptake by S. mutans was Fe(3+). The metabolic end product of citrate metabolism was aspartate, and a citrate transporter mutant was more citrate tolerant than the parent

Structure-based protein-protein docking suggests RIG-I CARD1 interacts with IPS1 CARD through a novel antiparallel α1-α3-α4 interface.

<p>We used ClusPro (A) and Dot2 (B) to predict the most likely structural orientation of human RIG-I CARD1 bound to IPS1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). We show the best-scoring complex (top panels) as well as the 20 top-scoring orientations (bottom panels) from each analysis. (C) We calculated the surface electrostatic charge (kT/e) of human RIG-I CARD1 and IPS1 CARD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). Top panel displays IPS1 (left) and RIG-I CARD1 (right) in the same antiparallel α1-α3-α4 orientation as A and B. Arrows indicate regions of inferred shape and electrostatic complementarity. Gray Xs indicate locations of mutations examined in D. In the bottom panel, IPS1 is shown in mirror image and made 35% transparent; regions of electrostatic complementarity appear purple in the overlay (also highlighted by dotted ovals). (D) We created mutant RIG-I CARD1 (Glu66,67—Arg) and IPS1 (Arg66,67—Glu) and measured the affinities with which mutant proteins bind their wild-type partners. We plot the mean and standard error–log steady-state dissociation (pKd) and initial binding rate (pKm) of each interaction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s006" target="_blank">S6 Fig</a>). Wild-type RIG-I CARD1 and CASP9 interacting with wild-type IPS1 were used as positive and negative controls, respectively (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g001" target="_blank">Fig 1B</a>).</p

Evolution of a Novel Antiviral Immune-Signaling Interaction by Partial-Gene Duplication

<div><p>The RIG-like receptors (RLRs) are related proteins that identify viral RNA in the cytoplasm and activate cellular immune responses, primarily through direct protein-protein interactions with the signal transducer, IPS1. Although it has been well established that the RLRs, RIG-I and MDA5, activate IPS1 through binding between the twin caspase activation and recruitment domains (CARDs) on the RLR and a homologous CARD on IPS1, it is less clear which specific RLR CARD(s) are required for this interaction, and almost nothing is known about how the RLR-IPS1 interaction evolved. In contrast to what has been observed in the presence of immune-modulating K63-linked polyubiquitin, here we show that—in the absence of ubiquitin—it is the first CARD domain of human RIG-I and MDA5 (CARD1) that binds directly to IPS1 CARD, and not the second (CARD2). Although the RLRs originated in the earliest animals, both the IPS1 gene and the twin-CARD domain architecture of RIG-I and MDA5 arose much later in the deuterostome lineage, probably through a series of tandem partial-gene duplication events facilitated by tight clustering of RLRs and IPS1 in the ancestral deuterostome genome. Functional differentiation of RIG-I CARD1 and CARD2 appears to have occurred early during this proliferation of RLR and related CARDs, potentially driven by adaptive coevolution between RIG-I CARD domains and IPS1 CARD. However, functional differentiation of MDA5 CARD1 and CARD2 occurred later. These results fit a general model in which duplications of protein-protein interaction domains into novel gene contexts could facilitate the expansion of signaling networks and suggest a potentially important role for functionally-linked gene clusters in generating novel immune-signaling pathways.</p></div

RIG-like receptor (RLR) and IPS1 CARD domains structurally diverged in early deuterostomes, and this structural divergence was associated with protein-coding adaptation.

<p>We inferred the 3-dimensional structures of ancestral RLR and IPS1 CARD domains at key points on the RLR-IPS1 CARD phylogeny (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">Fig 3</a>). We plot the electrostatic surface potential (kT/e) across the α1-α3-α4 surface of each CARD domain, with large acidic and basic patches indicated by dotted red and blue ovals, respectively. We additionally used phylogenetic techniques to infer the presence of adaptive protein-coding substitutions on each branch of the phylogeny (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>). Stars indicate significant support for protein-coding adaptation on the indicated branch (<i>p</i><0.05 after correcting for multiple tests). Large dotted vertical line indicates the approximate time of RLR-IPS1 CARD proliferation in early deuterostomes.</p

Deuterostome RIG-like receptors (RLRs) and IPS1 cluster with other potential CARD-signaling immune receptors in the <i>Branchiostoma floridae</i> genome.

<p>(A) We plot the physical location of each identified RLR and IPS1 gene along the assembled <i>B</i>. <i>floridae</i> chromosome (bottom). Other genes in the cluster are shown along the top. Functional domains are indicated by colors. Genes with significant BLAST hits to the <i>B</i>. <i>floridae</i> expressed sequence tag (EST) database are shaded according to e-value. (B) We measured the kinetics of <i>B</i>. <i>floridae</i> RIG-I CARD1+2 and MDA5 CARD1+2 domains binding to <i>B</i>. <i>floridae</i> IPS1 CARD in vitro (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s001" target="_blank">S1 Fig</a>). We plot the mean and standard error of–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm), with human CASP9 used as a negative control. Dotted vertical line indicates approximate level of non-specific binding, with longer bars indicating tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s012" target="_blank">S12 Fig</a>).</p

In the absence of ubiquitin, the RIG-like receptors (RLRs), RIG-I and MDA5, bind their signaling partner, IPS1, via a direct interaction between the first RLR CARD domain (CARD1) and the CARD domain of IPS1.

<p>(A) After binding viral RNA, RIG-I and MDA5 interact directly with IPS1 via CARD-CARD interactions. K63 Polyubiquitin chains—either covalently linked or noncovalently bound to RLR CARDs—can potentiate RLR-IPS1 signaling. In the case of noncovalent polyubiquitin binding, studies suggest RIG-I CARD2 interacts with IPS1 (bottom middle). Our results suggest that, in the absence of ubiquitin, RLR CARD1 interacts with IPS1 (bottom right). (B) We measured the kinetics of human RIG-I and MDA5 CARD1, CARD2 and CARD1+2 domains bound to IPS1 CARD in vitro (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#sec009" target="_blank">Materials and Methods</a>) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s001" target="_blank">S1 Fig</a>). We plot the mean and standard error in–log-transformed steady-state dissociation (pKd) and initial binding rate (pKm), with longer bars indicating tighter binding (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.s002" target="_blank">S2 Fig</a>). Human CASP9 CARD was used as a negative control to indicate an approximate level of nonspecific binding (dotted line); CASP9 is the CARD domain most closely related to RLR/IPS1 CARDs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137276#pone.0137276.g003" target="_blank">Fig 3</a>), and direct interactions between RLRs and CASP9 have not been reported.</p