Biology subjects

Walker, A. R.

Publications and source records attributed to Walker, A. R..

16 recordsLinked to original sources

bioRxiv2026

mCranberry: A rationally engineered monomeric red fluorescent protein with 85% quantum yield

Fluorescent proteins (FPs) are the workhorse of modern-day cellular imaging and biosensing. However, rationally predicting and engineering the photophysical properties of FPs remain challenging, as these are governed by complex three-dimensional microenvironments and post-translational modifications. Here, we present a novel surface-mutation-guided engineering strategy combined with iterative rounds of rational mutagenesis to improve key photophysical properties of red fluorescent proteins (RFPs). This approach generated two bright, monomeric RFP variants from the mFruits lineage, mCranberry and mCranberry2, with quantum yields of 85% and 80%, respectively. Strategic incorporation of charged amino acid residues on the protein surface enhanced fluorescence quantum yield without compromising photostability. Comprehensive photophysical characterization and live-cell microscopy demonstrate enhanced performance of these variants and establish their utility as robust probes for fluorescence imaging. However, the exact mechanism of such improvements is subject to further investigation. Collectively, this work introduces a strategy for optimizing fluorescence properties and provides a framework that may be broadly applicable to other pigment-protein systems such as genetically encoded metal-ion indicators and photosynthetic light-harvesting complexes.

biophysics↗

bioRxiv2026

Dark state-mediated photobleaching in mCherry-basedred fluorescent proteins

Developing bright and photostable red fluorescent proteins (RFPs) is one of the holy grails of the protein engineering community. Despite several attempts, finding such fluorescent proteins (FPs) has remained elusive. One bottleneck to engineering next generation RFPs is our lack of understanding of non-fluorescent or dark state properties in such constructs. Here, we develop a theoretical and experimental framework that describes how photobleaching decays in FPs relates to dark state conversion and ground state recovery. Our systematic photophysical investigation of mCherry and mCherry-d, an RFP with enhanced dark state behavior, showed the presence of photodestructive dark states in such FPs. Molecular dynamics simulations reveal enhanced fluctuation around the imidazolinone-end of the chromophore in mCherry-d, potentially facilitating conversion to non-fluorescent states. Collectively, this work quantifies dark state kinetics and gives insights into engineering dark states in RFPs to develop bright yet photostable molecular probes.

biophysics↗

bioRxiv2025

Environmental Contributions to Proton Sharing in Protein Low-Barrier Hydrogen Bonds

Hydrogen bonds (H-bonds) are central to biomolecular structure and dynamics. Although H-bonds are typically characterized by well-defined proton positions, proton delocalization can play a key role in facilitating enzyme catalysis and allostery in some systems. Experimentally locating protons is difficult, hampering the study of the proton mobility in H-bonds. We used neutron crystallography and large quantum mechanics/molecular mechanics-Born Oppenheimer molecular dynamics (QM/MM-BOMD) simulations of human DJ-1 and its bacterial homolog YajL to validate atomic resolution X-ray crystallographic bond length analysis, directly visualizing the shared deuteron in a low-barrier hydrogen bond (LBHB) between Glu14-Asp23 in YajL that is a conventional H-bond in DJ-1. In addition, X-ray bond length analysis of protiated and perdeuterated DJ-1 and YajL shows no significant effect of deuteron substitution on these carboxylic acid-carboxylate H-bonds but does reveal an effect at the active site glutamic acid. Residues in the vicinity of Glu14-Asp23 that might favor LBHB formation in YajL were interrogated by mutagenesis of homologous residues in DJ-1. X-ray bond length analysis and QM/MM-BOMD simulations demonstrate that a distal I21T DJ-1 substitution increases proton delocalization in the Glu-Asp H-bond. In addition, simulations show that the extent of proton mobility in the H-bond influences correlated dimer-spanning motions in YajL and DJ-1. In total, we show that mutations within extended H-bonding networks can modulate proton transfer barriers in carboxylic acid-carboxylate H-bonds, allowing proton delocalization to be engineered using combined bioinformatic, structural, and computational information.

biochemistry↗

bioRxiv2024

Genetically Encoded Red Fluorescent Indicators for Imaging Intracellular and Extracellular Potassium Ions

Potassium ion (K+) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K+ has impeded a comprehensive understanding of the physiological roles of K+ in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in E. coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 3.55 mM and 14.81 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K+ dynamics in various cell types and cellular environments.

biochemistry↗

bioRxiv2024

FRUCTOSE ACTIVATES A STRESS RESPONSE SHARED BY METHYLGLYOXAL AND HYDROGEN PEROXIDE IN STREPTOCOCCUS MUTANS

Fructose catabolism by Streptococcus mutans is initiated by three PTS transporters yielding fructose-1-phoshate (F-1-P) or fructose-6-phosphate. Deletion of one such F-1-P-generating PTS, fruI, was shown to reduce the cariogenicity of S. mutans in rats fed a high-sucrose diet. Moreover, a recent study linked fructose metabolism in S. mutans to a reactive electrophile species (RES) methylglyoxal. Here, we conducted a comparative transcriptomic analysis of S. mutans treated briefly with 50 mM fructose, 50 mM glucose, 5 mM methylglyoxal, or 0.5 mM hydrogen peroxide (H2O2). The results revealed a striking overlap between the fructose and methylglyoxal transcriptomes, totaling 176 genes, 61 of which were also shared with the H2O2 transcriptome. This core of 61 genes encompassed many of the same pathways affected by exposure to low pH or zinc intoxication. Consistent with these findings, fructose negatively impacted metal homeostasis of a mutant deficient in zinc expulsion and the growth of a mutant of the major oxidative stress regulator SpxA1. Importantly, fructose metabolism lowered culture pH at a faster pace, allowed better survival under acidic and nutrient-depleted conditions, and enhanced the competitiveness of S. mutans against Streptococcus sanguinis, although a moderated level of F-1-P might further boost some of these benefits. Conversely, several commensal streptococcal species displayed a greater sensitivity to fructose that may negatively affect their persistence and competitiveness in dental biofilm. In conclusion, fructose metabolism is integrated into the stress core of S. mutans and regulates critical functions required for survival and its ability to induce dysbiosis in the oral cavity. Importance.Fructose is a common monosaccharide in the biosphere, yet its overconsumption has been linked to various health problems in humans including insulin resistance, obesity, diabetes, non-alcoholic liver diseases, and even cancer. These effects are in large part attributable to the unique biochemical characteristics and metabolic responses associated with the degradation of fructose. Yet, an understanding of the effects of fructose on the physiology of bacteria and its implications to the human microbiome is severely lacking. Here we performed a series of analyses on the gene regulation of a dental pathogen Streptococcus mutans by exposing it to fructose and other important stress agents. Further supported by growth, persistence, and competition assays, our findings revealed the ability of fructose to activate a set of stress-related functions that may prove critical to the ability of the bacterium to persist and cause diseases both within and without of the oral cavity.

microbiology↗

bioRxiv2024

Glycerol Metabolism Contributes to Competition by Oral Streptococci through Production of Hydrogen Peroxide

As a biological byproduct from both humans and microbes, glycerols contribution to microbial homeostasis in the oral cavity remains understudied. Here we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source of H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate for growth, benefit from catabolism of glycerol through production of both ATP and H2O2. ImportanceGlycerol is an abundant carbohydrate found in oral cavity, both due to biological activities of humans and microbes, and as a common ingredient of foods and health care products. However, very little is understood regarding the metabolism of glycerol by some of the most abundant oral bacteria, commensal streptococci. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. Here we show that Streptococcus sanguinis, an oral commensal associated with dental health, can degrade glycerol for persistence and competition through two independent pathways, one of which generates hydrogen peroxide at levels capable of inhibiting a dental pathobiont, Streptococcus mutans. Preliminary studies suggest that several other commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are being actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis which warrants further exploration.

microbiology↗

bioRxiv2024

A spatiotemporally resolved GPCR interactome reveals novel mediators of receptor agonism

Cellular signaling by membrane G protein-coupled receptors (GPCRs) is orchestrated by a complex and diverse array of mechanisms. The dynamics of a GPCR interactome as it evolves over time and space in response to an agonist can offer a unique window on pleiotropic signaling decoding and functional selectivity at a cellular level. In this study, we employed proximity-based APEX2 proteomics to interrogate the interaction network of the GPCR for luteinizing hormone (LHR) on a minute-to-minute timescale. We developed an analytical approach integrating quantitative multiplexed proteomics and temporal reference profiles, providing a platform to identify the proteomic environment of APEX2-tagged LHR at the nanometer scale. LHR activity is exquisitely regulated at a spatial level, leading to identification of novel putative interactors including the Ras-related GTPase RAP2B that modulate both receptor signaling and post-endocytic trafficking, and providing a resource for spatiotemporal nanodomain mapping of LHR interactors across subcellular compartments. SignificanceG-protein coupled receptors (GPCRs) are the largest family of membrane proteins in the human body. GPCRs have established themselves as key drug targets, playing central roles in health and disease. The activity of this superfamily of receptors is highly dynamic, integrating diverse signaling pathways over time and across multiple subcellular compartments. Quantitative proximity proteomics using engineered ascorbate peroxidase (APEX2) has emerged as a powerful tool to map GPCR-protein interaction landscapes in both time and space. However, resolving these networks at a sub-minute timescale remains challenging, particularly for poorly characterized GPCRs with limited pharmacological tools or complex trafficking patterns. In this study, we applied APEX2 proximity proteomics to resolve at nanoscale the agonist (LH)-induced interactome of the luteinizing hormone receptor (LHR), a key GPCR in human reproduction. LHR undergoes rapid endocytosis upon LH stimulation, trafficking to a poorly understood compartment termed the very early endosome (VEE), where it engages in G protein signaling and post-endocytic sorting. Harnessing the temporal profile of GIPC1 protein, a key known interactor of LHR in the VEE pathway, we developed an analytical pipeline to identify novel endocytic interactors of LHR. Using our platform, we identified RAP2B and RAB38 as potential modulators of LHR signaling and trafficking. Overall, we present an APEX2 pipeline optimized for interrogating complex datasets, particularly for GPCRs, and potentially other membrane receptors, with limited known interactome data and minimal pharmacological tools. Our findings highlight both the complexity of GPCR interaction networks and an effective strategy to deconvolute them, providing a comprehensive resource to advance understanding of this large family of receptors. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/599010v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@13badf7org.highwire.dtl.DTLVardef@11a5182org.highwire.dtl.DTLVardef@dd5453org.highwire.dtl.DTLVardef@cb2c27_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Posteruptive Loss of Enamel Proteins Concurs with Gain in Enamel Hardness

Tooth enamel maturation requires the removal of proteins from the mineralizing enamel matrix to allow for crystallite growth until full hardness is reached to meet the mechanical needs of mastication. While this process takes up to several years in humans before the tooth erupts, it is greatly accelerated in in the faster developing pig. As a result, pig teeth erupt with softer, protein-rich enamel that is similar to hypomineralized human enamel but continues to harden quickly after eruption.Proteins, such as albumin, that bind to enamel crystals and prevent crystal growth and enamel hardening have been suggested as cause for hypomineralized human enamel that does not naturally harden after eruption. However, albumin is abundant in pig enamel. It is unclear whether fast posteruptive enamel hardening in pigs occurs despite the high protein content or requires a facilitated protein loss to allow for crystal growth. This study asked how the protein content in porcine enamel changes after eruption in relation to saliva. Based on previous data demonstrating the high albumin content in erupted porcine enamel, we hypothesize that following pre-eruptive maturation, enamel and saliva derived enzymes facilitate protein removal from porcine enamel after eruption. We analyzed enamel and the saliva proteome at three critical timepoints: at the time of tooth eruption, 2 weeks after eruption, and enamel 6 weeks after eruption. We used only fourth deciduous premolars and saliva samples from animals sacrificed at the respective time points to determine the organic content in tooth enamel, saliva, and saliva proteins within enamel. We found a decrease in the number of proteins and their abundancy in enamel with posteruptive time, including a decrease in serum albumin within enamel. The rapid decrease in the first two weeks is in line with previously reported rapid increase in mineral density of porcine enamel after eruption. In addition to the enamel proteases KLK-4 and MMP-20, we identified serine-, cysteine-, aspartic-, and metalloproteases. Some of these were only identified in enamel, while almost half of the enzymes are in common with saliva at all timepoints. Our findings suggest that the fast posteruptive enamel maturation in the porcine model coincides with saliva exchange and influx of saliva enzymes into porous enamel.

physiology↗

bioRxiv2024

Spatial targeting of the prostaglandin receptor EP2 to very early endosomes co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

Decidualization denotes the differentiation of endometrial stromal cells into specialized decidual cells, essential for embryo implantation and pregnancy. The process requires coordinated activation of both progesterone and cAMP signaling pathways, which converge on downstream transcription factors. PGE2 and relaxin, acting respectively through their Gs-coupled GPCRs EP2 and RXFP1, are putative candidates responsible for generating cAMP in differentiating stromal cells. Here, we show that PGE2 is less efficacious than relaxin in elevating intracellular cAMP levels in primary stromal cells but more effective at driving the expression of canonical decidual genes. Both PGE2- and relaxin-induced cAMP generation involves receptor internalization, but EP2 is endocytosed into very early endosomes (VEEs). Perturbation of the VEE machinery dysregulates PGE2-dependent cAMP profiles and disrupts key decidual signaling pathways, resulting in a disordered differentiation response. We demonstrate that the spatial location of EP2 is essential for coordinated activation of the downstream signaling cascades that govern decidualization.

cell biology↗

bioRxiv2022

Glucose PTS Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci

Spontaneous mutants with defects in the primary glucose phosphotransferase (PTS) permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. The observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of pyruvate and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide (IPS) metabolism, arginine deiminase, and pathways for metabolism of acetoin, ethanolamine, ascorbate and formate; along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm co-cultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis co-cultures increased the proportions of UA159 in the biofilms. Reduction of the buffer capacity or increasing the concentration of glucose benefited UA159 when co-cultured with SK36, but not with SK36/manL; likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that PTS-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. ImportanceDental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia, and improved persistence under acidic conditions. Significant metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

microbiology↗

bioRxiv2022

Activation of TnSmu1, an integrative and conjugative element, is lethal to Streptococcus mutans

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immRSmu (immunity repressor) and a metalloprotease immASmu (anti-repressor). It is not possible to create an in-frame deletion mutant of immRSmu and repression of immRSmu with CRISPRi causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immRSmu mutant. Deletion of immRSmu also leads to production of a circular intermediate form of TnSmu1, that is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems, and a Type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRASmu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

microbiology↗

bioRxiv2022

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

CRISPR-Cas is a bacterial immune system that restricts the acquisition of mobile DNA elements. These systems provide immunity against foreign DNA by encoding CRISPR spacers that help target DNA if it re-enters the cell. In this way, CRISPR spacers are a type of molecular tape recorder of foreign DNA encountered by the host microorganism. Here, we extracted [~]8,000 CRISPR spacers from a collection of over three hundred Streptococcus mutans genomes. Phage DNA is a major target of S. mutans spacers. Strains have also generated immunity against mobile DNA elements such as plasmids and integrative and conjugative elements. There may also be considerable immunity generated against bacterial DNA, although the relative contribution of self-targeting versus bona fide intra- or inter-species targeting needs to be investigated further. While there was clear evidence that these systems have acquired immunity against foreign DNA, there appeared to be minimal impact on horizontal gene transfer (HGT) constraints on a species-level. There was little or no impact on genome size, GC content and openness of the pangenome when comparing between S. mutans strains with low or high CRISPR spacer loads. In summary, while there is evidence of CRISPR spacer acquisition against self and foreign DNA, CRISPR-Cas does not act as a barrier on the expansion of the S. mutans accessory genome. Impact StatementCRISPR-Cas is a widespread bacterial immune system that has been repurposed as a molecular biology tool. This study investigates the role of these systems in the biology and evolution of the dental caries pathogen Streptococcus mutans. CRISPR spacers, that encode immunity against foreign DNA, were extracted from over three hundred S. mutans isolates. Sequence analysis showed that the CRISPR spacers match against phage, mobile element, and bacterial DNA. This shows that S. mutans is actively acquiring immunity against horizontally acquired DNA. However, additional analysis revealed little to no impact of CRISPR-Cas systems on diversification of the S. mutans genome. This suggests that while these systems are actively acquiring CRISPR spacers to defend against foreign DNA, the overall impact on the S. mutans genome might be small. Data SummarySupporting data provided on the Github platform: https://github.com/theshieldslab/Streptococcus-mutans-CRISPR-Spacers-Analysis The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2021

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage ([~]10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. ImportanceA health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

microbiology↗

bioRxiv2020

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources that are continually supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each bacterium when they were cultured alone. Likewise, co-cultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different than the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernates. Differing from a previous report, growth of S. mutans alone with GlcN inhibited expression of multiple operons required for mutacin production. Co-cultivation with S. gordonii consistently increased the expression by S. mutans of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes. Conversely, S. gordonii appeared to be less affected by the presence of S. mutans, but did show increases in genes for biosynthetic processes in the co-cultures. In conclusion, amino sugars profoundly alter the interactions between a pathogenic and commensal streptococcus by reprogramming central metabolism. IMPORTANCECarbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii) in a RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

microbiology↗

bioRxiv2019

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

A recent genome-wide screen identified [~]300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3 end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as the Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

microbiology↗

bioRxiv2019

Exploring novel probiotic mechanisms of Streptococcus A12 with functional genomics

Health-associated biofilms in the oral cavity are composed of a diverse group of microbial species that can foster an environment that is less favorable for the outgrowth of dental caries pathogens, like Streptococcus mutans. A novel oral bacterium, designated Streptococcus A12, was previously isolated from supragingival dental plaque of a caries-free individual, and was shown to interfere potently with the growth and virulence properties of S. mutans. Here, we apply functional genomics to begin to identify molecular mechanisms used by A12 to antagonize, and to resist the antagonistic factors of, S. mutans. Using bioinformatics, genes that could encode factors that enhance the ability of A12 to compete with S. mutans were identified. Selected genes, designated as potential competitive factors (pcf), were deleted. Certain mutant derivatives showed a reduced capacity to compete with S. mutans compared to the parental strain. The A12 pcfO mutant lost the ability to inhibit comX-inducing peptide (XIP) signaling by S. mutans, while mutants in the pcfFEG locus were impaired in sensing of, and were more sensitive to, the lantibiotic nisin. Loss of PcfV, annotated as a colicin V biosynthetic protein, resulted in diminished antagonism of S. mutans. Collectively, the data provide new insights into the complexities and variety of factors that affect biofilm ecology and virulence. Continued exploration of the genomic and physiologic factors that distinguish commensals from truly beneficial members of the oral microbiota will lead to a better understanding of the microbiome and new approaches to promote oral health.\n\nImportanceAdvances in defining the composition of health-associated biofilms have highlighted the important role for beneficial species in maintaining health. Comparatively little, however, has been done to address the genomic and physiological basis underlying the probiotic mechanisms of beneficial commensals. In this study, we explored the ability of a novel oral bacterial isolate, Streptococcus A12, to compete with the dental pathogen Streptococcus mutans, using various gene products with diverse functions. A12 displayed enhanced competitiveness by: i) disrupting intercellular communication pathways of S. mutans, ii) sensing and resisting antimicrobial peptides, and iii) producing factors involved in the production of a putative antimicrobial compound. Research on the probiotic mechanisms employed by Streptococcus A12 is providing essential insights into how beneficial bacteria may help maintain oral health, which will aid in the development of biomarkers and therapeutics that can improve the practice of clinical dentistry.

microbiology↗