Biology subjects

Baker, J. L.

Publications and source records attributed to Baker, J. L..

18 recordsLinked to original sources

bioRxiv2026

Scaling up polarization-sensitive optical coherence tomography to image the whole macaque brain

Polarization-sensitive optical coherence tomography (PS-OCT) is a label-free imaging technique that exploits birefringence to visualize myelinated axons at micrometer resolution. However, serial PS-OCT imaging has been limited to small volumes, including tissue blocks from larger species, owing to constraints in acquisition speed, system stability, and data processing. These limitations have prevented its application to whole-brain mapping in large mammals. Here we present a scalable PS-OCT acquisition system and computational pipeline for whole-brain imaging in the rhesus macaque. The framework integrates high-throughput serial imaging with automated reconstruction and processing, enabling volumetric imaging at micrometer-scale resolution across decimeter-scale brain volumes. Using this approach, we acquired two complete macaque brains at a voxel size of 5.5 x 5.5 x 3.4 m and an effective resolution of approximately 10 x 10 x 5.5 m, generating multi-terabyte datasets consisting of multiple contrasts including fiber orientation information. The datasets and associated processing tools are made publicly available. This platform establishes a method for large-scale, high-resolution mapping of white matter architecture in primate brains. The resulting datasets provide a reference for validating MRI models and support the development of neurotechnological applications, including deep brain stimulation, where accurate characterization of axonal organization is required.

neuroscience↗

bioRxiv2026

Epigenomics identifies three sources of DNA methylation in Streptococcus mutans UA159

DNA methylation is a widespread but incompletely characterized regulatory feature of bacterial genomes. While restriction-modification systems represent well-studied sources of DNA methylation, the full complement of methyltransferases shaping bacterial epigenomes and their physiological consequences remain poorly understood. Here, we used Oxford Nanopore sequencing to comprehensively map DNA methylation in the model oral pathogen Streptococcus mutans UA159. Genome-wide analysis identified extensive N6-methyladenosine (6mA) modification and revealed three predominant methylation motifs. Using targeted deletion mutants, we demonstrate that methylation at GATC sites is mediated by the conserved DpnII restriction-modification system, while a novel bipartite CGANNNNNNNTCY/RGANNNNNNNTCA motif is methylated by the HsdM component of the type I Hsd restriction-modification system. The remaining 6mA sites corresponded to a CTGNAG/CTNCAG motif, defining the activity of a third methyltransferase. Genetic and epigenomic analyses identified SMU.43 as the enzyme responsible for this modification, which we designate DnmA, a novel orphan adenine methyltransferase with homology to regulatory methyltransferases rather than defense-associated systems. Functional characterization of single and double mutants revealed that distinct methylation systems differentially influence biofilm formation and antagonistic interactions with the commensal, Streptococcus sanguinis. Notably, loss of dnmA reversed biofilm and aggregation defects associated with deletion of dpnII, indicating epistatic interactions between methylation pathways. Together, this study resolves the major sources of DNA methylation in S. mutans UA159, identifies a novel regulatory methyltransferase, and highlights the utility of nanopore sequencing for bacterial epigenome discovery. These findings expand our understanding of bacterial DNA methylation and suggest that epigenomic enzymes may represent targets for modulation of microbial physiology and virulence.

microbiology↗

bioRxiv2026

Monounsaturated fatty acid biosynthesis is critical for streptococcal envelope homeostasis and stress tolerance

AbstractThe genus Streptococcus contains some of the most important commensals and pathogens of the human microbiome. To obtain the fatty acids required for cell membranes, Streptococcus either produce fatty acids de novo through the fatty acid biosynthesis (fab) pathway or uptake host fatty acids through the fatty acid kinase (fak) pathway. Although both the fab and fak pathways represent potential therapeutic targets to prevent or treat infection, progress is limited because of an incomplete understanding of taxon-to-taxon variability in streptococcal lipid metabolism. Here, we examined the role of de novo monounsaturated fatty acid (MUFA) synthesis in physiology and virulence-associated traits in Streptococcus mutans, Streptococcus pyogenes, and Streptococcus pneumoniae, three major pathogens that cause disease at distinct body sites. In all three species, deletion of fabM abolished MUFA production and caused severe growth defects, decreased stress tolerance, increased antibiotic susceptibility, and defects in cell viability, morphology, and division. In S. mutans, loss of fabM also markedly reduced competence signaling and production of the mutacin IV bacteriocin. Deletion of fabM increased susceptibility to killing by human neutrophils in S. mutans and S. pneumoniae, but not S. pyogenes. Together, these findings illustrate that MUFA synthesis is broadly important for streptococcal physiology and cell membrane homeostasis, while its contribution to pathogenesis is strongly species- and context-dependent, providing leads to guide development of novel therapeutic and/or preventative strategies.

microbiology↗

bioRxiv2025

A clinical grade neurostimulation implant for hierarchical control of physiological activity

Bioelectronic implants for neurostimulation aim to steer disordered neurophysiological processes back towards a healthy state. However, physiology is subject to biological rhythms, including the circadian rhythm and the sleep-wake cycle. These predictable rhythms affect disease symptomatology, biomarkers used in closed-loop therapies, and a physiological systems expected response to stimulation. Therefore, therapeutic devices should incorporate feedforward elements to align algorithm parameters with predictable changes in physiological state, as a parallel of physiological rheostatic control. Here we introduce the DyNeuMo-2c, the first clinical-grade implant capable of delivering closed-loop neurostimulation while flexibly changing its functional configuration according to time of day. The device can chronically measure brain activity and motion state to track potential biomarker patterns in natural, out-of-clinic settings, allowing identification and targeting of patient-specific chronotypes. The system implements a hierarchical control flow, with baseline therapy set by a circadian scheduler, and adaptive policies layered to take effect based on specific biomarkers indicating patient and disease state. Using a benchtop validation setup, we demonstrate that the system has the required capabilities for delivering time-contingent closed-loop therapy in two established clinical use cases: Parkinsons disease and epilepsy. Next, we deploy the system in vivo to deliver closed-loop deep brain stimulation in a healthy non-human primate model of vigilance, highlighting the importance of synchronisation between device operation and physiological state in various conditions (task performance, unconstrained behaviour, and sleep). Time-of-day-dependent adaptation of closed-loop stimulation enabled modulation of both vigilance and behaviour. Overall, the novel device architecture provides a proof-of-concept for delivering time-contingent therapy in chronic therapeutic settings where biological rhythms are of key importance.

neuroscience↗

bioRxiv2025

Comparative Characterization Reveals Conserved and Divergent Ecological Traits of Oral Corynebacteria

Corynebacteria are abundant members of the oral microbiome and increasingly recognized as key structural organizers of supragingival biofilms. Despite their prevalence, the ecological roles and phenotypic traits of many oral corynebacterial species remain poorly defined. Here, we isolated and characterized two new strains, Corynebacterium durum JJ2 and Corynebacterium argentoratense MB1, and compared them with reference strains Corynebacterium durum JJ1 and Corynebacterium matruchotii ATCC14266. Phenotypic assays revealed that C. durum strains displayed robust aggregation, thick biofilm formation, and extensive extracellular polymeric substance (EPS) networks, whereas C. argentoratense and C. matruchotii formed thinner biofilms with minimal EPS production. All four strains secreted extracellular membrane vesicles (EMVs) capable of inducing chain elongation in Streptococcus sanguinis, underscoring a conserved interspecies signaling function. Genomic analysis demonstrated close relatedness between C. durum and C. matruchotii, while C. argentoratense was more distantly related, with a reduced genome, fewer metabolic pathways, and absence of nitrate reductase genes, consistent with its inability to grow under anaerobic conditions. These findings suggest that C. argentoratense may represent a less specialized or transient inhabitant of the oral cavity, whereas C. durum and C. matruchotii are well adapted to the oral niche. Together, this study expands our understanding of phenotypic diversity, metabolic capacity, and interspecies interactions among oral corynebacteria, highlighting their potential importance as biofilm organizers and contributors to oral microbial ecology. ImportanceOral Corynebacteria contribute to the structural and ecological stability of supragingival communities. Yet, their species-level functions remain poorly defined. By isolating and characterizing new strains of Corynebacterium durum and Corynebacterium argentoratense, and comparing them with reference strains including C. matruchotii, we provide new insight into their phenotypic diversity, metabolic capacity, and ecological roles. Our results demonstrate that C. durum strains form robust biofilms enriched in extracellular polymeric substances, while C. argentoratense produces thinner biofilms and lacks the genomic features required for anaerobic growth, suggesting a less specialized or transient role in the oral cavity. Importantly, we show that extracellular membrane vesicles secreted by all tested strains promote chain elongation in Streptococcus sanguinis, highlighting a conserved mechanism of interspecies communication. These findings advance our understanding of how oral corynebacteria contribute to biofilm organization and microbial homeostasis, and position them as critical but understudied players in oral microbial ecology.

microbiology↗

bioRxiv2025

Multi-omics profiling reveals atypical sugar utilization and identifies a key membrane composition regulator in Streptococcus pneumoniae

The human body comprises many different microenvironments, each with their own challenges for microorganisms to overcome in order to survive, and possibly cause infection. The human pathogen Streptococcus pneumoniae is notoriously flexible in this regard, and can adapt to a wide range of host niches, including the nasopharynx, lungs, and cerebrospinal fluid. However, the molecular and genetic underpinnings of this ability remain largely obscure. In this work, using infection-mimicking growth conditions we demonstrate that niche adaptation imposes genome-wide changes on multiple levels, including gene essentiality, expression and membrane lipid composition. In general, we show that gene expression and fitness profiling couple orthogonal sets of genes to environmental stimuli. For instance, N-acetylglucosamine (GlcNAc) import (manLMN) and catabolism (nagAB) genes were required for growth on this sugar, but not differentially expressed in its presence, whereas other amino sugar metabolism pathways were upregulated, but not essential. Surprisingly, we found that pneumococci do not necessarily prefer glucose over GlcNAc and that uptake of GlcNAc in absence of subsequent catabolism was toxic. Moreover, we identified a previously overlooked fatty acid saturation regulator, FasR, controlling membrane composition, rendering it important during heat stress. A fundamental understanding of how genes contribute to bacterial niche adaptation, including nutrient availability or temperature fluctuations, is crucial for understanding successful antibiotic therapy and vaccination strategies and the development of novel anti-infectives.

microbiology↗

bioRxiv2024

The salivary virome during childhood dental caries

While many studies have examined the bacterial taxa associated with dental caries, the most common chronic infectious disease globally, little is known about the caries-associated virome. In this study, the salivary viromes of 21 children with severe caries (>2 dentin lesions) and 23 children with healthy dentition were examined. 2,485 viral metagenome-assembled genomes (vMAGs) were identified, binned, and quantified from the metagenomic assemblies. These vMAGs were mostly phage, and represented 1,547 unique species-level vOTUs, 247 of which appear to be novel. The metagenomes were also queried for all 3,835 unique species-level vOTUs of DNA viruses with a human host on NCBI Virus, however all but Human betaherpesvirus 7 were at very low abundance in the saliva. The oral viromes of the children with caries exhibited significantly different beta diversity compared to the oral virome of the children with healthy dentition; several vOTUs predicted to infect Pauljensenia and Neisseria were strongly correlated with health, and two vOTUs predicted to infect Saccharibacteria and Prevotella histicola, respectively, were correlated with caries. Co-occurrence analysis indicated that phage typically co-occurred with both their predicted hosts and with bacteria that were themselves associated with the same disease status. Overall, this study provided the sequences of 53 complete or nearly complete novel oral phages and illustrated the significance of the oral virome in the context of dental caries, which has been largely overlooked. This work represents an important step towards the identification and study of phage therapy candidates which treat or prevent caries pathogenesis. ImportanceDental caries is the most common chronic infectious disease, worldwide, and is caused by a dysbiosis of the oral microbiome featuring an increased abundance of acid-tolerant, acid-producing, and biofilm-forming bacteria. The oral microbiome also contains viruses; however, very little is known about the the caries-associated virome. In this study, the salivary virome of children with severe caries was compared to the salivary virome of children with healthy dentition. The metagenomes contained a total of 1,547 unique species-level vOTUs, 247 of which appeared to be novel. The viromes from the children with caries were significantly different than the viromes from the children with healthy teeth, and several health- and disease-associated vOTUs were identified. This study illustrated the importance of the oral virome in the context of dental caries, and serves as a step towards a better understanding of oral inter-kingdom interactions and identification of potential phage-based caries therapeutics.

microbiology↗

bioRxiv2024

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacteriums poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets. Significance statementWhile GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

microbiology↗

bioRxiv2023

Persistent enrichment of multidrug resistant Klebsiella in oral and nasal communities during long-term starvation

The human oral and nasal cavities can act as reservoirs for opportunistic pathogens capable of causing acute infection. These microbes asymptomatically colonize the human oral and nasal cavities which facilitates transmission within human populations via the environment, and they routinely possess a clinically-significant antibiotic-resistance genes. Among these opportunistic pathogens, the Klebsiella genus stands out as a notable example, with its members frequently linked to nosocomial infections and multidrug resistance. As with many colonizing opportunistic pathogens, how Klebsiella transitions from an asymptomatic colonizer to a pathogen remains unclear. Here, we explored a possible explanation by investigating the ability of oral and nasal Klebsiella to outcompete their native microbial community members under in vitro starvation conditions, which could be analogous to external hospital environments. When Klebsiella was present within a healthy human oral or nasal sample, the bacterial community composition shifted dramatically under starvation conditions and typically became dominated by Klebsiella. Furthermore, introducing K. pneumoniae exogenously into a native microbial community lacking K. pneumoniae, even at low inoculum, led to repeated dominance under starvation. K.pneumoniae strains isolated from healthy individuals oral and nasal cavities also exhibited resistance to multiple classes of antibiotics and were genetically similar to clinical and gut isolates. In addition, we found that in the absence of Klebsiella, other understudied opportunistic pathogens, such as Peptostreptococcus, dominate under starvation conditions. Our findings establish an environmental circumstance that allows for the outgrowth of Klebsiella and other opportunistic pathogens. The ability to outcompete other commensal bacteria and to persist under harsh environmental conditions may contribute to the colonization-to-infection transition of these opportunistic pathogens.

microbiology↗

bioRxiv2022

Using nanopore sequencing to obtain complete genomes from saliva samples

Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e. multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (aka Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, 1 Lancefieldella parvula, and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing many additional draft genomes including many which are likely near-complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the specific taxa that have never been isolated or cultivated.

microbiology↗

bioRxiv2022

mucG, mucH,and mucI modulate production of mutanocyclin and reutericyclins in Streptococcus mutans B04Sm5

Streptococcus mutans is considered a primary etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans B04Sm5 was recently shown to produce reutericyclins and mutanocyclin through the muc biosynthetic gene cluster, and to utilize reutericyclins to inhibit the growth of neighboring commensal Streptococci. In this study, examination of S. mutans and muc phylogeny suggested evolution of an ancestral S. mutans muc into three lineages within one S. mutans clade, and then horizontal transfer of muc to other S. mutans clades. The roles of the mucG and mucH transcriptional regulators, and the mucI transporter, were also examined. mucH was demonstrated to encode a transcriptional activator of muc. mucH deletion reduced production of mutanocyclin and reutericyclins, and eliminated the impaired growth and inhibition of neighboring Streptococci phenotypes which are associated with reutericyclin production. {Delta}mucG had increased mutanocyclin and reutericyclin production, which impaired growth and increased the ability to inhibit neighboring Streptococci. However, deletion of mucG also caused reduced expression of mucD, mucE, and mucI. Deletion of mucI reduced mutanocyclin and reutericylin production, but enhanced growth, suggesting that mucI may not transport reutericyclin as its homolog does in Limosilactobacillus reuteri. Further research is needed to determine the roles of mucG and mucI, and to identify any co-factors affecting the activity of the mucG and mucH regulators. Overall, this study provided pangenome and phylogenetic analysis that serves as a resource for S. mutans research, and began elucidation of the regulation of reutericyclins and mutanocyclin production in S. mutans. ImportanceS. mutans must be able to outcompete neighboring organisms in its ecological niche in order to cause dental caries. S. mutans B04Sm5 inhibited the growth of neighboring commensal Streptococci through production of reutericyclins via the muc biosynthetic gene cluster. In this study, carriage of muc was examined across S. mutans, which showed that 35 of 244 RefSeq S. mutans genomes encoded muc and provided a valuable update to the S. mutans pangenome and phylogeny. The roles of the mucG and mucH transcriptional regulators, and the mucI transporter, were also examined. All 3 genes impacted production of mutanocyclin and reutericyclins, which affected the growth rates, transcriptomes, and the ability of the S. mutans strains to inhibit the growth of neighboring commensals.

microbiology↗

bioRxiv2022

Unveiling molecular interactions that stabilize bacterial adhesion pili

Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal "staple" region of P pili significantly stabilizes the helical filament structure, consistent with previous structural data, suggesting more layer-to-layer interactions could compensate for the lack of a staple in Type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the biophysical function of pili is niche-adapted rather than a direct consequence of genetic similarity or diversity.

biophysics↗

bioRxiv2021

Analysis of the Streptococcus mutans proteome during acid and oxidative stress reveals modules of co-expression and an expanded role for the TreR transcriptional regulator

Streptococcus mutans promotes a tooth-damaging dysbiosis in the oral microbiota because it can form biofilms and survive acid stress better than most of its ecological competitors, which are typically health-associated. Many of these commensals produce hydrogen peroxide, therefore S. mutans must manage both oxidative stress and acid stress with coordinated and complex physiological responses. In this study, the proteome of S. mutans was examined during regulated growth in acid and oxidative stresses, as well as in deletion mutants with impaired oxidative stress phenotypes, {Delta}nox and {Delta}treR. 607 proteins exhibited significantly different abundance levels across the conditions tested, and correlation network analysis identified modules of co-expressed proteins that were responsive to the deletion of nox and/or treR, as well as acid and oxidative stress. The data provided evidence explaining the ROS-sensitive and mutacin-deficient phenotypes exhibited by the {Delta}treR strain. SMU.1069-1070, a poorly understood LytTR system, had elevated abundance in the {Delta}treR strain. S. mutans LytTR systems regulate mutacin production and competence, which may explain how TreR affects mutacin production. Furthermore, the gene cluster that produces mutanobactin, a lipopeptide important in ROS tolerance, displayed reduced abundance in the {Delta}treR strain. The role of Nox as a keystone in the oxidative stress response was also emphasized. Crucially, this dataset provides oral health researchers with a proteome atlas that will enable a more complete understanding of the S. mutans stress responses that are required for pathogenesis, and facilitate the development of new and improved therapeutic approaches for dental caries. ImportanceDental caries is the most common chronic infectious disease worldwide, and disproportionally affects marginalized socioeconomic groups. Streptococcus mutans is a considered a primary etiologic agent of caries, with its pathogenicity dependent on coordinated physiologic stress responses that mitigate the damage caused by the oxidative and acid stress common within dental plaque. In this study, the proteome of S. mutans was examined during growth in acidic and oxidative stresses, as well in nox and treR deletion mutants. 607 proteins were differentially expressed across the strains/growth conditions, and modules of co-expressed proteins were identified, which enabled mapping the acid and oxidative stress responses across S. mutans metabolism. The presence of TreR was linked to mutacin production via LytTR system signaling and to oxidative stress via mutanobactin production. The data provided by this study will guide future research elucidating S. mutans pathogenesis and developing improved preventative and treatment modalities for dental caries.

microbiology↗

bioRxiv2021

Complete genomes of clade G6 Saccharibacteria suggest a divergent ecological niche and lifestyle

AO_SCPLOWBSTRACTC_SCPLOWSaccharibacteria (formerly TM7) have reduced genomes, a small size, and appear to have a parasitic lifestyle dependent on a bacterial host. Although there are at least 6 major clades of Saccharibacteria inhabiting the human oral cavity, cultured isolates or complete genomes of oral Saccharibacteria have been previously limited to the G1 clade. In this study, nanopore sequencing was used to obtain three complete genome sequences from clade G6. Phylogenetic analysis suggested the presence of at least 3-5 distinct species within G6, with two discrete taxa represented by the 3 complete genomes. G6 Saccharibacteria were highly divergent from the more well-studied clade G1, and had the smallest genomes and lowest GC-content of all Saccharibacteria. Pangenome analysis showed that although 97% of shared pan-Saccharibacteria core genes and 89% of G1-specific Core Genes had putative functions, only 50% of the 244 G6-specific Core Genes had putative functions, highlighting the novelty of this group. Compared to G1, G6 encoded divergent metabolic pathways. G6 genomes lacked an F1F0 ATPase, the pentose phosphate pathway, and several genes involved in nucleotide metabolism, which were all core genes for G1. G6 genomes were also unique compared to G1 in that they encoded lactate dehydrogenase, adenylate cyclase, limited glycerolipid metabolism, a homolog to a lipoarabinomannan biosynthesis enzyme, and the means to degrade starch. These differences at key metabolic steps suggest a distinct lifestyle and ecological niche for clade G6, possibly with alternative hosts and/or host-dependencies, which would have significant ecological, evolutionary, and likely pathogenic, implications. IO_SCPLOWMPORTANCEC_SCPLOWSaccharibacteria are ultrasmall, parasitic bacteria that are common members of the oral microbiota and have been increasingly linked to disease and inflammation. However, the lifestyle and impact on human health of Saccharibacteria remains poorly understood, especially for the 5 clades (G2-G6) with no complete genomes or cultured isolates. Obtaining complete genomes is of particular importance for Saccharibacteria, because they lack many of the "essential" core genes used for determining draft genome completeness and few references exist outside of clade G1. In this study, complete genomes of 3 G6 strains, representing two candidate species, were obtained and analyzed. The G6 genomes were highly divergent from G1, and enigmatic, with 50% of the G6 core genes having no putative functions. The significant difference in encoded functional pathways is suggestive of a distinct lifestyle and ecological niche, probably with alternative hosts and/or host-dependencies, which would have major implications in ecology, evolution, and pathogenesis.

microbiology↗

bioRxiv2021

Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces that are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow force, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1 pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Looking comprehensively at structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an -helical motif that can unfold when exposed to force. A disulfide bond cross-linking {beta}-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient shear forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili. Significance StatementAdhesion pili are often essential virulence factors for attachment of pathogenic bacteria in specific environmental niches. We provide mechanistic details of structural differences impacting the biophysical properties of pili found on bacteria in the urinary and intestinal tracts. We see that pili from urinary tract bacteria are composed of subunits optimized for their microenvironment. First, they can tolerate higher forces than intestinal pili due to a disulfide bond that limits subunit unfolding. Second, their greater flexibility is due to an -helical motif that can unfold, absorbing force that could otherwise lead to bacteria detachment. Our work provides insight into the central role of pilus structural and biophysical properties for the sustained bacterial adherence necessary to initiate disease.

biophysics↗

bioRxiv2020

Characterization of structural and energetic differences between conformations of the SARS-CoV-2 spike protein

The novel coronavirus disease 2019 (COVID-19) pandemic has disrupted modern societies and their economies. The resurgence in COVID-19 cases as part of the second wave is observed across Europe and the Americas. The scientific response has enabled a complete structural characterization of the Severe Acute Respiratory Syndrome - novel Coronavirus 2 (SARS-CoV-2). Among the most relevant proteins required by the novel coronavirus to facilitate the cell entry mechanism is the spike protein trimer. This protein possesses a receptor-binding domain (RBD) that binds the cellular angiotensin-converting enzyme 2 (ACE2) and then triggers the fusion of viral and host cell membranes. In this regard, a comprehensive characterization of the structural stability of the spike protein is a crucial step to find new therapeutics to interrupt the process of recognition. On the other hand, it has been suggested the participation of more than one RBD as a possible mechanism to enhance cell entry. Here we discuss the protein structural stability based on the computational determination of the dynamic contact map and the energetic difference of the spike protein conformations via the mapping of the hydration free energy by the Poisson-Boltzmann method. We expect our result to foster the discussion of the number of RBD involved during recognition and the repurposing of new drugs to disable the recognition by discovering new hotspots for drug targets apart from the flexible loop in the RBD that binds the ACE2.

biophysics↗

bioRxiv2019

Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules

Dental caries is the most common chronic infectious disease globally. The microbial communities associated with caries have mainly been examined using relatively low-resolution 16S rRNA gene amplicon sequencing and/or using downstream analyses that are unsound for the compositional nature of the data provided by sequencing. Additionally, the relationship between caries, oral microbiome composition, and host immunological markers has not been explored. In this study, the oral microbiome and a panel of 38 host markers was analyzed across the saliva from 23 children with dentin caries and 24 children with healthy dentition. Metagenomic sequencing, followed by investigation using tools designed to be robust for compositional data, illustrated that several Prevotella spp. were prevalent in caries, while Rothia spp. were associated with the health. The contributional diversity (extent to which multiple taxa contribute to each pathway) of functional pathways present in the oral microbiome was decreased in the caries group. This decrease was especially noticeable in several pathways known to impede caries pathogenesis, including arginine and branched-chain amino acid biosynthesis. 10 host immunological markers were found to be significantly elevated in the saliva of the caries group, and microbe-metabolite co-occurrence analysis provided an atlas of relationships contributing to the bi-directional influence between the oral microbiome and the host immune system. Finally, 527 metagenome-assembled genomes were obtained from the metagenomics data, representing 151 species. 23 taxa were novel genera/species and a further 20 taxa were novel species. This study thus serves as a model analysis pipeline that will tremendously expand our knowledge of the oral microbiome and its relationship to dental caries once applied to large populations.

microbiology↗

bioRxiv2018

Identification of the bacterial biosynthetic gene clusters of the oral microbiome illuminates the unexplored social language of bacteria during health and disease

Small molecules are the primary communication media of the microbial world. Recent bioinformatics studies, exploring the biosynthetic gene clusters (BGCs) which produce many small molecules, have highlighted the incredible biochemical potential of the signaling molecules encoded by the human microbiome. Thus far, most research efforts have focused on understanding the social language of the gut microbiome, leaving crucial signaling molecules produced by oral bacteria, and their connection to health versus disease, in need of investigation. In this study, a total of 4,915 BGCs were identified across 461 genomes representing a broad taxonomic diversity of oral bacteria. Sequence similarity networking provided a putative product class for over 100 unclassified novel BGCs. The newly identified BGCs were cross-referenced against 254 metagenomes and metatranscriptomes derived from individuals with either good oral health, dental caries, or periodontitis. This analysis revealed 2,473 BGCs, which were differentially represented across the oral microbiomes associated with health versus disease. Co-abundance network analysis identified numerous inverse correlations between BGCs and specific oral taxa. These correlations were present in health, but greatly reduced in dental caries, which may suggest a defect in colonization resistance. Finally, corroborating mass spectrometry identified several compounds with homology to products of the predicted BGC classes. Together, these findings greatly expand the number of known biosynthetic pathways present in the oral microbiome and provide an atlas for experimental characterization of these abundant, yet poorly understood, molecules and socio-chemical relationships, which impact the development of caries and periodontitis, two of the worlds most common chronic diseases.\n\nIMPORTANCEThe healthy oral microbiome is symbiotic with the human host, importantly providing colonization resistance against potential pathogens. Dental caries and periodontitis are two of the worlds most common and costly chronic infectious diseases, and are caused by a localized dysbiosis of the oral microbiome. Bacterially produced small molecules, often encoded by BGCs, are the primary communication media of bacterial communities, and play a crucial, yet largely unknown, role in the transition from health to dysbiosis. This study provides a comprehensive mapping of the BGC repertoire of the human oral microbiome and identifies major differences in health compared to disease. Furthermore, BGC representation and expression is linked to the abundance of particular oral bacterial taxa in health versus dental caries and periodontitis. Overall, this study provides a significant insight into the chemical communication network of the healthy oral microbiome, and how it devolves in the case of two prominent diseases.

microbiology↗