Biology subjects

Rice, P. A.

Publications and source records attributed to Rice, P. A..

13 recordsLinked to original sources

bioRxiv2026

Structural basis for recognition of a gonococcal lipooligosaccharide epitope by monoclonal antibody 2C7 informs vaccine and immunotherapeutic design

Neisseria gonorrhoeae causes gonorrhea and poses a growing global health threat driven by antimicrobial resistance and the lack of an effective vaccine. The lipooligosaccharide (LOS) epitope recognized by monoclonal antibody (mAb) 2C7 is expressed by most clinical isolates, making it an attractive therapeutic target. To determine the molecular basis for mAb 2C7 recognition, we conducted structural studies of mAb 2C7 in complex with an octasaccharide derived from LOS. NMR epitope mapping demonstrated that mAb 2C7 contacts two of three glycan chains: the {beta}-chain (lactose) and the {gamma}-chain (N-acetylglucosamine). The 1.6-[A] resolution crystal structure of the Fab 2C7-octasaccharide complex revealed that these chains make extensive hydrogen bonds and hydrophobic contacts within a cleft of the Fab. Molecular dynamics (MD) simulations showed that the {beta} and {gamma} chains adopt stable, specific conformations, contrasted with transient interactions of the chain. Informed by these structural data, we synthesized a tetrasaccharide comprising the {beta} and {gamma} chains joined by heptose II. The tetrasaccharide conjugated to a carrier protein bound mAb 2C7 by ELISA and Western blotting. Isothermal titration calorimetry showed that the tetrasaccharide and octasaccharide bound mAb 2C7 with similar affinities, indicating that the smaller oligosaccharide retains the key binding determinants. Binding was enthalpically driven, consistent with the polar interactions observed crystallographically and by MD. NMR experiments with the tetrasaccharide confirmed interactions with all four residues, establishing it as the minimal 2C7 epitope. Together, these studies provide a structural framework for rational design of glycan-based vaccines and antibody therapeutics against antimicrobial-resistant N. gonorrhoeae.

microbiology↗

bioRxiv2025

Antagonism by the Type VI secretion system of Bacteroides fragilis is controlled by a TetR family regulator and released small molecule

Antagonistic systems of bacteria are often tightly regulated. The human gut Bacteroidales harbor three distinct antagonistic Type VI secretion systems (T6SS), one of which is present only in Bacteroides fragilis, known as the GA3 T6SS. Although this is the best studied of the three T6SSs, little is known about how it is regulated. The gene upstream of the GA3 T6SS locus encodes a TetR family transcriptional regulator (TetRGA3), which we show represses expression of the GA3 T6SS locus. The gene immediately upstream and divergently transcribed from tetRGA3, designated here as lgsGA3, encodes a product of the -oxoamine synthase family of pyridoxal phosphate-dependent enzymes with structural homology to the CqsA autoinducer synthase of the CAI-1 quorum sensing system of Vibrio spp. When lgsGA3 is deleted, transcription of the GA3 T6SS locus is repressed in a TetR-dependent manner. Strains synthesizing LgsGA3 produce a molecule present in the supernatant that likely serves as the TetRGA3 ligand, overcoming TetR transcriptional repression of the GA3 T6SS. We show that GA3 T6SS-specific immunity genes present on two acquired immunity defense islands are also regulated by LgsGA3 coordinating expression of GA3 T6SS antagonism with protection from competitors GA3 T6SS toxins. Production and firing of the GA3 T6SS and subsequent antagonism occurs in bacteria deleted for lgsGA3 when grown with bacteria containing this gene or their supernatants. These data show that the GA3 T6SS is regulated by a small molecule acting through TetRGA3 allowing the bacteria to coordinate antagonistic and protective systems. Significance statementThere are numerous external and intrinsic signals that dictate when bacteria become aggressive and when they activate their defensive systems. We show that B. fragilis strains with a GA3 T6SS synthesize a small molecule released from bacterial cells that acts through TetR family regulators to coordinate transcription of both the antagonistic GA3 T6SS and arrays of immunity genes to competitors GA3 T6SS toxins. Bacteria can respond to this molecule when released from non-kin bacteria, allowing them to sense and respond to a threat from a nearby competitor. The coordinated regulation of the GA3 T6SS and arrays of immunity genes is the second example of regulatory crosstalk between the GA3 T6SS and genes of MGEs through TetR family regulators.

microbiology↗

bioRxiv2025

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

bioRxiv2024

Large serine integrases utilise scavenged phage proteins as directionality cofactors

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favor excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinases second DNA binding domain and the base of the coiled coil domain.

molecular biology↗

bioRxiv2024

Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown

Integrases from the "large serine" family are simple, highly directional site-specific DNA recombinases that have great promise as synthetic biology and genome editing tools. Integrative recombination (mimicking phage or mobile element insertion) requires only integrase and two short ([~]40 - 50) DNA sites. The reverse reaction, excisive recombination, does not occur until it is triggered by the presence of a second protein termed a Recombination Directionality Factor (RDF), which binds specifically to its cognate integrase. Identification of RDFs has been hampered due to their lack of sequence conservation and lack of synteny with the phage integrase gene. Here we use Alphafold2-multimer to identify putative RDFs for more than half of a test set of 98 large serine recombinases, and experimental methods to verify predicted RDFs for 4 of 5 integrases chosen as test cases. We find no universally conserved structural motifs among known and predicted RDFs, yet they are all predicted to bind a similar location on their cognate integrase, suggesting convergent evolution of function. Our methodology greatly expands the available genetic toolkit of cognate integrase - RDF pairs.

synthetic biology↗

bioRxiv2023

Flagellar switch inverted repeat sequence variation impacts Clostridioides difficile RT027/MLST1 virulence.

Clostridioides difficile RT027 strains cause infections that vary in severity from asymptomatic to lethal, but the molecular basis for this variability is poorly understood. Through comparative analyses of RT027 clinical isolates, we determined that isolates that exhibit greater variability in their flagellar gene expression exhibit greater virulence in vivo. C. difficile flagellar genes are phase-variably expressed due to the site-specific inversion of the flgB 5UTR region, which reversibly generates ON vs. OFF orientations for the flagellar switch. We found that longer inverted repeat (IR) sequences in this switch region correlate with greater disease severity, with RT027 strains carrying 6A/6T IR sequences exhibiting greater phenotypic heterogeneity in flagellar gene expression (60%-75% ON) and causing more severe disease than those with shorter IRs (> 99% ON or OFF). Taken together, our results reveal that phenotypic heterogeneity in flagellar gene expression may contribute to the variable disease severity observed in C. difficile patients.

microbiology↗

bioRxiv2023

A highly conserved and globally prevalent cryptic plasmid is among the most numerous mobile genetic elements in the human gut

Plasmids are extrachromosomal genetic elements that often encode fitness enhancing features. However, many bacteria carry cryptic plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes, and is 14 times as numerous as crAssphage, currently established as the most abundant genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from ones mother. pBI143 can transfer between Bacteroidales and although it does not appear to impact bacterial host fitness in vivo, can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an inexpensive alternative for detecting human colonic inflammatory states.

microbiology↗

bioRxiv2022

Dynamic genetic adaptation of Bacteroides thetaiotaomicron murine gut colonization

To understand how a bacterium ultimately succeeds or fails in adapting to a new environment, it is essential to assess the temporal dynamics of its fitness over the course of colonization. The mammalian gut, into which exogenous microorganisms are regularly introduced, represents a biologically and clinically relevant system to explore microbial adaptational processes. In this study, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron, into the guts of germ-free mice to 1) determine whether the genetic requirements for colonization shift over time and, if so, 2) characterize the biological functions required for microbial survival at different points of colonization. The results of a high-throughput functional genetics assay (BarSeq), transcriptomics, and metabolomics converge on several conclusions. First, adaptation to the host gut occurs in distinct stages. We observed drastic changes in gene usage during the first week, shifting from high expression of amino acid biosynthesis to polysaccharide utilization genes. These changes were sustained thereafter, except for the continued upregulation of a single polysaccharide utilization locus responsible for the degradation of raffinose-family oligosaccharides rich in the standard chow diet fed to our mice. Spontaneous mutations in wildtype Bt also evolve around this locus, highlighting the importance of efficient carbohydrate metabolism in long-term persistence within a monoassociated gut. To improve microbiome-based therapies, it will be important to appreciate and meet the distinct needs of the organism during each stage of colonization. ImportanceMicrobes regularly disperse across and adapt to new environments and ecological niches. A clinically significant microbial niche home to trillions of microbes is the mammalian gut. Temporal processes of microbial adaptation over the course of gut colonization are poorly understood on a genetic, transcriptional, and metabolite level. In this study, we leverage a three-pronged approach to characterize gut colonization as a dynamic process with shifting genetic determinants of microbial fitness. This study sheds light on host colonization by Bacteroides thetaiotaomicron, an organism that is prevalent and dominant across healthy human microbiomes, and not only identifies key pathways involved in colonization, but determines the timing of when these pathways are most vital to colonization success. By demonstrating that the key determinants of colonization success in the gut change over time, the results of this study highlight the importance of considering ecological dynamics in developing more effective microbiome-based therapies.

microbiology↗

bioRxiv2022

In vitro activity of ertapenem against Neisseria gonorrhoeae clinical isolates with decreased susceptibility or resistance to extended spectrum cephalosporins in Nanjing, China (2013-2019)

ObjectiveNeisseria gonorrhoeae isolates collected in Nanjing, China, that possessed decreased susceptibility (or resistance) to extended spectrum cephalosporins (ESCs), were examined for susceptibility to ertapenem and their sequence types determined. MethodsCeftriaxone and cefixime minimum inhibitory concentrations (MICs) [≥] 0.125 mg/L and [≥] 0.25 mg/L, respectively, were first determined in 259 strains isolated between 2013 and 2019 and then MICs of ertapenem were measured using the antimicrobial gradient epsilometer test (Etest). Genetic determinants of ESC resistance and multi-antigen sequence typing (NG-MAST) were also determined to analyze associations with ertapenem susceptibility. ResultsAll isolates displayed ertapenem MICs between 0.006 mg/L-0.38 mg/L; the overall MIC50 and MIC90 were 0.032 mg/L and 0.125 mg/L. 44 (17.0%) isolates displayed ertapenem MICs of [≥] 0.125 mg/L; 10 (3.9%) had MICs [≥] 0.25 mg/L. The proportion of isolates with ertapenem MICs [≥] 0.125 mg/L increased from 4.0% in 2013, to 20.0% in 2019 ({chi}2= 24.144, P<0.001; Chi square test for linear trend). The penA mosaic allele was present in a significantly higher proportion of isolates with ertapenem MICs [≥] 0.125 mg/L compared to isolates with MICs [≤] 0.094 mg/L) (97.7% vs. 34.9%, respectively; {chi}2=58.158, P<0.001). ST5308 was the most prevalent NG-MAST type (8.5%); ST5308 was also significantly more common among isolates with ertapenem MICs [≥] 0.125 mg/L vs. isolates with MICs [≤] 0.094mg/L (22.7% and 5.6% respectively; {chi}2=13.815, P=0.001). ConclusionsErtapenem may be effective therapy for gonococcal isolates with decreased susceptibility or resistance to ESCs and isolates with identifiable genetic resistance determinants.

microbiology↗

bioRxiv2021

Structural basis for topological regulation of Tn3 resolvase

Site-specific DNA recombinases play a variety of biological roles, often related to the dissemination of antibiotic resistance, and are also useful synthetic biology tools. The simplest sitespecific recombination systems will recombine any two cognate sites regardless of context. Other systems have evolved elaborate mechanisms, often sensing DNA topology, to ensure that only one of multiple possible recombination products is produced. The closely-related resolvases from the Tn3 and {gamma}{delta} transposons have historically served as paradigms for the regulation of recombinase activity by DNA topology. However, despite many proposals, models of the multi-subunit protein-DNA complex (termed the synaptosome) that enforces this regulation have been unsatisfying due to a lack of experimental constraints and incomplete concordance with experimental data. Here we present new structural and biochemical data that lead to a new, detailed model of the Tn3 synaptosome, and discuss how it harnesses DNA topology to regulate the enzymatic activity of the recombinase.

biochemistry↗

bioRxiv2021

In vitro efficacy of gentamicin against multidrug-resistant Neisseria gonorrhoeae: synergy of three gentamicin antimicrobial combinations

ObjectivesTo determine in vitro activities of gentamicin alone and in combination with ceftriaxone, ertapenem and azithromycin against multidrug-resistant (MDR) N. gonorrhoeae isolates. Methods407 isolates from Nanjing, China, obtained in 2016 and 2017, had minimum inhibitory concentrations (MICs) determined for gentamicin using the agar dilution method. Antimicrobial combinations were also tested in 97 MDR strains using the antimicrobial gradient epsilometer test (Etest); results ranging from synergy to antagonism were interpreted using the fractional inhibitory concentration (FICI). ResultsAll 407 gonococcal isolates were susceptible to gentamicin. MICs ranged from 2 mg/L to 16 mg/L. Synergy was demonstrated in 16.5%(16/97), 27.8%(27/97) and 8.2%(8/97) MDR strains when gentamicin was combined with ceftriaxone [geometric mean (GM) FICI; 0.747], ertapenem (GM FICI; 0.662) and azithromycin (GM FICI; 1.021), respectively. No antimicrobial antagonism was observed with any combination. The three antimicrobial combinations were indifferent overall. The overall GM MICs of gentamicin were reduced by 2.63-, 3.80- and 1.98-fold when tested in combination with ceftriaxone, ertapenem and azithromycin, respectively. The GM MICs of the three antimicrobials by themselves were reduced by 3-, 2.57- and 1.98-fold respectively, when each was tested in combination with gentamicin. No antimicrobial antagonism was observed with any combination. ConclusionsGentamicin alone was effective in vitro against MDR N. gonorrhoeae and in combination with ceftriaxone, ertapenem or azithromycin. Combination testing of resistant strains, overall, showed lower effective MICs against gentamicin itself and each of the three antimicrobials when used in combination with gentamicin.

microbiology↗

bioRxiv2020

Susceptibility trends of zoliflodacin against multidrug-resistant Neisseria gonorrhoeae clinical isolates in NanJing, China (2014-2018)

Previously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 gonococcal isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC and parE genes were determined by PCR and DNA sequencing. The MIC of zoliflodacin for N. gonorrhoeae ranged from [≤]0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P[≤]0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC [≥]1 g/ml); 21.2% (209/986) were resistant to azithromycin ([≥]1 g/ml), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 143 isolates with higher zoliflodacin MICs (0.125-0.25 mg/L), all had quinolone resistance associated double or triple mutations in gyrA; 139/143 (97.2%) also had mutations in parC. There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin has excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.

microbiology↗

bioRxiv2018

Static kinks or flexible hinges: conformational distributions of bent DNA bound to integration host factor mapped by fluorescence lifetime measurements

Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H) into a U-turn by kinking DNA at two sites. We utilized fluorescence lifetime based FRET spectroscopy to map the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 80% of the IHF-H population exhibited FRET efficiency consistent with the crystal structure, 20% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks an A-tract of H on one side of the binding site, the population in the fully U-bent conformation decreased to 36%, as did the extent of bending. A similar decrease in the U-bent population was observed with a single base mutation in H in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not), and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda \"intasome\" (the complex that integrates phage lambda into the E. coli chromosome).

biophysics↗