Antigenic operon fragmentation and diversification mechanism in Bacteroidota impacts gut metagenomics and pathobionts in Crohn's disease microlesions.

Comensal Bacteroidota (Bacteroidota) and Enterobacteriacea are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in Bacteroidota remain unclear. By using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-rfb-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification, we characterized the integrity and conservancy of the entire rfb operon in Bacteroidota. Through exploratory analysis of complete genomes and metagenomes, we discovered that most Bacteroidota have the rfb operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed 'rfb-gene-clusters', or rfb-'minioperons' if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in gut-wall specific micro-niches or micropathologies. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid (Bacteroides) species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating 'extra-species' abundance. Of disease relevance, Bacteroidota species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn's Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT Enterobacteriaceae. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn's disease microlesions.

Candida tropicalis Affects Candida albicans Virulence by Limiting Its Capacity to Adhere to the Host Intestinal Surface, Leading to Decreased Susceptibility to Colitis in Mice.

Candida (C.) infections represent a serious health risk for people affected by inflammatory bowel disease. An important fungal virulence factor is the capacity of the fungus to form biofilms on the colonized surface of the host. This research study aimed to determine the effect of a C. tropicalis and C. albicans co-infection on dextran sodium sulfate (DSS)-induced colitis in mice. The colitis severity was evaluated using histology and a colonoscopy. The mice were mono-inoculated with C. albicans or C. tropicalis or co-challenged with both species. The mice were administered 3% DSS to induce acute colitis. The biofilm activity was assessed using (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] 2H-tetrazoliumhydroxide (XTT) and dry-weight assays. The abundance of C. albicans in the colon tissues was assessed by immunohistochemistry. The co-challenged mice showed a decreased colitis severity compared to the mono-inoculated mice. The dry-weight assay demonstrated a marked decrease in C. albicans biofilm production in a C. albicans culture incubated with C. tropicalis supernatant. Immunohistochemical staining showed that C. albicans was more abundant in the mucosa of C. albicans mono-inoculated mice compared to the co-inoculated group. These data indicate an antagonistic microbial interaction between the two Candida species, where C. tropicalis may produce molecules capable of limiting the ability of C. albicans to adhere to the host intestinal surface, leading to a reduction in biofilm formation.

The basis of antigenic operon fragmentation in Bacteroidota and commensalism.

The causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism within the phylum Bacteroidota remain unclear (1, 2). Using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification (3), we characterized the architecture/conservancy of the entire rfb operon in Bacteroidota. Analyzing complete genomes, we discovered that most Bacteroidota have the rfb operon fragmented into non-random gene-singlets and/or doublets/triplets, termed 'minioperons'. To reflect global operon integrity, duplication, and fragmentation principles, we propose a five-category (infra/supernumerary) cataloguing system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in specific micro-niches. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes (4). DNA insertions overrepresenting DNA-exchange-avid species, impact functional metagenomics by inflating gene-based pathway inference and overestimating 'extra-species' abundance. Using bacteria from inflammatory gut-wall cavernous micro-tracts (CavFT) in Crohn's Disease (5), we illustrate that bacteria with supernumerary-fragmented operons cannot produce O-antigen, and that commensal/CavFT Bacteroidota stimulate macrophages with lower potency than Enterobacteriaceae, and do not induce peritonitis in mice. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism offers potential for novel diagnostics and therapeutics.

The Weissella Genus: Clinically Treatable Bacteria with Antimicrobial/Probiotic Effects on Inflammation and Cancer.

Weissella is a genus earlier considered a member of the family Leuconostocaceae, which was reclassified into the family Lactobacillaceae in 1993. Recently, there have been studies emphasizing the probiotic and anti-inflammatory potential of various species of Weissella, of which W. confusa and W. cibaria are the most representative. Other species within this genus include: W. paramesenteroides, W. viridescens, W. halotolerans, W. minor, W. kandleri, W. soli, W. ghanensis, W. hellenica, W. thailandensis, W. fabalis, W. cryptocerci, W. koreensis, W. beninensis, W. fabaria, W. oryzae, W. ceti, W. uvarum, W. bombi, W. sagaensis, W. kimchi, W. muntiaci, W. jogaejeotgali, W. coleopterorum, W. hanii, W. salipiscis, and W. diestrammenae. Weissella confusa, W. paramesenteroides, W. koreensis, and W. cibaria are among the few species that have been isolated from human samples, although the identification of these and other species is possible using metagenomics, as we have shown for inflammatory bowel disease (IBD) and healthy controls. We were able to isolate Weissella in gut-associated bacteria (post 24 h food deprivation and laxatives). Other sources of isolation include fermented food, soil, and skin/gut/saliva of insects/animals. With the potential for hospital and industrial applications, there is a concern about possible infections. Herein, we present the current applications of Weissella on its antimicrobial and anti-inflammatory mechanistic effects, the predisposing factors (e.g., vancomycin) for pathogenicity in humans, and the antimicrobials used in patients. To address the medical concerns, we examined 28 case reports focused on W. confusa and found that 78.5% of infections were bacteremia (of which 7 were fatal; 1 for lack of treatment), 8 were associated with underlying malignancies, and 8 with gastrointestinal procedures/diseases of which 2 were Crohn's disease patients. In cases of a successful resolution, commonly administered antibiotics included: cephalosporin, ampicillin, piperacillin-tazobactam, and daptomycin. Despite reports of Weissella-related infections, the evolving mechanistic findings suggest that Weissella are clinically treatable bacteria with emerging antimicrobial and probiotic benefits ranging from oral health, skin care, obesity, and inflammatory diseases to cancer.

An Immunocompromised Mouse Model of Candida auris Systemic Infection.

With the recent emergence of multidrug-resistant Candida auris, there is an urgent need for new antifungal compounds with novel pharmacodynamic and pharmacokinetic properties that can treat systemic fungal infections caused by this emerging yeast. Historically, testing the efficacy of treatment for disseminated candidiasis was accomplished using a diverse array of in vivo animal models, including mice which offer an advantage both in their similarities to humans and their lower cost of maintenance. However, in order to create effective in vivo models for testing new antifungal compounds designed to treat systemic infections, it is important that these models also mimic several of the relevant predisposing conditions that can lead to disseminated candidiasis. Here, we describe an immunocompromised mouse model of hematogenously disseminated C. auris infection, which may have utility to test the efficacy of candidate antifungal compounds.

Fast search algorithms for computational protein design.

One of the main challenges in computational protein design (CPD) is the huge size of the protein sequence and conformational space that has to be computationally explored. Recently, we showed that state-of-the-art combinatorial optimization technologies based on Cost Function Network (CFN) processing allow speeding up provable rigid backbone protein design methods by several orders of magnitudes. Building up on this, we improved and injected CFN technology into the well-established CPD package Osprey to allow all Osprey CPD algorithms to benefit from associated speedups. Because Osprey fundamentally relies on the ability of A* to produce conformations in increasing order of energy, we defined new A* strategies combining CFN lower bounds, with new side-chain positioning-based branching scheme. Beyond the speedups obtained in the new A*-CFN combination, this novel branching scheme enables a much faster enumeration of suboptimal sequences, far beyond what is reachable without it. Together with the immediate and important speedups provided by CFN technology, these developments directly benefit to all the algorithms that previously relied on the DEE/ A* combination inside Osprey* and make it possible to solve larger CPD problems with provable algorithms.

The Relationship of Foot Strike Pattern, Shoe Type, and Performance in a 50-km Trail Race.

Recent "in-race" studies have observed the foot strike patterns of runners in traditional road marathon races. However, similar studies have not been conducted for trail runners, which have been estimated to account for 11% of all runners. The purpose of this study was to (a) determine the rear-foot strike (RFS) prevalence in a 50-km trail race and compare with traditional road marathon races; (b) determine if there is a relationship between foot strike and sex in a 50-km trail race; and (c) determine if there is a relationship between foot strike, shoe type, and performance in a 50-km trail race. One hundred sixty-five runners were videotaped at the 8.1-km mark of the 2012 Ice Age Trail 50-km race. Foot strike analysis revealed RFS prevalence of 85.1%, less than previously reported in traditional road marathon races. There was no relationship found between sex and foot strike (p = 0.60). A significant effect of shoe type on foot strike (RFS was less common among runners in minimalist shoes, p < 0.01) and performance (faster runners were more likely to be wearing minimalist shoes, p < 0.01) was observed; however, no association between foot strike and performance was observed (p = 0.83). This study suggests that most trail runners, albeit less than road runners, prefer an RFS pattern, which is accompanied by biomechanical consequences unique from a non-RFS pattern and, therefore, likely carries a unique injury profile. In addition, the findings in this study suggest that minimalist shoes may represent a reasonable training modification to improve performance.

Fast gap-free enumeration of conformations and sequences for protein design.

Despite significant successes in structure-based computational protein design in recent years, protein design algorithms must be improved to increase the biological accuracy of new designs. Protein design algorithms search through an exponential number of protein conformations, protein ensembles, and amino acid sequences in an attempt to find globally optimal structures with a desired biological function. To improve the biological accuracy of protein designs, it is necessary to increase both the amount of protein flexibility allowed during the search and the overall size of the design, while guaranteeing that the lowest-energy structures and sequences are found. DEE/A*-based algorithms are the most prevalent provable algorithms in the field of protein design and can provably enumerate a gap-free list of low-energy protein conformations, which is necessary for ensemble-based algorithms that predict protein binding. We present two classes of algorithmic improvements to the A* algorithm that greatly increase the efficiency of A*. First, we analyze the effect of ordering the expansion of mutable residue positions within the A* tree and present a dynamic residue ordering that reduces the number of A* nodes that must be visited during the search. Second, we propose new methods to improve the conformational bounds used to estimate the energies of partial conformations during the A* search. The residue ordering techniques and improved bounds can be combined for additional increases in A* efficiency. Our enhancements enable all A*-based methods to more fully search protein conformation space, which will ultimately improve the accuracy of complex biomedically relevant designs.

Improved energy bound accuracy enhances the efficiency of continuous protein design.

Flexibility and dynamics are important for protein function and a protein's ability to accommodate amino acid substitutions. However, when computational protein design algorithms search over protein structures, the allowed flexibility is often reduced to a relatively small set of discrete side-chain and backbone conformations. While simplifications in scoring functions and protein flexibility are currently necessary to computationally search the vast protein sequence and conformational space, a rigid representation of a protein causes the search to become brittle and miss low-energy structures. Continuous rotamers more closely represent the allowed movement of a side chain within its torsional well and have been successfully incorporated into the protein design framework to design biomedically relevant protein systems. The use of continuous rotamers in protein design enables algorithms to search a larger conformational space than previously possible, but adds additional complexity to the design search. To design large, complex systems with continuous rotamers, new algorithms are needed to increase the efficiency of the search. We present two methods, PartCR and HOT, that greatly increase the speed and efficiency of protein design with continuous rotamers. These methods specifically target the large errors in energetic terms that are used to bound pairwise energies during the design search. By tightening the energy bounds, additional pruning of the conformation space can be achieved, and the number of conformations that must be enumerated to find the global minimum energy conformation is greatly reduced.

Enhanced potency of a broadly neutralizing HIV-1 antibody in vitro improves protection against lentiviral infection in vivo.

UNLABELLED: Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE: In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.

OSPREY: protein design with ensembles, flexibility, and provable algorithms.

UNLABELLED: We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) improved flexibility of the protein backbone, protein side-chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe OSPREY, a protein redesign suite that implements our protein design algorithms. OSPREY has been used prospectively, with experimental validation, in several biomedically relevant settings. We show in detail how OSPREY has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application. AVAILABILITY: OSPREY is free and open source under a Lesser GPL license. The latest version is OSPREY 2.0. The program, user manual, and source code are available at www.cs.duke.edu/donaldlab/software.php. CONTACT: osprey@cs.duke.edu.

Foot-strike pattern and performance in a marathon.

PURPOSE: To determine prevalence of heel strike in a midsize city marathon, if there is an association between foot-strike classification and race performance, and if there is an association between foot-strike classification and gender. METHODS: Foot-strike classification (forefoot, midfoot, heel, or split strike), gender, and rank (position in race) were recorded at the 8.1-km mark for 2112 runners at the 2011 Milwaukee Lakefront Marathon. RESULTS: 1991 runners were classified by foot-strike pattern, revealing a heel-strike prevalence of 93.67% (n = 1865). A significant difference between foot-strike classification and performance was found using a Kruskal-Wallis test (P < .0001), with more elite performers being less likely to heel strike. No significant difference between foot-strike classification and gender was found using a Fisher exact test. In addition, subgroup analysis of the 126 non-heel strikers found no significant difference between shoe wear and performance using a Kruskal-Wallis test. CONCLUSIONS: The high prevalence of heel striking observed in this study reflects the foot-strike pattern of most mid-distance to long-distance runners and, more important, may predict their injury profile based on the biomechanics of a heel-strike running pattern. This knowledge can help clinicians appropriately diagnose, manage, and train modifications of injured runners.

Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors"), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers") that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

Protein design using continuous rotamers.

UNLABELLED: Optimizing amino acid conformation and identity is a central problem in computational protein design. Protein design algorithms must allow realistic protein flexibility to occur during this optimization, or they may fail to find the best sequence with the lowest energy. Most design algorithms implement side-chain flexibility by allowing the side chains to move between a small set of discrete, low-energy states, which we call rigid rotamers. In this work we show that allowing continuous side-chain flexibility (which we call continuous rotamers) greatly improves protein flexibility modeling. We present a large-scale study that compares the sequences and best energy conformations in 69 protein-core redesigns using a rigid-rotamer model versus a continuous-rotamer model. We show that in nearly all of our redesigns the sequence found by the continuous-rotamer model is different and has a lower energy than the one found by the rigid-rotamer model. Moreover, the sequences found by the continuous-rotamer model are more similar to the native sequences. We then show that the seemingly easy solution of sampling more rigid rotamers within the continuous region is not a practical alternative to a continuous-rotamer model: at computationally feasible resolutions, using more rigid rotamers was never better than a continuous-rotamer model and almost always resulted in higher energies. Finally, we present a new protein design algorithm based on the dead-end elimination (DEE) algorithm, which we call iMinDEE, that makes the use of continuous rotamers feasible in larger systems. iMinDEE guarantees finding the optimal answer while pruning the search space with close to the same efficiency of DEE. AVAILABILITY: Software is available under the Lesser GNU Public License v3. Contact the authors for source code.

A Bayesian approach for determining protein side-chain rotamer conformations using unassigned NOE data.

A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein side-chain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which records the through-space dipolar interactions between protons nearby in three-dimensional (3D) space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A* search algorithms to provably find the global optimum solution that maximizes the posterior probability. We employ a Hausdorff-based measure to derive the likelihood of a rotamer or a pairwise rotamer interaction from unassigned NOESY data. In addition, we apply a systematic and rigorous approach to estimate the experimental noise in NMR data, which also determines the weighting factor of the data term in the scoring function derived from the Bayesian framework. We tested our approach on real NMR data of three proteins: the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), and human ubiquitin. The promising results indicate that our algorithm can be applied in high-resolution protein structure determination. Since our approach does not require any NOE assignment, it can accelerate the NMR structure determination process.

Position of helical kinks in membrane protein crystal structures and the accuracy of computational prediction.

The structural features of helical transmembrane (TM) proteins, such as helical kinks, tilts, and rotational orientations are important in modulation of their function and these structural features give rise to functional diversity in membrane proteins with similar topology. In particular, the helical kinks caused by breaking of the backbone hydrogen bonds lead to hinge bending flexibility in these helices. Therefore it is important to understand the nature of the helical kinks and to be able to reproduce these kinks in structural models of membrane proteins. We have analyzed the position and extent of helical kinks in the transmembrane helices of all the crystal structures of membrane proteins taken from the MPtopo database, which are about 405 individual helices of length between 19 and 35 residues. 44% of the crystal structures of TM helices showed a significant helical kink, and 35% of these kinks are caused by prolines. Many of the non-proline helical kinks are caused by other residues like Ser and Gly that are located at the center of helical kinks. The side chain of Ser makes a hydrogen bond with the main chain carbonyl of the i - 4th or i + 4th residue thus making a kink. We have also studied how well molecular dynamics (MD) simulations on isolated helices can reproduce the position of the helical kinks in TM helices. Such a method is useful for structure prediction of membrane proteins. We performed MD simulations, starting from a canonical helix for the 405 TM helices. 1 ns of MD simulation results show that we can reproduce about 79% of the proline kinks, only 59% of the vestigial proline kinks and 18% of the non-proline helical kinks. We found that similar results can be obtained from choosing the lowest potential energy structure from the MD simulation. 4-14% more of the vestigial prolines were reproduced by replacing them with prolines before performing MD simulations, and changing the amino acid back to proline after the MD simulations. From these results we conclude that the position of the helical kinks is dependent on the TM sequence. However the extent of helical kinking may depend on the packing of the rest of the protein and the lipid bilayer.