Deciphering complex antibiotic resistance patterns in Helicobacter pylori through whole genome sequencing and machine learning.

Introduction: Helicobacter pylori (H.pylori, Hp) affects billions of people worldwide. However, the emerging resistance of Hp to antibiotics challenges the effectiveness of current treatments. Investigating the genotype-phenotype connection for Hp using next-generation sequencing could enhance our understanding of this resistance. Methods: In this study, we analyzed 52 Hp strains collected from various hospitals. The susceptibility of these strains to five antibiotics was assessed using the agar dilution assay. Whole-genome sequencing was then performed to screen the antimicrobial resistance (AMR) genotypes of these Hp strains. To model the relationship between drug resistance and genotype, we employed univariate statistical tests, unsupervised machine learning, and supervised machine learning techniques, including the development of support vector machine models. Results: Our models for predicting Amoxicillin resistance demonstrated 66% sensitivity and 100% specificity, while those for Clarithromycin resistance showed 100% sensitivity and 100% specificity. These results outperformed the known resistance sites for Amoxicillin (A1834G) and Clarithromycin (A2147), which had sensitivities of 22.2% and 87%, and specificities of 100% and 96%, respectively. Discussion: Our study demonstrates that predictive modeling using supervised learning algorithms with feature selection can yield diagnostic models with higher predictive power compared to models relying on single single-nucleotide polymorphism (SNP) sites. This approach significantly contributes to enhancing the precision and effectiveness of antibiotic treatment strategies for Hp infections. The application of whole-genome sequencing for Hp presents a promising pathway for advancing personalized medicine in this context.

Pharmacological analysis of CFTR variants of cystic fibrosis using stem cell-derived organoids.

Cystic fibrosis (CF) is a life-shortening genetic disease caused by mutations of CFTR, the gene encoding cystic fibrosis transmembrane conductance regulator. Despite considerable progress in CF therapies, targeting specific CFTR genotypes based on small molecules has been hindered because of the substantial genetic heterogeneity of CFTR mutations in patients with CF, which is difficult to assess by animal models in vivo. There are broadly four classes (e.g., II, III, and IV) of CF genotypes that differentially respond to current CF drugs (e.g., VX-770 and VX-809). In this review, we shed light on the pharmacogenomics of diverse CFTR mutations and the emerging role of stem cell-based organoids in predicting the CF drug response. We discuss mechanisms that underlie differential CF drug responses both in organoid-based assays and in CF clinical trials, thereby facilitating the precision design of safer and more effective therapies for individual patients with CF.

AppA: a web server for analysis, comparison, and visualization of contact residues and interfacial waters of antibody-antigen structures and models.

The study of contact residues and interfacial waters of antibody-antigen (Ab-Ag) structures could help in understanding the principles of antibody-antigen interactions as well as provide guidance for designing antibodies with improved affinities. Given the rapid pace with which new antibody-antigen structures are deposited in the protein databank (PDB), it is crucial to have computational tools to analyze contact residues and interfacial waters, and investigate them at different levels. In this study, we have developed AppA, a web server that can be used to analyze and compare 3D structures of contact residues and interfacial waters of antibody-antigen complexes. To the best of our knowledge, this is the first web server for antibody-antigen structures equipped with the capability for dissecting the contributions of interfacial water molecules, hydrogen bonds, hydrophobic interactions, van der Waals interactions and ionic interactions at the antibody-antigen interface, and for comparing the structures and conformations of contact residues. Various examples showcase the utility of AppA for such analyses and comparisons that could help in the understanding of antibody-antigen interactions and suggest mutations of contact residues to improve affinities of antibodies. The AppA web server is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/appa.html.

The interfacial character of antibody paratopes: analysis of antibody-antigen structures.

SUMMARY: In this study, computational methods are applied to investigate the general properties of antigen engaging residues of a paratope from a non-redundant dataset of 403 antibody-antigen complexes to dissect the contribution of hydrogen bonds, hydrophobic, van der Waals contacts and ionic interactions, as well as role of water molecules in the antigen-antibody interface. Consistent with previous reports using smaller datasets, we found that Tyr, Trp, Ser, Asn, Asp, Thr, Arg, Gly, His contribute substantially to the interactions between antibody and antigen. Furthermore, antibody-antigen interactions can be mediated by interfacial waters. However, there is no reported comprehensive analysis for a large number of structured waters that engage in higher ordered structures at the antibody-antigen interface. From our dataset, we have found the presence of interfacial waters in 242 complexes. We present evidence that suggests a compelling role of these interfacial waters in interactions of antibodies with a range of antigens differing in shape complementarity. Finally, we carry out 296 835 pairwise 3D structure comparisons of 771 structures of contact residues of antibodies with their interfacial water molecules from our dataset using CLICK method. A heuristic clustering algorithm is used to obtain unique structural similarities, and found to separate into 368 different clusters. These clusters are used to identify structural motifs of contact residues of antibodies for epitope binding. AVAILABILITY AND IMPLEMENTATION: This clustering database of contact residues is freely accessible at http://mspc.bii.a-star.edu.sg/minhn/pclick.html. CONTACT: minhn@bii.a-star.edu.sg, chandra@bii.a-star.edu.sg or zhong_pingyu@immunol.a-star.edu.sg. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters.

A novel adapter-directed yeast display system with modular features was developed. This display system consists of two modules, a display vector and a helper vector, and is capable of displaying proteins of interest on the surface of Saccharomyces cerevisiae through the interaction of two small adapters that are expressed from the display and helper vectors. In this report, an anti-VEGF scFv antibody gene was cloned into the display vector and introduced alone into yeast S. cerevisiae cells. This led to the expression and secretion of a scFv antibody that was fused in-frame with the coiled-coil adapter GR1. For display purposes, a helper vector was constructed to express the second coiled-coil adapter GR2 that was fused with the outer wall protein Cwp2, and this was genetically integrated into the yeast genome. Co-expression of the scFv-GR1 and GR2-Cwp2 fusions in the yeast cells resulted in the functional display of anti-VEGF scFv antibodies on the yeast cell surfaces through pairwise interaction between the GR1 and GR2 adapters. Visualization of the co-localization of GR1 and GR2 on the cell surfaces confirmed the adapter-directed display mechanism. When the adapter-directed phage and yeast display modules are combined, it is possible to expand the adapter-directed display to a novel cross-species display that can shuttle between phage and yeast systems.

Adapter-directed display: a modular design for shuttling display on phage surfaces.

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.