Nonconservative Amino Acid Research Articles

Recent Molecular Epidemiology of Echovirus 11 Throughout North and West Africa Resulted in the First Identification of a Recombinant Strain from an Acute Flaccid Paralysis Case in West Africa

Echovirus 11 has emerged as a major public health concern, causing sepsis in neonates in many European countries in recent years. In Africa, especially West Africa, where resources and diagnostic capacities are limited, only sporadic cases have been reported. To better understand the recent molecular epidemiology of E11 in West Africa, we characterized twenty-three echovirus 11 strains isolated through the acute flaccid paralysis and environmental surveillance systems for polio from 2013 to 2023, using high-throughput sequencing. Our data are noteworthy due to identifying for the first time a recombinant strain from an acute flaccid paralysis case and represent the first focus to date on molecular characterization of echovirus 11 in West Africa. Moreover, our data show that echovirus 11 diverged from 1970 (95% HPD range, 1961–1979) and evolved into four distinct clades, with the virus spread from West Africa to Europe, exhibiting two introductions in France around 2017, from Senegal and Guinea. Furthermore, the in silico analysis reveals four non-conservative amino acid substitutions in the VP1 sequences of the European strains associated with neonatal sepsis in newborns and a conserved amino acid motif in the VP1 protein toward enterovirus genotypes. Our data provide new insights into the epidemiology of echovirus 11 and point to the crucial need to implement specific surveillance programs targeting non-polio enteroviruses for the rapid identification of emerging or re-emerging enterovirus species, particularly in Africa.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a β1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Docking interactions determine substrate specificity of members of a widespread family of protein phosphatases

How protein phosphatases achieve specificity for their substrates is a major outstanding question. PPM family serine/threonine phosphatases are widespread in bacteria and eukaryotes, where they dephosphorylate target proteins with a high degree of specificity. In bacteria, PPM phosphatases control diverse transcriptional responses by dephosphorylating anti-anti-sigma factors of the STAS domain family, exemplified by B. subtilis phosphatases SpoIIE, which controls cell-fate during endospore formation, and RsbU, which initiates the General Stress Response. Using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, we identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domains of SpoIIE and RsbU that recognize the three-dimensional structure of the substrate protein. Non-conserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the non-cognate substrate. Together, single-amino acid substitutions in these three elements cause an over five-hundred fold change in specificity. Our data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. We hypothesize that this is a generalizable mechanistic model for PPM family phosphatase substrate specificity. Importantly, the substrate docking interface with the phosphatase is only partially overlapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.

The Truncated Peptide AtPEP1(9-23) Has the Same Function as AtPEP1(1-23) in Inhibiting Primary Root Growth and Triggering of ROS Burst.

Currently, the widely used active form of plant elicitor peptide 1 (PEP1) from Arabidopsis thaliana is composed of 23 amino acids, hereafter AtPEP1(1-23), serving as an immune elicitor. The relatively less conserved N-terminal region in AtPEP family indicates that the amino acids in this region may be unrelated to the function and activity of AtPEP peptides. Consequently, we conducted an investigation to determine the necessity of the nonconserved amino acids in AtPEP1(1-23) peptide for its functional properties. By assessing the primary root growth and the burst of reactive oxygen species (ROS), we discovered that the first eight N-terminal amino acids of AtPEP1(1-23) are not crucial for its functionality, whereas the conserved C-terminal aspartic acid plays a significant role in its functionality. In this study, we identified a truncated peptide, AtPEP1(9-23), which exhibits comparable activity to AtPEP1(1-23) in inhibiting primary root growth and inducing ROS burst. Additionally, the truncated peptide AtPEP1(13-23) shows similar ability to induce ROS burst as AtPEP1(1-23), but its inhibitory effect on primary roots is significantly reduced. These findings are significant as they provide a novel approach to explore and understand the functionality of the AtPEP1(1-23) peptide. Moreover, exogenous application of AtPEP1(13-23) may enhance plant resistance to pathogens without affecting their growth and development. Therefore, AtPEP1(13-23) holds promise for development as a potentially applicable biopesticides.

Abstract 5292: CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies

Abstract Agonist antibodies targeting CD28 have proven to be effective against cancer, but also faced challenges due to severe adverse events triggered by its activation. Human (hCD28) and mouse CD28 (mCD28) have different signaling responses, with CD28 ligands and superagonists inducing pro-inflammatory cytokines upon stimulation in absence of TCR ligation in humans, but not in mice. Expression of CD28i amplifier isoform, which is thought to enhanced the production of cytokines in humans, could partially explain this difference, as it is not expressed in mice. In addition, evidence suggests that the different signaling between hCD28 and mCD28 relies on one amino acid change in the intracellular domain (ICD)[1]. Herein, we describe a CD28 humanized mouse model for assessment of CD28-targeting agents. To improve translatability, we decided to keep the expression of both canonical and CD28i human isoforms to avoid undermining the biological effects of the testing agents. Although keeping the human ICD could favor the evaluation of cytokine production and therefore the safety of the test agents, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signaling partners, allowing a physiological stimulation of CD28 in efficacy studies. The hCD28 model expresses a chimeric protein, with the whole human extracellular domain, and mouse intracellular domain. Human CD28 is expressed at physiological levels on T cells, and these cells can be activated and proliferate in response to hCD28 ligands and agonist antibodies. Tumor growth inhibition and complete tumor regressions are achieved in a MC38-TAA colorectal cancer model following prophylactic treatment with CD28-TAA bispecific antibody in combination with anti-PD-1. Extension of this study and re-challenged of the tumor-free surviving animals shows that re-challenged hCD28 mice have enhanced survival compared to naïve hCD28 mice inoculated with MC38-TAA, suggesting tumor immunological memory. Furthermore, TGN1412 administration in CD28 mice induces cytokine release, and a slightly body weight loss, but no major change in body temperature. Although the model does not fully reproduce the ability of human CD28 to produce cytokines upon activation due to the mouse ICD, the expression of the amplifier isoform CD28i might contribute to the response to TGN1412. Altogether, data suggest that hCD28 model enables assessment of efficacy and safety of CD28-targeting agents. The hCD28 model was intercrossed with CD3 humanized models to enable assessment of combination therapies and bispecific antibodies targeting both CD3 and CD28. [1] Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1-16 Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Yacine Cherifi, Patricia Isnard-Petit, Kader Thiam. CD28 humanized mouse model for efficacy and safety assessment of CD28-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5292.

Unveiling polymorphism and protein structure prediction insights in diacylglycerol O-acyltransferase 1 and telethonin genes of Egyptian buffalo

BackgroundThe Egyptian buffalo has a sizable impact on Egypt's agricultural sector and food supply. It is regarded as the main dairy animal and an important source of red meat. This study aimed to detect the polymorphisms of the DGAT1 and TCAP genes and assess the potential impact of the discovered nsSNPs on the stability of the tertiary structure polypeptides of selected genes in Egyptian buffalo.MethodsAllele identification was made by the restriction fragment length polymorphism (RFLP), and the single nucleotide polymorphisms (SNPs) were recognized by sequencing the purified PCR products. Protein translation indicated the synonymous and non-synonymous SNPs, and the peptides' 3D tertiary structure of selected genes, as well as the effect of amino acid substitution on the protein structure, was performed using bioinformatics tools.ResultsAnalysis of the data revealed that an nsSNP was detected in a tested region of the DGAT1 gene and caused an amino acid substitution in a polypeptide that was predicted to be neutral and located in the coiled part of the protein. The analysis of the TCAP gene showed four nsSNPs that caused four substitutions located in the α-helix region. Protein prediction analysis showed that the amino acid substitutions in DGAT1 and TCAP were non-conserved with low sensitivity to variation. The non-conservative amino acid substitutions result in amino acids with new properties different from the original amino acid that change the protein's structure and function.ConclusionWe can infer that the DGAT1 and TCAP genes' SNPs may affect meat-related traits and may improve meat quality.

Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

Using a novel structure/function approach to select diverse swine major histocompatibility complex 1 alleles to predict epitopes for vaccine development.

Swine leukocyte antigens (SLAs) (i.e. swine major histocompatibility complex proteins) conduct a fundamental role in swine immunity. To generate a protective vaccine across an outbred species, such as pigs, it is critical that epitopes that bind to diverse SLA alleles are used in the vaccine development process. We introduced a new strategy for epitope prediction. We employed molecular dynamics simulation to identify key amino acids for interactions with epitopes. We developed an algorithm wherein each SLA-1 is compared to a crystalized reference allele with unique weighting for non-conserved amino acids based on R group and position. We then performed homology modeling and electrostatic contact mapping to visualize how relatively small changes in sequences impacted the charge distribution in the binding site. We selected eight diverse SLA-1 alleles and performed homology modeling followed, by protein-peptide docking and binding affinity analyses, to identify porcine reproductive and respiratory syndrome virus matrix protein epitopes that bind with high affinity to these alleles. We also performed docking analysis on the epitopes identified as strong binders using NetMHCpan 4.1. Epitopes predicted to bind to our eight SLA-1 alleles had equivalent or higher energetic interactions than those predicted to bind to the NetMHCpan 4.1 allele repertoire. This approach of selecting diverse SLA-1 alleles, followed by homology modeling, and docking simulations, can be used as a novel strategy for epitope prediction that complements other available tools and is especially useful when available tools do not offer a prediction for SLAs/major histocompatibility complex. The data underlying this article are available in the online Supplementary Material.

The Bactericidal Activity of a Novel Aneurinibacillus aneurinilyticus Isolate Effectively Controls Foodborne Pathogens Campylobacter jejuni and Listeria monocytogenes

Human zoonotic infection with Campylobacter is a major cause of gastroenteritis in the United States and worldwide. Listeria monocytogenes causes a potentially fatal infection in humans and is often attributed to contaminated food. Genetic typing has demonstrated that Campylobacter infection is often associated with the consumption of contaminated poultry products, with Campylobacter often colonizing the poultry gastrointestinal tract, while listeriosis is commonly associated with the consumption of contaminated ready-to-eat (RTE) foods. In this study, a strain of endospore-forming bacterium (strain NH) that is bactericidal towards the human food pathogens Campylobacter jejuni and Listeria monocytogenes was identified and characterized. Transwell experiments demonstrated that the bactericidal effect on both C. jejuni and L. monocytogenes is due to secretions from the spore former. These foodborne pathogens consistently exhibited 7 log reductions in growth when exposed to the NH cell-free culture filtrate. Sequencing of the 16s rRNA gene V4 variable region and analysis of the full-length 16s rRNA gene sequence from the WGS indicated that strain NH belongs to the species Aneurinibacillus aneurinilyticus. A microplate bioassay demonstrated that a bactericidal substance that is sensitive to protease could be collected from cell-free filtrates by salting out with ammonium sulfate. Gel filtration chromatography indicated a native molecular weight for the bactericidal protein of ca. 50 kDa, consistent with a class III bacteriocin. The active protein bound strongly to a cation-exchange resin and with an isoelectric point of ten, suggesting a positively charged protein. Both cation-exchange chromatography and isoelectric focusing indicated the enrichment of an 11 kDa protein on SDS-PAGE. This protein was identified through mass spectroscopy as the flgM protein, an anti-sigma factor. Analysis of whole genome sequencing (WGS) of the strain NH genome indicated the presence of a number of non-conservative amino acid substitutions in the flgM-gene-derived amino acid sequence of strain NH and A. aneurinilyticus compared to other members of the Aneurinibacillus genus. Further investigation is needed to determine whether these substitutions are correlated with the bactericidal activity. The identified strain may be useful as a feed additive for the pre-harvest control of Campylobacter jejuni in poultry.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence

ABSTRACT Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

External amino acid residues of insect GABA receptor channels dictate the action of the isoxazoline ectoparasiticide fluralaner.

Fluralaner is the first isoxazoline ectoparasiticide developed to protect companion animals from fleas and ticks. Fluralaner primarily inhibits arthropod γ-aminobutyric acid receptors (GABARs), which are ligand-gated ion channels comprising five subunits arranged around the channel pore. We previously reported that the action site of fluralaner resides at the M1-M3 transmembrane interface between adjacent GABAR subunits. To investigate whether fluralaner interacts with the second transmembrane segment (M2) located deep in the interface, we generated four housefly RDL GABAR mutants with non-conservative amino acid substitutions in the M2 region. Electrophysiological analysis of GABARs expressed in Xenopus oocytes indicated that S313A and S314A mutants exhibited fluralaner sensitivities similar to that of the wild type. M312S mutant exhibited approximately seven-fold lower sensitivity than that of the wild type. Notably, the N316L mutant was almost insensitive to fluralaner. The findings of this study indicate that the conserved external amino acid residues of insect GABAR channels play a critical role in the antagonistic effect of fluralaner. © 2023 Society of Chemical Industry.

PO-01-158 MODELING AN ATRIAL FIBRILLATION-CAUSING TITIN MUTATION USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Atrial fibrillation (AF) affects more than 33 million people worldwide and, if left untreated, can lead to increased morbidity and mortality. Although AF has been classified as an ‘ion channelopathy’, next-generation sequencing has identified mutations in sarcomeric proteins like TTN, the gene encoding titin, that are associated with AF. Despite progress in understanding the genetic basis of AF, the translation of these discoveries to the bedside care of patients has been limited due to challenges in understanding the myocardial substrate for AF, especially that develops from the mutation in the structural protein. As human atrial tissue is rarely available and animal models are mechanistically limited, our goal is to model AF using mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) by a CRISPR-Cas9-mediated single amino acid substitution of a rare missense ethnic-specific mutation (TTN_T32756I) in an immunoglobulin (Ig)-like domain of the A-band of titin that is enriched in AF-causing pathogenic variants. Isogenic TTN_T32756I hiPSC-aCM mutants were generated using the CRISPR-Cas9 technique. Cells were matured either with metabolic conditioning or co-culturing with atrial fibroblasts in a micropatterned platform. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping to assess the electrophysiological properties of TTN_T32756I hiPSC-aCMs. T32756I results in a non-conservative amino acid change, which in-silico tools have previously predicted results in an impairment in titin function. Here, we show that TTN_T32756I hiPSC-aCM mutants display aberrant contractility (Fig. 1A-C) and Ca2+ homeostasis (Fig. 1D) leading to abnormal electrophysiology (Fig. 1E-F) at the cellular level without compromising the sarcomeric integrity of the atrial cardiomyocytes (Fig. 1G-H). Our results suggest that a single amino acid change could have a profound effect on titin function, further establishing titin as a major signaling hub for contractility. Ongoing studies will not only identify the key pathways and transcription factors involved in the pathogenesis of TTN_ T32756I mutant but also uncover the impact of the mutation on atrial action potential and cardiac ion channel activity. Our study will shed new light on the mechanisms by which TTN mutation cause AF and identify novel therapeutic targets for patients carrying AF-associated rare variants in the structural genes.

Genome-wide scan for oil quality reveals a coregulation mechanism of tocopherols and fatty acids in soybean seeds

Tocopherols (vitamin E) play essential roles in human health because of their antioxidant activity, and plant-derived oils are the richest sources of tocopherols in the human diet. Although soybean (Glycine max) is one of the main sources of plant-derived oil and tocopherol in the world, the relationship between tocopherol and oil in soybean seeds remains unclear. Here, we focus on dissecting tocopherol metabolism with the long-term goal of increasing α-tocopherol content and soybean oil quality. We first collected tocopherol and fatty acid profiles in a soybean population (>800 soybean accessions) and found that tocopherol content increased during soybean domestication. A strong positive correlation between tocopherol and oil content was also detected. Five tocopherol pathway–related loci were identified using a metabolite genome-wide association study strategy. Genetic variations in three tocopherol pathway genes were responsible for total tocopherol content and composition in the soybean population through effects on enzyme activity, mainly caused by non-conserved amino acid substitution or changes in gene transcription level. Moreover, the fatty acid regulatory transcription factor GmZF351 directly activated tocopherol pathway gene expression, increasing both fatty acid and tocopherol contents in soybean seeds. Our study reveals the functional differentiation of tocopherol pathway genes in soybean populations and provides a framework for development of new soybean varieties with high α-tocopherol content and oil quality in seeds.

Production of 2′-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain

2′-Fucosyllactose (2′-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2′-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2′-FL from glucose and lactose. Thus, the overproduction of 2′-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

Molecular evolution and population genetics of glutamate decarboxylase acid resistance pathway in lactic acid bacteria.

Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms' survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.

Structural characterization of protective non-neutralizing antibodies targeting Crimean-Congo hemorrhagic fever virus

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.

An expansion of phenotype: novel homozygous variant in the MED17 identified in patients with progressive microcephaly and global developmental delay

Global developmental delay (GDD) is a lifelong disability that affects 1–3% of the population around the globe. It is phenotypically variable and highly heterogeneous in terms of the underlying genetics. Patients with GDD are intellectually disabled (ID) manifesting cognitive impairment and deficient adaptive behavior. Here, we investigated a two-looped consanguineous family segregating severe ID, seizure, and progressive microcephaly. Magnetic resonance imaging (MRI) of the brain showed mild brain atrophy and myelination defect. Whole exome sequencing (WES) was performed on the DNA samples of two patients and a novel homozygous missense variant (Chr11:g0.93528085; NM_004268.5_c.871T > C; p. Trp291Gly) was identified in the MED17 gene. Sanger sequencing revealed that the identified variant is heterozygous in both parents and healthy siblings. This variant is conserved among different species, causes a non-conserved amino acid change, and is predicted deleterious by various in silico tools. The variant is not reported in population variant databases. MED17 (OMIM: 613668) encodes for the mediator of RNA polymerase II transcription complex subunit 17. Structure modeling of MED17 protein revealed that Trp291 is involved in different inter-helical interactions, providing structural stability. Replacement of Trp291Gly, a less hydrophobic amino acid loses the inter-helical interaction leading to a perturb variant of MED17 protein.

First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria.

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

Enhanced thermostability of formate dehydrogenase via semi-rational design

An NAD+-dependent formate dehydrogenase from Lodderomyces elongisporus (LeFDH) was expressed in Escherichia coli and then purified by nickel affinity chromatography. The analysis of enzymatic properties showed that the specific activity of LeFDH was much high (16.26 U·mg–1), while the thermal stability may be insufficient for industrial application. To further enhance the thermal stability by semi-rational design, sequences alignment, homologous modeling and molecular dynamic simulations were conducted. As a result, seven non-conserved amino acid residues far from the active center were selected for site-directed mutagenesis. Several mutants as well as a binary mutant displayed improved thermal stability and unreduced specific activity. Then temperature and pH stability, kinetic parameters and specific activity, and structural characteristics of wild-type and mutant LeFDH were compared and evaluated. The variant K21P/K22R exhibited an increased half-life by 211% at 60 °C and a 4.5 °C higher Tm than those of wild type, and good stability in alkaline environment. This binary mutant enhances the operational stability of LeFDH in the catalytic reaction and thus improves its potential for application in coenzyme regenerations.

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b

The N-glycans attached to proteins contain various GlcNAc branches, the aberrant formation of which correlates with various diseases. N-Acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and Gnt-IVb (or MGAT4B) are isoenzymes that catalyze the formation of the β1,4-GlcNAc branch in N-glycans. However, the functional differences between these isozymes remain unresolved. Here, using cellular and UDP-Glo enzyme assays, we discovered that GnT-IVa and GnT-IVb have distinct glycoprotein preferences both in cells and in vitro. Notably, we show that GnT-IVb acted efficiently on glycoproteins bearing an N-glycan premodified by GnT-IV. To further understand the mechanism of this reaction, we focused on the noncatalytic C-terminal lectin domain, which selectively recognizes the product glycans. Replacement of a nonconserved amino acid in the GnT-IVb lectin domain with the corresponding residue in GnT-IVa altered the glycoprotein preference of GnT-IVb to resemble that of GnT-IVa. Our findings demonstrate that the C-terminal lectin domain regulates differential substrate selectivity of GnT-IVa and GnT-IVb, highlighting a new mechanism by which N-glycan branches are formed on glycoproteins.