Amino Acid Polymorphisms Research Articles

A suite of selective pressures supports the maintenance of alleles of a Drosophila immune peptide.

The innate immune system provides hosts with a crucial first line of defense against pathogens. While immune genes are often among the fastest evolving genes in the genome, in Drosophila , antimicrobial peptides (AMPs) are notable exceptions. Instead, AMPs may be under balancing selection, such that over evolutionary timescales multiple alleles are maintained in populations. In this study, we focus on the Drosophila antimicrobial peptide Diptericin A, which has a segregating amino acid polymorphism associated with differential survival after infection with the Gram-negative bacteria Providencia rettgeri . Diptericin A also helps control opportunistic gut infections by common Drosophila gut microbes, especially those of Lactobacillus plantarum . In addition to genotypic effects on gut immunity, we also see strong sex-specific effects that are most prominent in flies without functional diptericin A . To further characterize differences in microbiomes between different diptericin genotypes, we used 16S metagenomics to look at the microbiome composition. We used both lab reared and wild caught flies for our sequencing and looked at overall composition as well as the differential abundance of individual bacterial families. Overall, we find flies that are homozygous for one allele of diptericin A are better equipped to survive a systemic infection from P. rettgeri , but in general have a shorter lifespans after being fed common gut commensals. Our results suggest a possible mechanism for the maintenance of genetic variation of diptericin A through the complex interactions of sex, systemic immunity, and the maintenance of the gut microbiome.

Comparative analysis on natural variants of fire blight resistance protein FB_MR5 indicates distinct effector recognition mechanisms

FB_MR5 is an NLR protein identified from wild apple species Malus × robusta 5 conferring disease resistance to bacterial fire blight. FB_MR5 (hereafter MrMR5) recognizes the cysteine protease effector EaAvrRpt2 secreted from the causal agent of bacterial fire blight, Erwinia amylovora. We previously reported that MrMR5 is activated by the C-terminal cleavage product (ACP3) of Malus domestica RIN4 (MdRIN4) produced by EaAvrRpt2-directed proteolysis. Here, we show that MbMR5 from a wild apple species Malus baccata share 99.4% amino acid sequence identity with MrMR5. Surprisingly, transient expression of MbMR5 in Nicotiana benthamiana showed auto-activity in contrast to MrMR5. Domain swap and mutational analyses revealed that one amino acid polymorphism in the MbMR5 CC domain is critical in enhancing auto-activity. We further demonstrated that MrMR5 carrying seven amino acid polymorphisms present in MbMR5 is not activated by MdRIN4 ACP3 but recognizes AvrRpt2 without MdRIN4 in Nicotiana benthamiana. Our findings indicate that naturally occurring polymorphisms of MR5 natural variants can confer its cell death-inducing activity and the effector recognition mechanism likely due to altered compatibility with RIN4.

Revealing the complete mtDNA genome sequence of Cemani chicken (Gallus gallus) by using Nanopore sequencing analysis.

This study aimed to identify, discover and explore the characteristics of the mtDNA genomes of Cemani chicken (Gallus gallus). This study used gDNA of Cemani chicken isolated from liver tissue. mtDNA sequencing was performed using WGS mtDNA analysis with nanopore technology by Oxford Nanopore Technologies GridION. Bioinformatics and data analysis were then performed. This study showed that the length of the mtDNA genome is 16,789 bp, consisting of two ribosomal RNA (12S rRNA, 16S rRNA), 22 transfer RNA genes (trnR, trnG, trnK, trnD, trnS, trnY, trnC, trnN, trnA, trnW, trnM, trnQ, trnl, trnL, trnV, trnF, trnP, trnT, trnE, trnL, trnS, trnH), 13 protein-coding genes (PCGs) (ND4l, ND3, COX3, ATP6, ATP8, COX2, COX1, ND2, ND1, CYTB, ND6, ND5, ND4), and a noncoding control region (Dloop). Furthermore, analysis showed there were polymorphic sites and amino acid alterations when mtDNA Cemani chicken was aligned with references from GenBank. Site (988T>*) in Dloop genes and (328A>G) in ND3 genes which alter glycine to stop codon, were specific markers found only in Cemani chicken.

Complete S1 glycoprotein analysis of two distinct groups of GI-19 (QX) infectious bronchitis virus from Indonesia: Molecular phylogenetic and in-silico approaches

Infectious bronchitis (IB) is an important viral disease that impacts poultry productivity, according to many studies. The main important infectious bronchitis virus (IBV) protein, spike glycoprotein subunit 1 (S1), plays a role in pathogenicity and immunogenicity. Conventional molecular detection and analysis of the S1 gene still face many challenges due to its hypervariability. In Indonesia, there were only a few reports on partial S1 gene analysis of IBVs; thus far, there have been no phylogenetic or in-silico approaches to their S1 complete gene. This study aims to reveal molecular pathogenicity and immunogenicity features on the S1 complete gene of IBVs from Indonesia. We analyzed fourteen IBV isolates using conventional RT-PCR and Sanger sequencing and then aligned their verified S1 complete sequences with other Indonesian GI-19 IBVs from Genbank for molecular phylogenetic and in-silico analysis. Phylogenetic analysis consisted of amino acid polymorphism and identity, as well as phylogenetic tree construction. As part of the in-silico analysis, we used proteins that have homology and immunoinformatic predictions (B-cell epitope and N-glycosylation). Molecular phylogenetic analysis revealed the division of the Indonesian GI-19 IBVs into two distinct groups. These groups had different amino acid variations at receptor binding sites, hypervariable antigenic sites, and cleavage sites. We also calculated the amino acid identity at 95.4–98.3%. In-silico studies revealed significant similarities in B-cell epitopes, distinct N-glycosylation sites, and the suggestion of a homotrimer protein structure. We found that the S1 full gene of two different groups of GI-19 IBVs (QX strain) from Indonesia predicted changes in their pathogenicity and immunogenicity.

Prediction of Deleterious Single Amino Acid Polymorphisms with a Consensus Holdout Sampler.

Single Amino Acid Polymorphisms (SAPs) or nonsynonymous Single Nucleotide Variants (nsSNVs) are the most common genetic variations. They result from missense mutations where a single base pair substitution changes the genetic code in such a way that the triplet of bases (codon) at a given position is coding a different amino acid. Since genetic mutations sometimes cause genetic diseases, it is important to comprehend and foresee which variations are harmful and which ones are neutral (not causing changes in the phenotype). This can be posed as a classification problem. Computational methods using machine intelligence are gradually replacing repetitive and exceedingly overpriced mutagenic tests. By and large, uneven quality, deficiencies, and irregularities of nsSNVs datasets debase the convenience of artificial intelligence-based methods. Subsequently, strong and more exact approaches are needed to address these problems. In the present work paper, we show a consensus classifier built on the holdout sampler, which appears strong and precise and outflanks all other popular methods. We produced 100 holdouts to test the structures and diverse classification variables of diverse classifiers during the training phase. The finest performing holdouts were chosen to develop a consensus classifier and tested using a k-fold (1 ≤ k ≤5) cross-validation method. We also examined which protein properties have the biggest impact on the precise prediction of the effects of nsSNVs. Our Consensus Holdout Sampler outflanks other popular algorithms, and gives excellent results, highly accurate with low standard deviation. The advantage of our method emerges from using a tree of holdouts, where diverse LM/AI-based programs are sampled in diverse ways.

Amino acid insertion in Bat MHC-I enhances complex stability and augments peptide presentation

Bats serve as reservoirs for numerous zoonotic viruses, yet they typically remain asymptomatic owing to their unique immune system. Of particular significance is the MHC-I in bats, which plays crucial role in anti-viral response and exhibits polymorphic amino acid (AA) insertions. This study demonstrated that both 5AA and 3AA insertions enhance the thermal stability of the bat MHC-I complex and enrich the diversity of bound peptides in terms of quantity and length distribution, by stabilizing the 310 helix, a region prone to conformational changes during peptide loading. However, the mismatched insertion could diminish the stability of bat pMHC-I. We proposed that a suitable insertion may help bat MHC-I adapt to high body temperatures during flight while enhancing antiviral responses. Moreover, this site-specific insertions may represent a strategy of evolutionary adaptation of MHC-I molecules to fluctuations in body temperature, as similar insertions have been found in other lower vertebrates.

Phenomic landscape and pharmacogenomic implications for HLA region in a Taiwan Han Chinese population

BackgroundThe human leukocyte antigen (HLA) genes, exhibiting significant genetic diversity, are associated with susceptibility to various clinical diseases and diverse in drug responses. High costs of HLA sequencing and the population-specific architecture of this genetic region necessitate the establishment of a population-specific HLA imputation reference panel. Moreover, there is a lack of understanding about the genetic and phenotypic landscape of HLA variations within the Taiwanese population.MethodsWe created models for a Taiwanese-specific HLA imputation reference panel. These models were trained with the array genotype data and HLA sequencing data from 845 Taiwanese subjects. HLA imputation was applied for 59,448 Taiwanese subjects to characterize the HLA allele and haplotype frequencies. Additionally, a phenome-wide association study (PheWAS) was conducted to identify the phenotypes associated with HLA variations. The association of the biallelic HLA variants with the binary and quantitative traits were evaluated with additive logistic and linear regression models, respectively. Furthermore, an omnibus test with likelihood-ratio test was applied for each HLA amino acid position in the multiallelic HLA amino acid polymorphisms to compare the difference between a fitted model and a null model following a χ2 distribution of n-1 degree of freedom at a position with n residues. Finally, we estimated the prevalence of adverse drug reactions (ADR)-related HLA alleles in the Taiwanese population.ResultsIn this study, the reference panel models displayed remarkable accuracy, with averages of 99.3%, 98.9%, and 99.1% for 2-, 4-, 6-digit alleles of the eight classical HLA genes, respectively. For PheWAS, a total of 18,136 significant associations with HLA variants across 26 phenotypes are identified (p < 5×10-8), highlighting the pleiotropy feature of the HLA region. Among the independent signals, 15 are novel, including the association of HLA-B pos 138 variation with ankylosing spondylitis (AS), and rs9266290 and rs9266292 with allergy. Through an analysis spanning the entire HLA region, we identified clusters of phenotype correlations. Finally, the carriers of pharmacogenomic related HLA alleles, including HLA-C*01:02 (35.86%), HLA-B*58:01 (20.9%), and HLA-B*15:02 (8.38%), were characterized in the Taiwanese general population.ConclusionsWe successfully delivered the HLA imputation for 59,448 Taiwanese subjects and characterized the genetic and phenotypic landscapes of the HLA variations. In addition, we quantified the estimated prevalence of the ADR-related HLA alleles in the Taiwanese population. The developed HLA imputation reference panel could be used for estimation of population HLA allele frequencies, which can facilitate further studies in the role of HLA variants in a wider range of phenotypes in the population.

Separating the Wheat from the Chaff among HLA-DQ Eplets.

In transplantation, anti-HLA Abs, especially targeting the DQ locus, are well-known to lead to rejection. These Abs identified by Luminex single Ag assays recognize polymorphic amino acids on HLA, named eplets. The HLA Eplet Registry included 83 DQ eplets, mainly deduced from amino acid sequence alignments, among which 66 have not been experimentally verified. Because eplet mismatch load may improve organ allocation and transplant outcomes, it is imperative to confirm the genuine reactivity of eplets to validate this approach. Our study aimed to confirm 29 nonverified eplets, using adsorption of eplet-positive patients' sera on human spleen mononuclear cells and on transfected murine cell clones expressing a unique DQα- and DQβ-chain combination. In addition, we compared the positive beads patterns obtained in the two commercially available Luminex single Ag assays. Among the 29 nonverified DQ eplets studied, 24 were confirmed by this strategy, including the 7 DQα eplets 40E, 40ERV, 75I, 76 V, 129H, 129QS, and 130A and the 17 DQβ eplets 3P, 23L, 45G, 56L, 57 V, 66DR, 66ER, 67VG, 70GT, 74EL, 86A, 87F, 125G, 130R, 135D, 167R, and 185I. However, adsorption results did not allow us to conclude for the five eplets 66IT, 75S, 160D, 175E, and 185T.

Predominant amino acid substitutions in NS5B gene of hepatitis C virus in blood donors and treatment-naïve hepatitis and HIV patients in Nigeria

Background: Hepatitis C virus (HCV) genome undergoes high rate of mutation, which results in generation of genetically diverse HCV isolates. There is paucity of data on mutations in the nonstructural 5b (NS5b) gene of circulating HCV and their implications in the Nigerian population. Here, we identified clinically-important mutations in HCV isolates, which may influence response to therapy and disease prognosis.&#x0D; Methodology: HCV RNA was extracted from a total of 301 blood samples collected from 99 symptomatic treatment-naïve hepatitis patients, 125 HIV-infected individuals and 77 asymptomatic blood donors in Ibadan, Nigeria. The RNA was reverse–transcribed to complimentary DNA and HCV NS5B gene amplified by nested PCR. The amplified products of 42 HCV were sequenced and sequences were aligned with those from GenBank and HCV databases in MEGA 7.0. Nucleotide sequences were translated to amino acids while substitutions in the amino acids were analyzed with reference to H77 prototype strain of HCV.&#x0D; Results: A total of 10 amino acid polymorphisms were observed from the 42 sequenced NS5B gene, with the major clinically-important amino acid mutations being S15G in 28 (66.7%) participants, T7N (24, 57.1%), G61R (23, 54.8%), S54L (22, 52.4%), G89E (14, 33.3%), T79M (12, 28.6%), and T711 (11, 26.2%). Others were Q67R (7, 16.7%), Q47H (7, 16.7%) and S84F (2, 4.8%). S15G/A/V mutations were more predominant in patients with HIV (76.9%, 10/13) followed by patients with clinical hepatitis (75.0%, 12/16) and blood donors (46.1%, 6/13). Q67R and T71I mutations were not predominant in patients with clinical hepatitis as they were detected in only 31.3% (5/16) and 43.8% (7/16) participants respectively, compared to S15G (75.0%, 12/16), S54L (68.8%, 11/16), G61R/E (68.8%, 11/16) and T7N/S (56.3%, 9/16). There was no statistically significant difference in the distribution of each of the 10 amino acid polymorphisms detected within patients with symptomatic clinical hepatitis (x2=9.311, p=0.409), HIV-infected patients (x2=13.431, p=0.1440) and asymp- tomatic blood donors (x2=3.775, p=0.9256). Similarly, there was no significant difference in the distribution between the 3 categories of the study participants except for T79M mutation, which was significantly higher in HIV-infected patients (61.5%, 8/13) compared to patients with clinical hepatitis (18.8%, 3/16) and asymp- tomatic blood donors (7.7%, 1/13) (x2=10.456, p=0.0054).&#x0D; Conclusion: Mutations in the NS5B gene could be associated with worse prognosis of the disease or antiviral failure due to viral resistance in patients undergoing therapy. The absence of Q47H mutations in majority of the study participants in our study implies that they will not respond well to daprevir and mericitabine. Screening of patients for pre-existing resistant mutations before commencement of therapy and monitoring during and after therapy are recommended.

LORE receptor homomerization is required for 3-hydroxydecanoic acid-induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus.

The S-domain-type receptor-like kinase (SD-RLK) LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) from Arabidopsis thaliana is a pattern recognition receptor that senses medium-chain 3-hydroxy fatty acids, such as 3-hydroxydecanoic acid (3-OH-C10:0), to activate pattern-triggered immunity. Here, we show that LORE homomerization is required to activate 3-OH-C10:0-induced immune signaling. Fluorescence lifetime imaging in Nicotiana benthamiana demonstrates that AtLORE homomerizes via the extracellular and transmembrane domains. Co-expression of AtLORE truncations lacking the intracellular domain exerts a dominant negative effect on AtLORE signaling in both N. benthamiana and A. thaliana, highlighting that homomerization is essential for signaling. Screening for 3-OH-C10:0-induced reactive oxygen species production revealed natural variation within the Arabidopsis genus. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3-OH-C10:0, although both possess a putative LORE ortholog. Both LORE orthologs have defective extracellular domains that bind 3-OH-C10:0 to a similar level as AtLORE, but lack the ability to homomerize. Thus, ligand binding is independent of LORE homomerization. Analysis of AtLORE and AlyrLORE chimera suggests that the loss of AlyrLORE homomerization is caused by several amino acid polymorphisms across the extracellular domain. Our findings shed light on the activation mechanism of LORE and the loss of 3-OH-C10:0 perception within the Arabidopsis genus.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (&lt;1h) and slow (&gt;5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.

A single amino acid polymorphism in natural Metchnikowin alleles of Drosophila results in systemic immunity and life history tradeoffs.

Antimicrobial peptides (AMPs) are at the interface of interactions between hosts and microbes and are therefore expected to be rapidly evolving in a coevolutionary arms race with pathogens. In contrast, previous work demonstrated that insect AMPs tend to evolve more slowly than the genome average. Metchikowin (Mtk) is a Drosophila AMP that has a single amino acid residue that segregates as either proline (P) or arginine (R) in populations of four different species, some of which diverged more than 10 million years ago. These results suggest that there is a distinct functional importance to each allele. The most likely hypotheses are driven by two main questions: does each allele have a different efficacy against different specific pathogens (specificity hypothesis)? Or, is one allele a more potent antimicrobial, but with a host fitness cost (autoimmune hypothesis)? To assess their functional differences, we created D. melanogaster lines with the P allele, R allele, or Mtk null mutation using CRISPR/Cas9 genome editing and performed a series of life history and infection assays to assess them. In males, testing of systemic immune responses to a repertoire of bacteria and fungi demonstrated that the R allele performs as well or better than the P and null alleles with most infections. Females show some results that contrast with males, with Mtk alleles either not contributing to survival or with the P allele outperforming the R allele. In addition, measurements of life history traits demonstrate that the R allele is more costly in the absence of infection for both sexes. These results are consistent with both the specificity hypothesis (either allele can perform better against certain pathogens depending on context), and the autoimmune hypothesis (the R allele is generally the more potent antimicrobial in males, and carries a fitness cost). These results provide strong in vivo evidence that differential fitness with or without infection and sex-based functional differences in alleles may be adaptive mechanisms of maintaining immune gene polymorphisms in contrast with expectations of rapid evolution. Therefore, a complex interplay of forces including pathogen species and host sex may lead to balancing selection for immune genotypes. Strikingly, this selection may act on even a single amino acid polymorphism in an AMP.

The impact of PA/I38 substitutions and PA polymorphisms on the susceptibility of zoonotic influenza A viruses to baloxavir

Genetic reassortment of avian, swine, and human influenza A viruses (IAVs) poses potential pandemic risks. Surveillance is important for influenza pandemic preparedness, but the susceptibility of zoonotic IAVs to the cap-dependent endonuclease inhibitor baloxavir acid (BXA) has not been thoroughly researched. Although an amino acid substitution at position 38 in the polymerase acidic protein (PA/I38) in seasonal IAVs reduces BXA susceptibility, PA polymorphisms at position 38 are rarely seen in zoonotic IAVs. Here, we examined the impact of PA/I38 substitutions on the BXA susceptibility of recombinant A(H5N1) viruses. PA mutants that harbored I38T, F, and M were 48.2-, 24.0-, and 15.5-fold less susceptible, respectively, to BXA than wild-type A(H5N1) but were susceptible to the neuraminidase inhibitor oseltamivir acid and the RNA polymerase inhibitor favipiravir. PA mutants exhibited significantly impaired replicative fitness in Madin-Darby canine kidney cells at 24 h postinfection. In addition, in order to investigate new genetic markers for BXA susceptibility, we screened geographically and temporally distinct IAVs isolated worldwide from birds and pigs. The results showed that BXA exhibited antiviral activity against avian and swine viruses with similar levels to seasonal isolates. All viruses tested in the study lacked the PA/I38 substitution and were susceptible to BXA. Isolates harboring amino acid polymorphisms at positions 20, 24, and 37, which have been implicated in the binding of BXA to the PA endonuclease domain, were also susceptible to BXA. These results suggest that monitoring of the PA/I38 substitution in animal-derived influenza viruses is important for preparedness against zoonotic influenza virus outbreaks.

Val43 residue of NsrR is crucial for the nitric oxide response of Salmonella Typhimurium.

In pathogenic bacteria, the flavohemoglobin Hmp is crucial in metabolizing the cytotoxic levels of nitric oxide (NO) produced in phagocytic cells, contributing to bacterial virulence. Hmp expression is predominantly regulated by the Rrf2 family transcription repressor NsrR in an NO-dependent manner; however, the underlying molecular mechanism in enterobacteria remains poorly understood. In this study, we identified Val43 of Salmonella Typhimurium NsrR (StNsrR) as a critical amino acid residue for regulating Hmp expression. The Val43-to-Ala-substituted mutant NsrR isolated through random and site-directed mutagenesis showed high binding affinity to the target DNA irrespective of NO exposure, resulting in a severe reduction in hmp transcription and slow NO metabolism in Salmonella under NO-producing conditions. Conversely, the Val43-to-Glu-substituted NsrR caused effects similar to nsrR null mutation, which directed hmp transcription and NO metabolism in a constitutive way. Comparative analysis of the primary sequences of NsrR and another NO-sensing Rrf2 family regulator, IscR, from diverse bacteria, revealed that Val43 of enterobacterial NsrR corresponds to Ala in Pseudomonas aeruginosa or Streptomyces coelicolor NsrR and Glu in enterobacterial IscR, all of which are located in the DNA recognition helix α3. The predicted structure of StNsrR in complex with the hmp DNA suggests dissimilar spatial stoichiometry in the interactions of Val43 and its substituted residues with the target DNA, consistent with the observed phenotypic changes in StNsrR Val43 mutants. Our findings highlight the discriminative roles of the NsrR recognition helix in regulating species-specific target gene expression, facilitating effective NO detoxification strategies in bacteria across diverse environments. The precise regulation of flavohemoglobin Hmp expression by NsrR is critical for bacterial fitness, as excessive Hmp expression in the absence of NO can disturb bacterial redox homeostasis. While the molecular structure of Streptomyces coelicolor NsrR has been recently identified, the specific molecular structures of NsrR proteins in enterobacteria remain unknown. Our discovery of the crucial role of Val43 in the DNA recognition helix α3 of Salmonella NsrR offers valuable insights into the Hmp modulation under NO stress. Furthermore, the observed amino acid polymorphisms in the α3 helices of NsrR proteins across different bacterial species suggest the diverse evolution of NsrR structure and gene regulation in response to varying levels of NO pressure within their ecological niches.

Significant Impact of AcrB Amino Acid Polymorphism at Residue 716 on Susceptibility to Tigecycline and Other Antibiotics in Klebsiella pneumoniae.

AcrAB-TolC is a multidrug RND-type efflux pump that is widespread in Gram-negative bacteria. As the substrate-binding subunit, AcrB was shown to modulate antimicrobial resistance in Escherichia coli, but the influence of AcrB mutation on Klebsiella pneumoniae, a major clinical pathogen, has not been well-studied. The finding of an R716L mutation in AcrB in a clinical tigecycline-nonsusceptible K. pneumoniae S1 strain inspired us to probe the role of AcrB residue 716 in antimicrobial resistance. This residue was subsequently subjected to saturation mutagenesis, followed by antibiotic susceptibility tests, survival assays, and antibiotic accumulation assays, showing strong influences of AcrB mutation on antimicrobial resistance. In particular, resistance levels to azithromycin, tetracycline, tigecycline, and cefoxitin were significantly changed by AcrB mutation at residue 716. Mutations to charged residues, polar residues, and residues that disrupt secondary structures have particularly reduced the antimicrobial susceptibility of bacteria, except for azithromycin, and the impact is not due to the abolishment of the efflux function of the pump. Therefore, it is concluded that residue 716 is an important residue that significantly influences antimicrobial resistance in K. pneumoniae, adding to our understanding of antimicrobial resistance mechanisms in this key clinical pathogen.

The adaptive potential of North American subtype H7N2 avian influenza viruses to mammals

Introduction. H7 subtype avian influenza viruses causing severe epizootics among birds are phylogenetically different in the Eastern and Western hemispheres. Numerous human infections caused by these viruses in the Eastern hemisphere indicate that H7 viruses can overcome the interspecies barrier and pose a potential threat of a new pandemic.The H7N2 viruses with deletion of amino acids 221–228 (H3 numbering) in hemagglutinin (HA) had been circulating among poultry in the Western Hemisphere during 1996–2006, and had once again been detected in 2016 in an animal shelter, where they caused cat diseases.&#x0D; The objective of this study is to elucidate the mechanism of adaptation to mammals of North American H7N2 influenza viruses with deletion in HA.&#x0D; Materials and methods. The A/chicken/New Jersey/294598-12/2004 (H7N2) virus was adapted to mice by the lung passages. Complete genomes of original and mouse-adapted viruses were analyzed. The receptor specificity and thermostability of viruses, HA activation pH and virulence for mice were determined.&#x0D; Results. The non-pathogenic H7N2 avian influenza virus became pathogenic after 10 passages in mice. Amino acid substitutions occurred in five viral proteins: one in PB2 (E627K), NA (K127N), NEP (E14Q), four in HA and six in NS1. Mutations in HA slightly changed receptor specificity but increased the pH of HA activation by 0.4 units. The NS1 protein undergone the greatest changes in the positions (N73T, S114G, K118R, G171A, F214L and G224R), where amino acid polymorphisms were observed in the original virus, but only minor amino acid variants have been preserved in the mouse adapted variant.&#x0D; Conclusion. The results show that H7N2 viruses have the potential to adapt to mammals. The increase in virulence is most likely due to the adaptive E627K mutation in PB2 and possibly in HA.

Relationship between acaricide resistance and acetylcholinesterase gene polymorphisms in the cattle tick Rhipicephalus microplus.

In this study, we aimed to develop a comprehensive methodology for identifying amino acid polymorphisms in acetylcholinesterase transcript 2 (AChE2) in acaricide-resistant Rhipicephalus microplus ticks. This included assessing AChE2 expression levels through qPCR and conducting 3D modeling to evaluate the interaction between acaricides and AChE2 using docking techniques. The study produced significant results, demonstrating that acaricide-resistant R. microplus ticks exhibit significantly higher levels of AChE expression than susceptible reference ticks. In terms of amino acid sequence, we identified 9 radical amino acid substitutions in AChE2 from acaricide-resistant ticks, when compared to the gene sequence of the susceptible reference strain. To further understand the implications of these substitutions, we utilized 3D acaricide-AChE2 docking modeling to examine the interaction between the acaricide and the AChE2 catalytic site. Our models suggest that these amino acid polymorphisms alter the configuration of the binding pocket, thereby contributing to differences in acaricide interactions and ultimately providing insights into the acaricide-resistance phenomenon in R. microplus.

Determination of the clinical relevance of donor epitope-specific HLA-antibodies in kidney transplantation.

In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.

Polymorphisms at amino acid positions 85 and 86 in succinate dehydrogenase subunit C of Colletotrichum siamense: Implications for fitness and intrinsic sensitivity to SDHI fungicides

Among succinate dehydrogenase inhibiter (SDHI) fungicides, penthiopyrad and benzovindiflupyr particularly inhibit Colletotrichum. Studying SDH amino acid polymorphism in Colletotrichum, along with its fungicide binding sites, is key to understanding their mechanisms of action. This study explores the SDH amino acid polymorphisms in Colletotrichum siamense strains from rubber trees in China and their interaction with SDHI fungicides, specifically penthiopyrad and benzovindiflupyr. Sequencing revealed most polymorphisms were in the SDHC subunit, particularly at positions 85 and 86, which are key to penthiopyrad resistance. Among 33 isolates, 33.3 % exhibited a substitution at position 85, and 9 % at position 86. A strain with W85L and T86N substitutions in SDHC showed reduced SDH activity, ATP content, mycelial growth, and virulence, and decreased sensitivity to penthiopyrad but not benzovindiflupyr. Molecular docking with Alphafold2 modeling suggested distinct binding modes of the two fungicides to C. siamense SDH. These findings underscore the importance of SDHC polymorphisms in C. siamense's fitness and sensitivity to SDHIs, enhancing our understanding of pathogen-SDHI interactions and aiding the development of novel SDHI fungicides.

Targeted amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial

Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (&lt;1h) and slow (&gt;5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.