Missense Research Articles

Genomic and Bioinformatics Analysis of Familial Partial Lipodystrophy Type 3 Identified in a Patient with Novel PPARγ Mutation and Robust Response to Pioglitazone

Familial partial lipodystrophies (FPLDs) are very rare inherited disorders characterized by partial loss of adipose tissue from the upper and lower extremities. At least seven subtypes of FPLD have been identified and are mostly dominantly inherited. FPLD type 3 is caused by mutations in the PPARγ gene, which encodes for the protein peroxisome proliferator-activated receptor gamma (PPARγ). We identified a Saudi female with PFLD3 presented with partial lipoatrophy, uncontrolled diabetes, severe hypertriglyceridemia, and recurrent pancreatitis. The clinical and biochemical findings in this proband were described before and after treatment with Pioglitazone in addition to the conventional treatment. DNA extraction and whole exome sequencing (WES) were performed to detect the variant. The mutant gene was subjected to Sanger analysis to confirm the results. We applied five specific computational prediction tools to assess the pathogenicity of variation, namely the MT, DANN, CADD, BayesDel, and fitCons tools. We assessed protein modeling and stability with the AlphaFold-generated structures for both wild-type and mutant proteins. Finally, we conducted molecular docking using the AutoDock Vina virtual docking. Upon whole exome sequencing, a c.1024C>T p.(Gln342Ter) missense mutation was detected in the PPARγ gene associated with FPLD3. This variant is a novel mutation that has not been described in all genome databases. Sanger analysis confirmed the heterogenicity and pathogenicity of this variant. All five computational prediction tools indicate that this variant is considered highly pathogenic. Our patient showed a dramatic response to Pioglitazone, a synthetic PPARγ agonist. From structural modeling, we found that the enhanced binding affinity of the mutant PPARγ protein to Pioglitazone likely improves the activation of PPARγ, enhancing its transcriptional activity and resulting in better clinical outcomes. These findings extend the spectrum of PPARγ mutations responsible for FPLD3 and highlight the potential for personalized treatment strategies based on genetic mutations.

Identification of novel ABCB4 variants and genotype-phenotype correlation in progressive familial intrahepatic cholestasis type 3

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe hepatic disorder characterized by cholestasis. Elucidating the genotype-phenotype correlations and expanding the mutational spectrum of the ABCB4 gene are crucial for enhancing diagnostic accuracy and therapeutic strategies.Clinical and genetic data from 2 original PFIC3 patients from our institution, along with 118 additional cases identified through a comprehensive literature review, were integrated for a comprehensive analysis. The study included statistical analysis of clinical information, genetic analysis, multi-species sequence alignment, protein structure modeling, and pathogenicity assessment. Machine learning techniques were applied to identify genotype-phenotype relationships. We identified three novel ABCB4 mutations: two missense mutations (c.904G > T and c.2493G > C) and one splicing mutation (c.1230 + 1G > A). Homozygous mutations were associated with significantly earlier disease onset compared to compound heterozygous mutations (p < 0.0001). Missense mutations were predominant (76.9%), with Exon 7 being the most frequently affected region. A random forest model indicated that Exon 10 had the highest feature importance score (9.9%). Liver transplantation remains the most effective treatment modality for PFIC3. This investigation broadens the known mutation spectrum of the ABCB4 gene and identifies key variant sites associated with clinical manifestations. These insights lay a foundation for early diagnosis, optimal treatment selection, and further research into PFIC3.

Molecular mechanism analysis of a family with hereditary coagulation FXI deficiency caused by compound heterozygous mutations.

The purpose of this study was to determine the molecular basis of a Chinese family with factor XI (FXI) deficiency. The qRT-PCR was used to detect the transcription of F11 mRNA in transfected cells. ELISAs and western blot were used to detect the expression of FXI protein in culture media and lysates. Genetic analysis revealed that the proband carried a heterozygous nonsense mutation c.1107C>A (p.Tyr351stop) in exon 10 and a heterozygous missense mutation c.1562A>G (p.Tyr503Cys) in exon 13. The expression study revealed that p.Tyr351stop mutation resulted in the degradation of F11 mRNA. The p.Tyr503Cys mutation, however, had no effects on biosynthesis and secretion of FXI protein, but it had affected the catalytic activity of FXI. The inherited FXI deficiency of this family is related to nonsense mutation p.Tyr351stop and missense mutation p.Tyr503Cys.

Omics based approaches to decipher the leaf ionome and transcriptome changes in Solanum lycopersicum L. upon Tomato Brown Rugose Fruit Virus (ToBRFV) infection.

The Tomato Brown Rugose Fruit Virus (ToBRFV) is a pathogen that mostly affects plants from the Solanaceae family. This virus severely affects the yield of tomato (Solanum lycopersicum L.) plants, thus creating an urgent need to research the basis of resistance to manage the disease. To understand the molecular basis of resistance, we employed omics-based approaches involving leaf ionomics and transcriptomics to help us decipher the interaction between elemental and nutritional composition and investigate its effect on the gene expression profile upon the ToBRFV infection in tomatoes. Ionomics was used to investigate the accumulation of trace elements in leaves, showcasing that the plants resistant to the virus had significantly higher concentrations of iron (p-value = 0.039) and nickel (p-value = 0.042) than the susceptible ones. By correlating these findings with transcriptomics, we identified some key genes involved in iron homeostasis and abscisic acid pathways, potentially responsible for conferring resistance against the pathogen. From the obtained list of differentially expressed genes, a panel of mutation profile was discovered with three important genes-Solyc02g068590.3.1 (K+ transporter), Solyc01g111890.3.1 (LRR), and Solyc02g061770.4.1 (Chitinase) showing persistent missense mutations. We ascertain the role of these genes and establish their association with plant resistance using genotyping assays in various tomato lines. The targeted selection of these genetic determinants can further enhance plant breeding and crop yield management strategies.

Conformational plasticity links structural instability of NAA10F128I and NAA10F128L mutants to their catalytic deregulation

The acetylation of proteins' N-terminal amino groups by the N-acetyltransferase complexes plays a crucial role in modulating the spatial stability and functional activities of diverse human proteins. Mutations disrupting the stability and function of NAA10 result in X-linked rare genetic disorders. In this study, we conducted a global analysis of the impact of fifteen disease-associated missense mutations in NAA10. The analyses revealed that mutations in specific residues, such as Y43, V107, V111, and F128, predictably disrupted interactions essential for NAA10 stability, while most mutations (except R79C, A111W, Q129P, and N178K) expectedly led to structural destabilization. Mutations in many conserved residues within short linear motifs and post-translational modification sites were predicted to affect NAA10 functionality and regulation. All mutations were classified as pathogenic, with F128I and F128L identified as the most destabilizing mutations. The findings show that the F128L and F128I mutations employ different mechanisms for the loss of catalytic activities of NAA10F128L and NAA10F128I due to their structural instability. These two mutations induce distinct folding energy states that differentially modulate the structures of different regions of NAA10F128L and NAA10F128I. Specifically, the predicted instability caused by the F128I mutation results in decreased flexibility within the substrate-binding region, impairing the substrate peptide binding ability of NAA10F128I. Conversely, F128L is predicted to reduce the flexibility of the region containing the acetyl-CoA binding residues in NAA10F128L. Our study provides insights into the mechanism of catalytic inactivation of mutants of NAA10, particularly elucidating the mechanistic features of the structural and functional pathogenicity of the F128L and F128I mutations.

Identification and in vivo functional analysis of a novel missense mutation in GATA3 causing hypoparathyroidism, sensorineural deafness and renal dysplasia syndrome in a Chinese family.

Hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome is a rare autosomal dominant genetic disease associated with mutations in the GATA3 gene, which encodes GATA3 that plays essential roles in vertebrate development. This study aimed to identify and report the pathogenic mutation in GATA3 in a Chinese family diagnosed with HDR syndrome and determine its functional impacts in vivo. The clinical features of a 25-year-old male patient with HDR syndrome and his parents were collected. GATA3 gene exome sequencing and Sanger sequencing were performed on the proband and his family, respectively. Functional analyses of GATA3 were performed using bioinformatics tools and zebrafish assays to determine pathogenicity and phenotype spectrum. A novel, heterozygous, missense mutation in exon 4 of the GATA3 gene, c.863 G > A, p.Cys288Tyr, in the proband and his mother who presented the complete HDR triad, was predicted to be deleterious by in silico tools. 3D structure modeling showed that the variant caused significant structural changes. In vivo studies using a zebrafish animal model revealed the deleterious impact of the variant on the gill buds, otoliths, and pronephros. We identified a novel missense mutation, GATA3 p.Cys288Tyr, within a family with HDR syndrome and delineated it as a loss-of-function variant in vivo. This expands the spectrum of GATA3 mutations associated with HDR syndrome in the Chinese population and mimics HDR-related changes in vivo.

Cryo-EM structures of the membrane repair protein dysferlin

Plasma membrane repair in response to damage is essential for cell viability. The ferlin family protein dysferlin plays a key role in Ca2+-dependent membrane repair in striated muscles. Mutations in dysferlin lead to a spectrum of diseases known as dysferlinopathies. The lack of a structure of dysferlin and other ferlin family members has impeded a mechanistic understanding of membrane repair mechanisms and the development of therapies. Here, we present the cryo-EM structures of the full-length human dysferlin monomer and homodimer at 2.96 Å and 4.65 Å resolution. These structures define the architecture of dysferlin, ferlin family-specific domains, and homodimerization mechanisms essential to function. Furthermore, biophysical and cell biology studies revealed how missense mutations in dysferlin contribute to disease mechanisms. In summary, our study provides a framework for the molecular mechanisms of dysferlin and the broader ferlin family, offering a foundation for the development of therapeutic strategies aimed at treating dysferlinopathies.

Clinicopathological Analysis of a European Cohort of MYOD1 Mutant Rhabdomyosarcomas in Children and Young Adults.

Patients with PAX3/7-FOXO1 fusion-negative rhabdomyosarcomas (fnRMS) harbouring the rare L122R MYOD1 mutation have significantly poorer prognosis than other fnRMS. We undertook a detailed clinicopathological evaluation of a cohort of patients with MYOD1 mutated fnRMS in order to improve risk stratification and treatment options. Histological, mutational and clinical data from a cohort of patients with MYOD1 mutant RMS treated in Europe were analysed. Thirty-two cases with mutant MYOD1 RMS were identified from patients enrolled in sequential European rhabdomyosarcoma clinical trials from 1992 to 2022 (n = 22) and non-trial cohorts (n = 10). Thirty cases had the recurrent L122R missense mutation, one case harboured a K124E mutation and one case had a truncating mutation (S63X). Increased MyoD1 and reduced MYF4 immunostaining were consistent features of MYOD1L122R-mutated RMS. Applying the risk stratification of the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS2005 trial, among 20 localised RMS cases that could be assigned a risk category, one was Very High Risk, 13 were High Risk and six were Standard Risk. Eight patients had distant metastases at diagnosis. Of the 25 patients with adequate clinical follow-up data, 15/25 (60%) patients had an event at a median time of 9months (12/15 included failure of local control) and 13/25 (52%) died of disease. This MYOD1 mutant cohort demonstrates increased MYOD and reduced MYF4 immunostaining, high risk of local failure and poor survival in agreement with other studies. Increased treatment intensity and improved local control should be considered for these patients.

Investigating the differential structural organization and gene expression regulatory networks of lamin A Ig fold domain mutants of muscular dystrophy.

Lamins form a proteinaceous meshwork as a major structural component of the nucleus. Lamins, along with their interactors, act as determinants for chromatin organization throughout the nucleus. The major dominant missense mutations responsible for autosomal dominant forms of muscular dystrophies reside in the Ig fold domain of lamin A. However, how lamin A contributes to the distribution of heterochromatin and balances euchromatin, and how it relocates epigenetic marks to shape chromatin states, remains poorly defined, making it difficult to draw conclusions about the prognosis of lamin A-mediated muscular dystrophies.In the first part of this report, we identified the in-vitro organization of full-length lamin A proteins due to two well-documented Ig LMNA mutations, R453W and W514R. We further demonstrated that both lamin A/C mutant cells predominantly expressed nucleoplasmic aggregates. Labelling specific markers of epigenetics allowed correlation of lamin A mutations with epigenetic mechanisms. In addition to manipulating epigenetic mechanisms, our proteomic studies traced diverse expressions of transcription regulators, RNA synthesis and processing proteins, protein translation components, and posttranslational modifications. These data suggest severe perturbations in targeting other proteins to the nucleus.

Preoperative prediction of occult lymph node metastasis in patients with non-small cell lung cancer: a simple and widely applicable model.

Lymph node metastasis (LNM) is one of the most common pathways of metastasis in non-small cell lung cancer (NSCLC). Preoperative assessment of occult lymph node metastasis (OLNM) in NSCLC patients is beneficial for selecting appropriate treatment plans and improving patient prognosis. A total of 370 NSCLC patients were included in the study. Univariate and multivariate logistic regression analysis were used to screen potential risk factors for OLNM in preoperative NSCLC patients. And establish a nomogram for OLNM in NSCLC patients before surgery. Receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the established nomogram. Both univariate and multivariate logistic regression analyses suggested that multiple tumors, ERBB2 missense mutation, CA125 levels, CA153 levels, tumor site, tumor length, and serum ferritin are potential risk factors for OLNM in NSCLC patients. The constructed nomogram was evaluated, and the consistency index (C-index) and area under the ROC curve of the model were both 0.846. The calibration curve showed that the predicted values of the model had a high degree of fit with the actual observed values, and DCA suggested that the above indicators had good utility. The personalized scoring prediction model constructed based on multiple tumors, ERBB2 miss mutation, CA125 levels, CA153 levels, tumor site, tumor length, and serum ferritin can screen NSCLC patients who may have OLNM.

De novo SCN1A missense variant in a patient with Parkinson’s disease

BackgroundVariants in a gene encoding sodium voltage-gated channel alpha subunit 1 (SCN1A) are known to cause a broad clinical spectrum of epilepsy and associated features, including Dravet syndrome (MIM 607208), non-Dravet developmental and epileptic encephalopathy (MIM 619317), familial febrile seizures (MIM 604403), familial hemiplegic migraine (MIM 609634), and generalized epilepsy with febrile seizures (MIM 604403).MethodsIn this study, we examined a patient with Parkinson’s disease (PD) without any clinical manifestations of epilepsy and associated features. Genomic nucleic acid was extracted, and a complete coding sequence of the human genome (whole-exome sequencing) was sequenced. Moreover, Sanger sequencing of variants of interest was performed to validate the exome-discovered variants.ResultsWe identified a heterozygous pathogenic missense mutation (c.1498C&amp;gt;T; p.Arg500Trp) in the SCN1A gene in the patient using the whole-exome sequencing approach. The onset of PD features in our patient occurred at the age of 30 years. Biochemical investigations were carried out to rule out any secondary cause of the disease, including Wilson's disease or another metabolic disorder. MRI of the brain and spinal images were unremarkable. Moreover, a dramatic response to carbidopa–levodopa treatment was also observed in the patient.ConclusionOur results suggest that the pathogenic variant in SCN1A may lead to PD features without epilepsy.

Multi-omics analysis provides insights into the mechanism underlying fruit color formation in Capsicum

Fruit color is a crucial attribute of fruit quality in peppers (Capsicum spp.). However, few studies have focused on the mechanism of color formation in immature pepper fruits. In this study, the light-yellow color observed in immature CSJ009 fruits compared to CSJ010 could be attributed to decreased chlorophyll and carotenoid pigments. Through integrated analysis of the transcriptome and metabolome of CSJ009 and CSJ010, we identified 23,930 differentially expressed genes (DEGs) and 345 differentially accumulated metabolites (DAMs). Furthermore, integrated analysis revealed a strong correlation between the HCT-like gene and metabolite MWS0178 (chlorogenic acid). Paraffin section assay revealed that the epidermal cells of immature CSJ010 fruits exhibited a more compact arrangement with significantly greater length than those of CSJ009. Quantitative determination of carotenoids showed that lutein emerged as the predominant carotenoid in immature pepper fruits. Additionally, missense mutation of LCYB2 is likely to lead to a decrease in β-carotene content in immature CSJ009 fruits, whereas CCS may directly catalyze the conversion of lycopene to β-carotene in mature fruits. The null mutation in CCS promoted the biosynthesis of β,ϵ-branch carotenoids leading to lutein being the most abundant carotenoid found in orange CSJ010 fruits. These findings provide important insights into the mechanism underlying color formation in pepper fruits and establish a foundation for the further exploration of color-related genes.

TP53 Mutation Status in Myelodysplastic Neoplasm and Acute Myeloid Leukemia: Impact of Reclassification Based on the 5th WHO and International Consensus Classification Criteria: A Korean Multicenter Study.

TP53 mutations are associated with poor prognosis in myelodysplastic neoplasm (MDS) and AML. The updated 5th WHO classification and International Consensus Classification (ICC) categorize TP53-mutated MDS and AML as unique entities. We conducted a multicenter study in Korea to investigate the characteristics of TP53-mutated MDS and AML, focusing on diagnostic aspects based on updated classifications. This study included patients aged ≥ 18 yrs who were diagnosed as having MDS (N=1,244) or AML (N=2,115) at six institutions. The results of bone marrow examination, cytogenetic studies, and targeted next-generation sequencing, including TP53, were collected and analyzed. TP53 mutations were detected in 9.3% and 9.2% of patients with MDS and AML, respectively. Missense mutation was the most common, with hotspot codons R248/R273/G245/Y220/R175/C238 accounting for 25.4% of TP53 mutations. Ten percent of patients had multiple TP53 mutations, and 78.4% had a complex karyotype. The median variant allele frequency (VAF) of TP53 mutations was 41.5%, with a notable difference according to the presence of a complex karyotype. According to the 5th WHO classification and ICC, the multi-hit TP53 mutation criteria were met in 58.6% and 75% of MDS patients, respectively, and the primary determinants were a TP53 VAF >50% for the 5th WHO classification and the presence of a complex karyotype for the ICC. Collectively, we elucidated the molecular genetic characteristics of patients with TP53-mutated MDS and AML, highlighting key factors in applying TP53 mutation-related criteria in updated classifications, which will aid in establishing diagnostic strategies.

Clinical and mutational signatures of CRB1-associated retinopathies: a multicentre study

BackgroundTo delineate the clinical and mutational signatures of patients with CRB1-associated retinopathies.MethodsThis multicentre retrospective cohort study involved 40 patients with CRB1 mutations and 40 age-matched and gender-matched inherited retinal diseases...

The molecular consequences of FOXF1 missense mutations associated with alveolar capillary dysplasia with misalignment of pulmonary veins

BackgroundAlveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a fatal congenital lung disorder strongly associated with genomic alterations in the Forkhead box F1 (FOXF1) gene and its regulatory region. However, little is known about how FOXF1 genomic alterations cause ACD/MPV and what molecular mechanisms are affected by these mutations. Therefore, the effect of ACD/MPV patient-specific mutations in the FOXF1 gene on the molecular function of FOXF1 was studied.MethodsEpitope-tagged FOXF1 constructs containing one of the ACD/MPV-associated mutations were expressed in mammalian cell lines to study the effect of FOXF1 mutations on protein function. EMSA binding assays and luciferase assays were performed to study the effect on target gene binding and activation. Immunoprecipitation followed by SDS‒PAGE and western blotting were used to study protein‒protein interactions. Protein phosphorylation was studied using phos-tag western blotting.ResultsAn overview of the localization of ACD/MPV-associated FOXF1 mutations revealed that the G91-S101 region was frequently mutated. A three-dimensional model of the forkhead DNA-binding domain of FOXF1 showed that the G91-S101 region consists of an α-helix and is predicted to be important for DNA binding. We showed that FOXF1 missense mutations in this region differentially affect the DNA binding of the FOXF1 protein and influence the transcriptional regulation of target genes depending on the location of the mutation. Furthermore, we showed that some of these mutations can affect the FOXF1 protein at the posttranscriptional level, as shown by altered phosphorylation by MST1 and MST2 kinases.ConclusionMissense mutations in the coding region of the FOXF1 gene alter the molecular function of the FOXF1 protein at multiple levels, such as phosphorylation, DNA binding and target gene activation. These results indicate that FOXF1 molecular pathways may be differentially affected in ACD/MPV patients carrying missense mutations in the DNA-binding domain and may explain the phenotypic heterogeneity of ACD/MPV.

Novel Loss-of-function Variants of ZP3 Associated with Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is one of the leading causes of female infertility. To date, the genetic etiology of POI has been elucidated in approximately 20-25% of the total cases. The human zona pellucida (ZP) plays an important role in the organization and differentiation of granulosa cells, follicle formation, and sperm recognition. Mutations in ZP1, ZP2, and ZP3 have been reported to cause female infertility due to oocyte degeneration, empty follicle, or in vitro fertilization failure. In this study, we identified three novel missense mutations in ZP3 (NM_001110354.2): c.643G > A (p.Asp215Asn), c.215C > T (p.Thr72Ile), and c.152T > C (p.Leu51Pro) in three sporadic Han Chinese POI patients through whole-exome sequencing. These variants are absent from population databases and were predicted to be deleterious by multiple in silico tools. Structure prediction analysis showed that the affected amino acid altered the ZP3 protein structure. Western blot further confirmed that these ZP3 variants reduced the expression and secretion of ZP components. In summary, this study reports three novel deleterious variants in ZP3 associated with POI, thereby broadening the mutation spectrum of ZP3 in POI patients.

Generation of a Vibrio-based platform for efficient conversion of raffinose through Adaptive Laboratory Evolution on a solid medium.

Raffinose, a trisaccharide abundantly found in soybeans, is a potential alternative carbon source for biorefineries. Nevertheless, residual intermediate di- or monosaccharides and low catabolic efficiency limit raffinose use through conventional microbial hosts. This study presents a Vibrio-based platform to convert raffinose efficiently. Vibrio sp. dhg was selected as the starting strain for the Adaptive Laboratory Evolution (ALE) strategy to leverage its significantly higher metabolic efficiency. We conducted ALE on a solid minimal medium supplemented with raffinose to prevent the enrichment of undesired phenotypes due to the shared effect of extracellular raffinose hydrolysis among multiple strains. As a result, we generated the VRA10 strain that efficiently utilizes raffinose without leaving behind degraded di- or monosaccharides, achieving a notable growth rate (0.40 h-1) and raffinose consumption rate (1.2g/gdcw/h). Whole genome sequencing and reverse engineering identified that a missense mutation in the melB gene (encoding a melibiose/raffinose:sodium symporter) and the deletion of the two galR genes (encoding transcriptional repressors for galactose catabolism) facilitated rapid raffinose utilization. The further engineered strain produced 6.2g/L of citramalate from 20g/L of raffinose. This study will pave the way for the efficient utilization of diverse raffinose-rich byproducts and the expansion of alternative carbon streams in biorefinery applications.

Lost in translation: Illuminating protein mislocalization through high-content screening microscopy

Establishing the subcellular distribution of all proteins encoded by the human genome remains a key objective of life science research. This is particularly important in the context of proteins that, through genetic sequencing of patients, have been identified as containing missense mutations. A recent publication in Cell1 highlights the prominence of protein mislocalization as a hallmark of dysfunctional proteins. The use of high-content subcellular phenotypic screens and allied technology by Lacoste and colleagues has enormous potential to change the landscape of how we approach both diagnostic and therapeutic decisions.

Impact of missense mutations on the structure–function relationship of human succinyl-CoA synthetase using in silico analysis

The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.

Molecular characterization of colistin-resistant Klebsiella pneumoniae isolates and their conjugative mcr-carrying plasmids

BackgroundThis study aimed to characterize colistin-resistant K. pneumoniae (CoRKp) strains isolated from patients with urinary tract infections and bacteremia between 1999 and 2022 at a tertiary teaching hospital in Taiwan. MethodsA total of 1,966K. pneumoniae isolates were collected, among which 21 strains were identified as CoRKp. The antimicrobial susceptibility of these CoRKp strains to 19 antibiotics was assessed. The genome characteristics of 21 CoRKp strains were determined by Nanopore-Illumina hybrid whole genome sequencing. Additionally, conjugation assays were conducted to determine the transferability of plasmids carrying mcr genes to K. pneumoniae ATCC BAA-1706 and E. coli C600. The larvae infection model was used to analyze the differences in virulence between transconjugants and recipient strains. ResultsAmong the 21 CoRKp, 12 were multidrug-resistant, and four were extensively drug-resistant. The distribution of sequence types (STs) and K types among the CoRKp strains was quite diverse, and ST307 (5 strains) and K64 (3 strains) dominated in CoRKp. The insertion elements IS903B and ISVsa5, were found to inactivate mgrB of 1 and 2 CoRKp isolates, respectively. Moreover, 1, 4, 6, and 1 missense mutations of PhoQ, PmrA, PmrB, and MgrB, were identified in 21 CoRKp. Only two isolates SC-KP169 and SC-KP585 carried mcr-1 and mcr-8, respectively. The plasmid pSC-KP169-1 could be transferred inter- and intra-genus and contributed to the virulence of K. pneumoniae to larvae. In contrast, the plasmid pSC-KP585-1 could be transferred to E. coli but could not affect its virulence to larvae. ConclusionsWe identified 21 CoRKp from 1,966 isolates and found a conjugative plasmid carrying mcr-1 gene that contributed to the virulence of K. pneumoniae to larvae.