Biology subjects

Bitar, I.

Publications and source records attributed to Bitar, I..

4 recordsLinked to original sources

bioRxiv2025

Long-Read Sequencing of the MUC1 VNTR: Genomic Variation, Mutational Landscape, and Its Impact on ADTKD Diagnosis and Progression

BackgroundADTKD-MUC1 is caused by frameshift mutations in MUC1 gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The genes variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations. MethodsWe employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the MUC1 VNTR with the rate of kidney function decline in affected individuals. ResultsWe identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units. ConclusionsSMRT sequencing outperforms current diagnostic methods for ADTKD-MUC1 and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest ([~]6% variance explained), it remains biologically and clinically meaningful. Key Points 3O_LISingle Molecule, Real-Time (SMRT) sequencing of MUC1 outperforms existing genetic and immunohistochemical methods for ADTKD-MUC1 diagnosis. C_LIO_LICLIA-approved mass spectrometry-based genotyping assay, complemented by SMRT sequencing, can detect nearly all ADTKD-MUC1 patients. C_LIO_LIDisease progression correlates with VNTR pattern: fast progressors have fewer WT repeats and more mutant/frameshifted repeats C_LI

genetics↗

bioRxiv2024

Does the host drive Wolbachia gene expression?

Wolbachia pipientis is an obligate intracellular bacterium, associated with several arthropods and filarial nematodes. Wolbachia establishes strict symbiotic relationships with its hosts, with the consequent loss of many genes and regulatory regions. Despite this, experimental studies show that Wolbachia gene expression is coordinated to host needs, but the mechanism is still unknown. The first published RNA-Seq study on Wolbachia evidenced a strong differential expression of a DNA methyltransferase (MTase). In bacteria, this enzyme methylates either adenines or cytosines on specific motifs, contributing to the regulation of gene expression. In this work, we tested the hypothesis that the activity of MTase modulates the expression of Wolbachia genes. We first determined the methylation motif of the Wolbachia MTase by expressing it in Escherichia coli. Surprisingly, the experiment revealed that the Wolbachia MTase methylates both adenine and cytosine, without recognising highly specific motifs. Then, re-analysing data from six RNA-Seq studies, we found that the nucleotide content of Wolbachia genes correlates with their expressions, with a pattern compatible to be a consequence of the DNA methylation. Lastly, we identified MTase as the Wolbachia gene with the most conserved binding site for the Ccka/CtrA signalling transduction system, a mechanism likely involved the host-bacterium communication. Overall, these findings suggest a cascade mechanism in which the host activates the Wolbachia Ccka/CtrA signalling system, thus inducing the expression of the MTase gene. Then, the subsequent DNA methylation will affect the expression of several Wolbachia genes on the basis of their cytosine and adenine content.

microbiology↗

bioRxiv2022

Whole genome-based characterization of multi-drug resistant Enterobacter and Klebsiella aerogenes isolates from Lebanon

BackgroundEnterobacter spp. are rod-shaped Gram-negative opportunistic pathogens belonging to Enterobacterales. This study aimed at the molecular and genomic characterization of multi-drug resistant Enterobacter spp. isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. MaterialsA total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by E-test were included in this study. Genotypic identification through whole-genome sequencing was performed and confirmed in silico. Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multi-locus sequence typing (MLST) was used to determine the isolates clonality. ResultsANI identified and confirmed that 47 (80%) isolates were E. hormaechei, 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC90 of [≥] 32 {micro}g/ml for ertapenem, imipenem, and meropenem. blaNDM-1 (58.5%), blaACT-16 (54%), and blaOXA-1 (54%) were the most common detected {beta}-lactamases, while blaCTX-M-15 gene (68%) was the main detected extended-spectrum {beta}-lactamase (ESBL) encoding gene. Chromosomal ampC gene, carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common amongst the studied E. hormaechei. ConclusionThe spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. ImportanceDrug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in healthcare settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

genomics↗

bioRxiv2021

Fitness effects of CTX-M-15-encoding IncF plasmids on their native Escherichia coli ST131 H30Rx hosts

ObjectivesThe objective of this study was to investigate effects of large CTX-M-15-encoding IncF plasmids on the fitness of their native E. coli ST131 H30Rx hosts in order to understand possible plasmid-host coevolution. MethodsWe selected five E. coli ST131 H30Rx strains of diverse origin, each carrying a multireplicon IncF plasmid encoding the gene blaCTX-M-15. The plasmid was eliminated from each isolate by displacement using an incompatible plasmid vector pMDP5_cureEC958. Whole-genome sequencing (WGS) was performed to obtain complete chromosome and plasmid sequences of wild-type isolates and to detect chromosomal mutations in plasmid-free strains. Competition assays were conducted to determine the relative fitness of plasmid-free clones compared to the corresponding wild-type isolates. ResultsWe were able to successfully eliminate the IncF plasmids from all of the wild-type strains using the curing vector pMDP5_cureEC958. The chromosomes of plasmid-free clones contained zero to six point mutations. Plasmid-free strains of three isolates showed no significant difference in relative fitness compared to the corresponding plasmid-free strains. In the two remaining isolates, the plasmids produced a small but significant fitness cost. ConclusionWe conclude that IncF plasmids produce moderate fitness effects in their E. coli ST131 H30Rx hosts. This fitness compatibility is likely to promote the maintenance of antibiotic resistance in this worrisome E. coli lineage.

evolutionary biology↗