Acquisition of a novel restriction modification system regulates genetic flux and gene expression in the hypervirulent and globally disseminated CC17 lineage of group B Streptococcus

A Conditional Plasmid System for Markerless Gene Deletion in Genetically Recalcitrant Fusobacterium nucleatum subsp. animalis.

This chapter provides a detailed, step-by-step protocol for implementing a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains and provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.

Comparative genomic analysis of Streptococcus parasuis and Streptococcus suis reveals mobile element-associated enrichment of antimicrobial resistance and lack of detectable same-MGE colocalization with virulence-associated genes within stable species boundaries.

Streptococcus suis is a major porcine pathogen and a zoonotic agent that causes meningitis and septicemia in humans. Streptococcus parasuis, a recently recognized close relative, remains poorly characterized with regard to its clinical significance and genomic features. In this study, we generated a single-contig closed genome assembly with genome-wide DNA methylation profiles for S. parasuis strain A1, isolated from a diseased pig in Xinjiang, China, and complemented in silico genomic predictions with isolate-level experimental validation of antimicrobial resistance (AMR) genotypes, virulence genotypes, and phenotypic susceptibility for this reference strain. Using this high-quality genome as a reference anchor, we performed comparative genomic analyses across 195 streptococcal genomes, comprising 15 S. parasuis and 180 S. suis strains, to distinguish genome-level co-occurrence of resistance and virulence determinants from their physical colocalization on the same mobile genetic element (MGE).Species boundaries remained clearly delineated at the genomic level, with a median interspecies average nucleotide identity (ANI) of approximately 86.0%, compared with intraspecies ANI medians of 97.5% for S. parasuis and 96.2% for S. suis. Pangenome analysis identified 12,693 gene clusters, of which 1086 were core clusters, and functional annotation revealed significant differences in accessory gene repertoires between the two species. Within this stable genomic framework, S. parasuis genomes carried a higher AMR gene burden; strain A1 harbored 10 AMR genes, multiple virulence-associated genes, three genomic islands, and eight prophage regions. For strain A1, PCR validation confirmed six AMR genes and six virulence genes, and disk diffusion testing demonstrated a multidrug-resistant phenotype consistent with the genotypic profile.Among 235 predicted mobile elements, 19 harbored AMR genes and seven carried Virulence Factor Database (VFDB) homologs, but none carried both categories simultaneously. This finding reflects a lack of detectable same-MGE colocalization under the applied annotation and assembly framework; it should not be interpreted as evidence of biological physical decoupling. Under a random-placement model, the expected number of co-carrying regions was only 0.57, and the probability of observing zero co-carrying regions was P = 0.55. This negative result should be interpreted with caution, given the limited number of cargo-bearing regions and the predominantly draft status of most genomes. Furthermore, the A1 genome contained multiple restriction-modification systems, showed depletion of several methylation motif families in mobile regions, and had limited CRISPR spacer matching evidence, suggesting prior exposure to the relevant sequence space. None of the genomes met our predefined criteria for whole-genome convergence.Collectively, our results support a model in which S. parasuis accumulates AMR-related genes in a modular fashion via mobile elements within stable species boundaries, with no detectable same-MGE colocalization of AMR and virulence determinants under our analytical pipeline. These findings imply that AMR surveillance strategies for this species should prioritize tracking mobile genetic elements rather than inferring wholesale genomic convergence toward S. suis.

Adaptive Evolution in Aminoacyl-tRNA Synthetases Drives Antibiotic Tolerance and Resistance in Clinical Klebsiella pneumoniae Isolates.

Mammalian-evolved strains of the fungal pathogen Cryptococcus neoformans show adaptation via heritable transcript level differences specific to mouse MHC haplotype.

BACKGROUND Organisms adapt to novel environments using changes to genome, gene expression, and protein functions. This study focused on changes that had occurred when a fungal pathogen previously encountered hosts that differed only at the major histocompatibility complex (MHC) region, loci that control immune recognition during the adaptive immune response. To investigate how this fungal pathogen adapted to the host environment, next generation sequencing data were examined from strains of Cryptococcus neoformans (C. neoformans) that had been previously passaged eight times through congenic mice that specifically differed at the MHC locus, H2. Transcript levels and the genomic sequence for each post-adapted fungal strain were examined to identify molecular adaptation strategies via heritable gene expression changes (epigenetic changes) and mutation (DNA changes). RESULTS The post-adapted strains displayed repeated changes in transcript levels, as determined by RNA-sequencing. Some of these epigenetically regulated genes (ERGs) only occurred in strains passaged in MHC specific hosts, suggesting possible prior adaptations to specific host MHCs. To our knowledge, this is the first time ERGs have been reported as possible pathogen adaptations to specific host MHC alleles. Additionally, of the total 47 single nucleotide polymorphisms (SNPs) identified, 7 SNPs each were found in 2 or more fungal strains (of the 6 analyzed strains) passaged through different MHC congenic hosts, suggesting much of the fungal mutation-based adaptation was to the mouse host, and not MHC-specific. CONCLUSIONS These data demonstrated that these passaged C. neoformans strains adapted to the MHC haplotype of a novel mammalian host environment primarily via epigenetic rather than mutation-based mechanisms.

A targeted mutational strategy aiding in generating antisense RNA to knockdown the Ehrlichia chaffeensis P28-outer membrane protein 19 expression

An innovative targeted mutational approach leading to expression of an antisense RNA facilitating the knockdown of p28-Omp19 protein expression from E. chaffeensis to express specific antisense RNA (asRNA) to knockdown protein synthesis from a gene.

Repeated mutation of a GT92 glycosyltransferase gene confers antiviral resistance in two Caenorhabditis species

Abstract Host–pathogen interactions evolve rapidly within species, providing natural genetic resources for the identification of specific ecological interaction factors. We previously identified RNA viruses that infect the nematodes Caenorhabditis elegans and Caenorhabditis briggsae in a species-specific manner. Wild strains of both host species demonstrate ample variation in viral sensitivity. Here we use recombinant inbred lines and pool-sequencing approaches to genetically map a major resistance locus in the C. elegans MY10 strain, narrowing down its position by CRISPR/Cas9-mediated recombination and testing candidates by genome editing. A rare non-synonymous polymorphism in the gtnt-1 gene, encoding a putative glycosyltransferase of the GT92 family, causes resistance to viral infection in MY10. Loss-of-function gtnt-1 alleles conferred host protection only at late developmental stages, highlighting the importance of multigenerational assays capturing the full course of viral infection and transmission. Viral resistance through gtnt-1 mutation occurred repeatedly in C. elegans, with diverse alleles each remaining at low frequency (<1%). Furthermore, leveraging closely related C. briggsae strains differing in viral susceptibility, we find that repeated loss-of-function alleles of the Cbr-gtnt-1 ortholog similarly enhanced resistance and host fitness upon infection. In conclusion, recurrent evolution in two host species of loss-of-function alleles of gtnt-1 orthologs leads to viral resistance. The gtnt-1 gene being conserved between Caenorhabditis species, these repeated inactivation events provide a case of transient ecological adaptation to a pathogen through recurrent mutation of the same gene in two species. The low population frequencies of resistant alleles point to a changing eco-evolutionary context that prevents their spread in populations.