• Complete Genome Sequencing Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes

      Ramli, Siti Roszilawati; Bunk, Boyke; Spröer, Cathrin; Geffers, Robert; Jarek, Michael; Bhuju, Sabin; Goris, Marga; Mustakim, Sahlawati; Pessler, Frank; HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. (PLOS, 2021-09-15)
      The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence.
    • Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Typhimurium ST313.

      Ondari, Edna M; Klemm, Elizabeth J; Msefula, Chisomo L; El Ghany, Moataz Abd; Heath, Jennifer N; Pickard, Derek J; Barquist, Lars; Dougan, Gordon; Kingsley, Robert A; MacLennan, Calman A; et al. (F1000Research, 2019-01-01)
      Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313. Methods: Six S. Typhimurium ST313 bloodstream isolates, three of which were antibody resistant, were studied. Genomic content (single nucleotide polymorphisms and larger chromosomal modifications) of the strains was determined by Illumina and PACBIO sequencing, and functionally characterized using RNA-seq, transposon directed insertion site sequencing (TraDIS), targeted gene deletion and transfer of selected point mutations in an attempt to identify features associated with serum resistance.   Results: Sequence polymorphisms in genes from strains with atypical serum susceptibility when transferred from strains that were highly resistant or susceptible to a strain that exhibited intermediate susceptibility did not significantly alter serum killing phenotype. No large chromosomal modifications typified serum resistance or susceptibility. Genes required for resistance to serum identified by TraDIS and RNA-seq included those involved in exopolysaccharide synthesis, iron scavenging and metabolism. Most of the down-regulated genes were associated with membrane proteins. Resistant and susceptible strains had distinct transcriptional responses to serum, particularly related to genes responsible for polysaccharide biosynthesis. There was higher upregulation of wca locus genes, involved in the biosynthesis of colanic acid exopolysaccharide, in susceptible strains and increased expression of fepE, a regulator of very long-chain lipopolysaccharide in resistant strains. Conclusion: Clinical isolates of S. Typhimurium ST313 exhibit distinct antibody susceptibility phenotypes that may be associated with changes in gene expression on exposure to serum.