Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

Bloodstream infections caused by nontyphoidal Salmonella are a major public health concern in Africa, causing ~49,600 deaths every year. The most common Salmonella enterica pathovariant associated with invasive nontyphoidal Salmonella disease is Salmonella Typhimurium sequence type (ST)313. It has been proposed that antimicrobial resistance and genome degradation has contributed to the success of ST313 lineages in Africa, but the evolutionary trajectory of such changes was unclear. Here, to define the evolutionary dynamics of ST313, we sub-sampled from two comprehensive collections of Salmonella isolates from African patients with bloodstream infections, spanning 1966 to 2018. The resulting 680 genome sequences led to the discovery of a pan-susceptible ST313 lineage (ST313 L3), which emerged in Malawi in 2016 and is closely related to ST313 variants that cause gastrointestinal disease in the United Kingdom and Brazil. Genomic analysis revealed degradation events in important virulence genes in ST313 L3, which had not occurred in other ST313 lineages. Despite arising only recently in the clinic, ST313 L3 is a phylogenetic intermediate between ST313 L1 and L2, with a characteristic accessory genome. Our in-depth genotypic and phenotypic characterization identifies the crucial loss-of-function genetic events that occurred during the stepwise evolution of invasive S. Typhimurium across Africa.

An anaerobic, Gram-stain-positive, rod-shaped, motile and spore-forming bacterium, designated strain LBM18003T, was isolated from pit clay used for making Chinese strong aroma-type liquor. Growth occurred at 20–40 °C (optimum, 30–37 °C), pH 4.5–9.5 (optimum, pH 6.5–7.0) and in the presence of 0.0–1.0 % (w/v) sodium chloride (optimum, 0 %). The predominant fatty acids were C16:0, C14:0, C14:0 DMA and C16:0 3-OH, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids and nine unidentified glycolipids. Phylogenetic analysis revealed that strain LBM18003T is a novel member of the family Oscillospiraceae . The 16S rRNA gene sequence similarities of strain LBM18003T to its two most closely related species were less than 94.5 % for distinguishing genera, i.e. closely related to Caproiciproducens galactitolivorans JCM 30532T (94.1 %) and Caproicibacter fermentans DSM 107079T (93.2 %). The genome size of strain LBM18003T was 2 996 201 bp and its DNA G+C content was 48.48 mol%. Strain LBM18003T exhibited 67.8 and 68.1% pairwise-determined whole-genome average nucleotide identity values to Caproiciproducens galactitolivorans JCM 30532T and Caproicibacter fermentans DSM 107079T, respectively; and showed 62.2 and 61.0 % the average amino acid identity values to Caproiciproducens galactitolivorans JCM 30532T and Caproicibacter fermentans DSM 107079T, respectively; and demonstrated 46.1 and 41.5 % conserved genes to Caproiciproducens galactitolivorans JCM 30532T and Caproicibacter fermentans DSM 107079T, respectively. The comparisons of 16S rRNA gene and genome sequences confirmed that strain LBM18003T represented a novel genus of the family Oscillospiraceae . Based on morphological, physiological, biochemical, chemotaxonomic, genotypic and phylogenetic results, strain LBM18003T represents a novel species of a novel genus of the family Oscillospiraceae , for which the name Caproicibacterium amylolyticum gen. nov., sp. nov. is proposed. The type strain is LBM18003T (=GDMCC 1.1626T=JCM 33783T).

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both “Shell first” and “Cargo first” assembly pathways, unlike the β-carboxysome structural analog which only involves the “Cargo first” strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB. This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

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SUMMARYMucosal-associated invariant T (MAIT) cells are a subset of innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defence. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with a selective absence of MAIT cells have not been identified. We report that typhoidal and non-typhoidal S. enterica strains generally activate MAIT cells. However, African invasive disease-associated multidrug-resistant S. Typhimurium sequence type 313 lineage 2 strains escape MAIT cell recognition through overexpression of ribB, a bacterial gene that encodes the 4-dihydroxy-2-butanone 4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. This MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium sequence type 313 lineage 2 in vivo.