Biology subjects

Canals, R.

Publications and source records attributed to Canals, R..

8 recordsLinked to original sources

bioRxiv2023

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi, and we performed an intensive comparative genomic analysis of 608 isolates obtained from fever surveillance at the Queen Elizabeth Hospital, Blantyre between 1996 and 2018. We discovered that following the upsurge of the well-characterised S. Typhimurium ST313 lineage 2 from 1999 onwards, two new multidrug-resistant sublineages designated 2.2 and 2.3, emerged in Malawi in 2006 and 2008, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineage 2.2 or 2.3. To identify factors that characterised the emergence of the prevalent ST313 sublineage 2.2, we performed genomic and functional analysis of two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). Comparative genomic analysis showed that the chromosome of ST313 lineage 2 and sublineage 2.2 were broadly similar, only differing by 29 SNPs and small indels and a 3kb deletion in the Gifsy-2 prophage region that spanned the sseI pseudogene. Lineage 2 and sublineage 2.2 have unique plasmid profiles that were verified by long read sequencing. The transcriptome was initially explored in 15 infection-relevant conditions and within macrophages. Differential gene expression was subsequently investigated in depth in the four most important in vitro growth conditions. We identified up-regulation of SPI2 genes in non-inducing conditions, and down-regulation of flagellar genes in D37712, compared to D23580. Following phenotypic confirmation of transcriptional differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed-growth in minimal media. We speculate that this competitive advantage is contributing to the continuing presence of sublineage 2.2 in Malawi.

bioinformatics↗

bioRxiv2022

Genome-wide fitness analysis identifies genes required for in vitro growth and macrophage infection by African and Global Epidemic pathovariants of Salmonella Enteritidis

Salmonella Enteritidis is the second most common serovar associated with invasive non-typhoidal Salmonella (iNTS) disease in sub-Saharan Africa. Previously, genomic and phylogenetic characterisation of S. Enteritidis isolates from human bloodstream led to the discovery of the Central/Eastern African (CEAC) and West African clades, which were distinct from the gastroenteritis-associated Global Epidemic clade (GEC). The African S. Enteritidis clades have unique genetic signatures that include genomic degradation, novel prophage repertoires and multi-drug resistance, but the molecular basis for the enhanced propensity of African S. Enteritidis to cause bloodstream infection is poorly understood. We used transposon insertion sequencing (TIS) to identify the genetic determinants of the GEC representative strain P125109 and the CEAC representative strain D7795 for growth in three in vitro conditions (LB or minimal NonSPI2 and InSPI2 growth media), and for survival and replication in RAW 264.7 murine macrophages. We identified 207 in vitro-required genes that were common to both S. Enteritidis strains and also required by S. Typhimurium, S. Typhi and Escherichia coli, and 63 genes that were only required by individual S. Enteritidis strains. Similar types of genes were required by both P125109 and D7795 for optimal growth in particular media. Screening the transposon libraries during macrophage infection identified 177 P125109 and 201 D7795 genes that contribute to bacterial survival and replication in mammalian cells. The majority of these genes have proven roles in Salmonella virulence. Our analysis also revealed candidate strain-specific macrophage fitness genes, some of which represent potential novel Salmonella virulence factors. IMPACT STATEMENTInvasive non-typhoidal Salmonella (iNTS) disease is a systemic infection that has a high case fatality rate of 15% and is responsible for an estimated 66,500 deaths/year in sub-Saharan Africa. The main causative agents are pathovariants of Salmonella Typhimurium, known as S. Typhimurium ST313, and Salmonella Enteritidis (S. Enteritidis), known as Central/Eastern African (CEAC) and West African S. Enteritidis. Whilst the African S. Typhimurium pathovariant has been an active focus of research over the past decade, studies on African S. Enteritidis have been lacking. We used transposon insertion sequencing (TIS) to identify the genetic requirements of both African and Global Epidemic S. Enteritidis to grow in vitro and to infect murine macrophages. To our knowledge, this is the first genome-wide functional analysis of African S. Enteritidis under conditions relevant to infection of a mammalian host. We show that the gene sets required for growth under laboratory conditions and macrophage infection by African and Global Epidemic S. Enteritidis were broadly similar, and that the majority of the genes that contribute to survival and replication in macrophage already have proven roles in Salmonella virulence. Our analysis did identify candidate strain-specific macrophage fitness genes, some of which could be novel Salmonella virulence factors.

microbiology↗

bioRxiv2019

A window into lysogeny: Revealing temperate phage biology with transcriptomics

Integrated phage elements, known as prophages, are a pervasive feature of bacterial genomes. Prophages can enhance the fitness of their bacterial hosts by conferring beneficial functions, such as virulence, stress tolerance or phage resistance, which are encoded by accessory loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, novel prophage accessory loci are challenging to identify based on DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and often represent functionally-important accessory loci. Using this approach we identify a novel anti-sense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1 and immunity to closely-related phages. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.

microbiology↗

bioRxiv2019

African Salmonella Typhimurium sequence type 313 lineage 2 evades MAIT cell recognition by overexpressing RibB

Mucosal-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.

immunology↗

bioRxiv2019

The fitness landscape of the African Salmonella Typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of gram-negative and gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated.

microbiology↗

bioRxiv2019

The diversity, evolution and ecology of Salmonella in venomous snakes

BackgroundReptile-associated Salmonella are a major, but often neglected cause of both gastrointestinal and bloodstream infection globally. The diversity of Salmonella enterica has not yet been determined in venomous snakes, however other cold-blooded animals have been reported to carry a broad range of Salmonella bacteria. We investigated the prevalence and assortment of Salmonella in a collection of venomous snakes in comparison with non-venomous reptiles.\n\nMethodology/Principle FindingsWe used a combination of selective enrichment techniques and whole-genome sequencing. We established a unique dataset of reptilian isolates to study Salmonella enterica species-level evolution and ecology and investigated differences between phylogenetic groups. We observed that 91% of venomous snakes carried Salmonella, and found substantial diversity between the serovars (n=58) carried by reptiles. The Salmonella serovars belonged to four of the six Salmonella enterica subspecies: diarizonae, enterica, houtanae and salamae. Subspecies enterica isolates were distributed among two distinct phylogenetic clusters, previously described as clade A (52%) and clade B (48%). We identified metabolic differences between S. diarizonae, S. enterica clade A and clade B involving growth on lactose, tartaric acid, dulcitol, myo-inositol and allantoin.\n\nSignificanceWe present the first whole genome-based comparative study of the Salmonella bacteria that colonise venomous and non-venomous reptiles and shed new light on Salmonella evolution. The findings raise the possibility that venomous snakes are a reservoir for human Salmonellosis in Africa. The proximity of venomous snakes to human dwellings in rural Africa may result in contaminated faecal matter being shed on surfaces and in water sources used for human homes and to irrigate salad crops. Because most of the venomous snakes had been captured in Africa, we conclude that the high level of Salmonella diversity reflects the African environmental niches where the snakes have inhabited.\n\nAuthor SummarySalmonella enterica is a remarkable bacterial species that causes Neglected Tropical Diseases globally. The burden of disease is greatest in some of the most poverty-afflicted regions of Africa, where salmonellosis frequently causes bloodstream infection with fatal consequences. The bacteria have the ability to colonise the gastrointestinal tract of a wide range of animals including reptiles. Direct or indirect contact between reptiles and humans can cause Salmonellosis. In this study, we determined the prevalence and diversity of Salmonella in a collection of African venomous snakes for the first time. Using the power of genomics, we showed that the majority of venomous snakes (91%) carry Salmonella, two thirds of which belonged to a subspecies of S. enterica called enterica, which is associated with most cases of human salmonellosis. Within the S. enterica subspecies we identified two evolutionary groups which display distinct growth patterns on infection relevant carbon sources. Our findings could have particular significance in Africa where venomous snakes wander freely around human dwellings and potentially shed contaminated faecal matter in water sources and on surfaces in rural homes.

microbiology↗

bioRxiv2018

Adding function to the genome of African Salmonella ST313

Salmonella Typhimurium ST313 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 sixteen 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: bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by 3 independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolise this carbon source has been negatively selected during ST313 evolution. The data revealed a novel plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multi-condition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

microbiology↗

bioRxiv2017

The role of a single non-coding nucleotide in the evolution of an epidemic African clade of Salmonella

Introductory ParagraphSalmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely-related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by just 1000 single-nucleotide polymorphisms (SNPs). We hypothesised that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes.\n\nHere we identified 3,597 transcriptional start sites (TSS) of the ST313 strain D23580, and searched for a gene expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance and modulated virulence in the chicken infection model. The PgtE protease is known to mediate systemic infection in animal models. We propose that high levels of expression PgtE of by African S. Typhimurium ST313 promotes bacterial survival and bacterial dissemination during human infection.\n\nOur finding of a functional role for an extra-genic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on non-coding regions of the genome.

molecular biology↗