The Fitness Landscape of the African \u3cem\u3eSalmonella\u3c/em\u3e 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

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella

Salmonella 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 ofSalmonellagenomes has revealed that ST313 is closely related to the ST19 group ofSTyphimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish AfricanSalmonellafrom ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis ofSalmonellaWe identified a SNP in the promoter of thepgtEgene that caused high expression of the PgtE virulence factor in AfricanS.Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by AfricanSTyphimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome

Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease

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

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

AbstractInvasive non-typhoidalSalmonella(iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa.Salmonella entericaserovar 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-characterisedS.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 ofS.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 thesseIpseudogene. 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 importantin vitrogrowth 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.</jats:p

<i>Salmonella enterica</i> 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. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic 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 emergence of sublineage 2.2 in Malawi

A window into lysogeny: revealing temperate phage biology with transcriptomics

Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by 'accessory' or 'moron' loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from 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 represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages

Changing fitness effects of mutations through long-term bacterial evolution

13 Pág.The distribution of fitness effects of new mutations shapes evolution, but it is challenging to observe how it changes as organisms adapt. Using Escherichia coli lineages spanning 50,000 generations of evolution, we quantify the fitness effects of insertion mutations in every gene. Macroscopically, the fraction of deleterious mutations changed little over time whereas the beneficial tail declined sharply, approaching an exponential distribution. Microscopically, changes in individual gene essentiality and deleterious effects often occurred in parallel; altered essentiality is only partly explained by structural variation. The identity and effect sizes of beneficial mutations changed rapidly over time, but many targets of selection remained predictable because of the importance of loss-of-function mutations. Taken together, these results reveal the dynamic-but statistically predictable-nature of mutational fitness effects.M.B. acknowledges support from the NIGMS of the National Institutes of Health (R35GM133700), the David and Lucile Packard Foundation, the Pew Charitable Trusts, and the Alfred P. Sloan Foundation. This work was partially supported by the European Commission under the 7th Framework Program (ERC grant 310944 to O.T.), Foundation pour le Recherche Médicale (EQU201903007848) (to O.T.). This research was also funded in whole or in part by Agence Nationale pour la Recherche ANR GeWiEp (ANR-18-CE35-0005-0) (to O.T.) and Horizon 2020 Framework Programme (MSCA-IF 750129), cOAlition S organizations. The author will make the Author Accepted Manuscript (AAM) version available under a CC BY public copyright license. A.C. acknowledges support from a Comunidad de Madrid “Talento” Fellowship (2019-T1/BIO-12882) and the Agencia Estatal de Investigación (Proyectos de I+D+i, PID2019-110992GA-I00; Centros de Excelencia “Severo Ochoa”, SEV-2016-0672 and CEX2020-000999-S). A.L. acknowledges support from the Molecules, Cells, and Organisms Graduate Program, Harvard University. R.E.L. acknowledges support from the US National Science Foundation (DEB-1951307) and the John Hannah endowment at Michigan State University.Peer reviewe

The use of chicken and insect infection models to assess the virulence of African Salmonella Typhimurium ST313.

Over recent decades, Salmonella infection research has predominantly relied on murine infection models. However, in many cases the infection phenotypes of Salmonella pathovars in mice do not recapitulate human disease. For example, Salmonella Typhimurium ST313 is associated with enhanced invasive infection of immunocompromised people in Africa, but infection of mice and other animal models with ST313 have not consistently reproduced this invasive phenotype. The introduction of alternative infection models could help to improve the quality and reproducibility of pathogenesis research by facilitating larger-scale experiments. To investigate the virulence of S. Typhimurium ST313 in comparison with ST19, a combination of avian and insect disease models were used. We performed experimental infections in five lines of inbred and one line of outbred chickens, as well as in the alternative chick embryo and Galleria mellonella wax moth larvae models. This extensive set of experiments identified broadly similar patterns of disease caused by the African and global pathovariants of Salmonella Typhimurium in the chicken, the chicken embryo and insect models. A comprehensive analysis of all the chicken infection experiments revealed that the African ST313 isolate D23580 had a subtle phenotype of reduced levels of organ colonisation in inbred chickens, relative to ST19 strain 4/74. ST313 isolate D23580 also caused reduced mortality in chicken embryos and insect larvae, when compared with ST19 4/74. We conclude that these three infection models do not reproduce the characteristics of the systemic disease caused by S. Typhimurium ST313 in humans

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BackgroundInvasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment.MethodologySixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment.Findings1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates.ConclusionsThe finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment

The transcriptional organization of SPI2 in intra-macrophage <i>Salmonella</i>.

<p>Horizontal arrows represent individual SPI2 genes in scale with the whole Island. The <i>ssrA</i>-<i>B</i> gene products regulate SPI2 expression, the <i>sseA</i>-<i>E</i> genes encode effector proteins, the <i>sscA</i>-<i>B</i> genes encode chaperone proteins and the <i>ssaB</i>-<i>E</i> and <i>ssaG</i>-<i>U</i> loci encode components of the T3SS apparatus. The colour of each gene represents its relative expression, macrophage versus ESP (Dataset 4 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.s003" target="_blank">S1 Table</a>) based on the colour scale given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.g004" target="_blank">Fig 4</a>. Each track above and below the island shows the mapping of the RNA-seq or dRNA-seq reads against the plus or minus strand of the 4/74 chromosome visualized in IGB. Each curved arrow indicates the location of a TSS upstream of the respective gene; the width and height of each curved arrow is proportional to the TSS expression, based on relative PUV, macrophage versus ESP (Dataset 3 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.s003" target="_blank">S1 Table</a>) (Materials and Methods). The red hash on P<i>ssaR</i> indicates that the TSS was reported previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.ref017" target="_blank">17</a>], and confirmed in this study by 5’RACE (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.s001" target="_blank">S1 Fig</a>). The blue hashes indicate that the location of promoters P<i>ssaB</i>, P<i>sseA</i>, P<i>ssaG</i>, P<i>ssaM</i> and P<i>ssrB</i> that have been confirmed independently by 5’RACE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.ref107" target="_blank">107</a>] (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005262#ppat.1005262.s001" target="_blank">S1 Fig</a>).</p