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Final Report Summary - DISTINCT (Determining how invasive S. Typhimurium infects human cells by transposon-insertion sequencing)

Salmonella enterica serovar Typhimurium (S. Typhimurium) infects a wide range of animal hosts, and generally causes a self-limiting gastroenteritis in humans. However, some variants of this serovar, sequence-type ST313, are causing an emerging invasive Salmonella disease in sub-Saharan Africa that targets susceptible HIV+, malarial and malnourished individuals. These ST313 isolates show multiple-antibiotic resistance, necessitating the replacement of conventional therapies with alternative antibiotics. The genome sequence of one representative of ST313, D23580, was published and compared to the genomes of other Salmonella, such as S. Typhimurium SL1344, S. Typhi and S. Paratyphi A [1]. The fact that 60% of the genome degradation identified in the D23580 isolate was shared with typhoidal Salmonellae, host-restricted serovars that infect humans, suggested that the ST313 isolates have evolved to adapt to a unique niche in Africa and become human-adapted [1].
The goal of this project was to understand the factors that have allowed S. Typhimurium ST313 isolates to cause invasive disease, and to become a major public health problem in sub-Saharan Africa. The study used the latest advances in transposon-insertion sequencing to identify bacterial genes responsible for survival inside monocyte-derived macrophages from human blood (huMDM), and to discover how these S. Typhimurium isolates might have become “human-adapted”.
During the first stages of this transposon-insertion sequencing project, the fellow constructed a transposon library in S. Typhimurium ST313 (strain D23580) and optimized the preparation of the DNA libraries for Illumina sequencing. Competitive fitness experiments of the pool of transposon mutants were carried out in two different laboratory media to study the selection effects of growing bacteria in these media between passages in the eukaryotic cells. One of the two media was a rich medium, Lennox broth (LB), and the other one was a phosphate carbon nitrogen (PCN) minimal medium that reflects the intra-macrophage environment [2]: SPI2-inducing PCN (an acidic phosphate-limiting minimal medium that induces Salmonella pathogenicity island (SPI) 2 expression) [3]. The analysis of the sequencing data was used to determine the complexity of the library, verify the robustness of the improved transposon-insertion sequencing strategy and identify Salmonella genes required for growth on those conditions.
Data analysis at this stage highlighted the importance of having a good reference genome and annotation for mapping the transposon-insertion sequence data. The fellow discovered that the public annotation for the S. Typhimurium D23580 strain (chromosome and pSLT-BT plasmid: accession nos. FN424405 and FN432031, respectively) was missing some important essential genes that were actually present on the genome. Thus, the fellow improved the annotation of this strain using a combination of comparative genomic analysis and RNA-seq data. In addition, the D23580 strain was sequenced using the state-of-the-art PacBio technology, to determine the exact nucleotide sequence of the chromosome and the two large plasmids, pSLT-BT and pBT1. Two more plasmids were known to be carried by this strain, pBT2 and pBT3, but their DNA sequences were not publically available [1]. Draft sequences of plasmids pBT1, pBT2, and pBT3 were kindly provided by Dr. Rob Kingsley (Institute of Food Research, Norwich, UK), and these were used to obtain a complete nucleotide sequence of plasmids pBT2 and pBT3 using a primer walking strategy.
The results of the transposon-insertion sequencing experiments were visualised with a user-friendly online tool. Also, a pipeline for transposon-insertion sequencing data analysis was implemented and improved. Results were compared to RNA-seq data obtained from S. Typhimurium D23580 grown in different infection-relevant conditions and interesting findings were revealed by this correlative analysis. Previous transposon-insertion sequencing published studies for other Salmonella strains (a derivative of S. Typhimurium SL1344, S. Typhimurium 14028 and S. Typhi [4,5,6]) were used to identify specific fitness features for D23580.
Different human cell infection models were explored prior to screening the S. Typhimurium D23580 transposon library. A first methodology based on a Ficoll-Paque Plus density gradient (GE Healthcare Life Sciences) was used to isolate human peripheral blood mononuclear cells (PBMCs) and determine the number of viable cells obtained from one individual. A second methodology was based on a recent publication that showed that replication of S. Typhimurium in huMDMs depends on the phenotype of the infected macrophages: M1 macrophages (classically activated) do not permit bacterial replication, while M0 macrophages (nonactivated) or M2a (alternatively activated by IL-4) do permit bacterial replication [7]. This second methodology involved the enrichment of CD14+ monocytes from human peripheral blood, and polarization into the different macrophage phenotypes by addition of IFNγ and LPS for M1, or IL-4 for M2.
Two monocyte-screening panels based on surface markers and a compensation panel for flow cytometry were designed to validate the phenotype of the huMDMs before performing Salmonella infections. The compensation matrix that was used for the experiments was created using human blood from a healthy individual.
Experiments based on the optimization of Salmonella infections were carried out. Several infection assays using RAW264.7 macrophages and the wild-type S. Typhimurium D23580 strain were performed to determine the replication rate of this strain inside this cell line. These results were used to finalise the parameters for huMDM infection with Salmonella.
M0, M1 and M2 huMDMs were infected with S. Typhimurium D23580 using the same gentamicin exclusion assay protocol that was used for murine RAW264.7 macrophages. Counts of intracellular bacteria showed that Salmonella only replicated in M2 macrophages. This observation was crucial for setting up the conditions for the competitive screening of the transposon library in huMDMs.
The S. Typhimurium D23580 transposon library was passaged in murine RAW264.7 macrophages and genomic DNA samples were isolated from three replicates of each passage. The same library will be passaged in huMDMs to identify genes important for survival in human primary macrophages but not in murine macrophages.
Candidate genes were identified in the first part of the project that showed a putative role in bacteria survival in laboratory media and/or proliferation in murine macrophages. Knockout and SNP mutants were constructed and used for various fitness and phenotypic assays.
The growing number of organisms resistant to currently available antibiotics has become a major public health threat worldwide. There is a need to develop effective therapeutics based on new targets and approaches. Multiple-antibiotic resistant nontyphoidal Salmonella (NTS) are causing a major public health problem in sub-Saharan Africa and we are obliged to find new therapeutic strategies to combat and prevent these infections. To achieve this goal, the mechanisms of pathogenesis of these invasive isolates must be understood. The “human-specific” genes identified in this study will represent important targets for the development of novel therapeutics to combat these multidrug resistant isolates and to improve current Salmonella vaccines to protect the population of Africa. This global mutagenesis approach will also identify virulence-associated genes in the murine and human macrophages that may be used as potential targets for developing new antibacterial therapies.

REFERENCES: [1] Kingsley RA, et al (2009) Genome Res 19:2279-87; [2] Löber S, et al (2006) Int J Med Microbiol 296: 435-447; [3] Kröger C, et al (2013) Cell Host Microbe 14:683-95; [4] Langridge GC, et al (2009) Genome Res 19:2308-16; [5] Canals R, et al (2012) BMC Genomics 13:212; [6] Barquist L, et al (2013) Nucleic Acids Res 41:4549-64; [7] Lathrop SK, et al (2015) Infect Immun 83:2661-71.

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