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

Date Published: September 27, 2019

Publisher: Public Library of Science

Author(s): Rocío Canals, Roy R. Chaudhuri, Rebecca E. Steiner, Siân V. Owen, Natalia Quinones-Olvera, Melita A. Gordon, Michael Baym, Michael Ibba, Jay C. D. Hinton, Andreas J. Baumler.


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.

Partial Text

Salmonella spp. are important pathogens of humans and animals. In humans, salmonellosis is classified as either a typhoidal or non-typhoidal Salmonella (NTS) disease. Typhoidal salmonellosis involves systemic spread through the body that causes enteric fever, and is associated with the S. enterica serovars Typhi (S. Typhi) and Paratyphi (S. Paratyphi). In contrast, NTS disease normally involves a self-limiting gastroenteritis that is transmitted via food, involving approximately 94 million human cases and about 155,000 deaths [1]. The S. enterica serovar Typhimurium (S. Typhimurium) sequence type ST19 causes the majority of gastroenteritis in immuno-competent individuals worldwide via pathogenic mechanisms that induce mucosal inflammatory responses in the gut. S. Typhimurium can thrive in this inflamed gut whilst other key members of the gut microbiota cannot survive [2,3]. The remarkable ability of this pathovariant to enter, survive, and proliferate in mammalian macrophages and epithelial cells in a “Salmonella-containing vacuole” (SCV) is responsible for systemic disease in both animals and humans [4].




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