Genome-wide analysis exploring mechanisms used by Shigella sonnei to survive long-term nutrient starvation

Shigella is a major cause of severe diarrhoea with S. flexneri and S. sonnei accounting for over 90% of infections. As economies grow, S. sonnei replaces S. flexneri as the dominant cause of shigellosis, however the basis of this epidemiological shift remains unclear. Here we investigated whether S. sonnei is better equipped to survive nutrient starvation, a crucial condition for persistence both outside the host and within the colonic lumen. S. sonnei exhibited greater survival under long-term nutrient starvation (LTNS) than S. flexneri, rapidly activating survival mechanisms. We interrogated the genome of S. sonnei using Transposon Directed Insertion-site Sequencing (TraDIS) revealing that metabolic pathways (ATP, nucleotide, and amino acid synthesis), and envelope homeostasis complexes (e.g., Tol-Pal, Bam) are conditionally essential for LTNS. TraDIS findings were validated by non-competitive and competitive survival of wild-type and deletion mutant strains. We compared the homology of conditionally essential genes between S. sonnei and S. flexneri to identify putative genetic markers of differential interspecies LTNS survival. Analysis of ldcA (a peptidoglycan carboxypeptidase) and rseA (the anti-sigma factor regulator) indicated a major role in sustaining survival in LTNS in S. sonnei; however, allele-swap with S. flexneri alleles restored wild-type survival in S. sonnei suggesting that monogenic changes may not explain the divergent survival of these two species. Together, these data define the molecular adaptations of starvation resistance in S. sonnei and provide insights into its epidemiological dominance in high-income countries. Author summaryUnderstanding why S. sonnei has a higher prevalence over S. flexneri as a country undergoes economic growth is one of the most important challenges in Shigella research. The investigation of their biological and genetic differences is key to tackle the impact of the disease. We discovered that S. sonnei resists nutrient deprivation better than S. flexneri, suggesting a better adaptation to an extracellular lifestyle and a greater preservation of metabolic capabilities. Using a genome-wide transposon sequencing approach we uncovered the key pathways behind the survival of S. sonnei facing nutrient starvation, which include ATP, nucleotide, and amino acid synthesis as well as maintenance of cell envelope integrity. Comparative analysis between S. sonnei and S. flexneri did not identify a single gene responsible for the differing survival and we suggest that the differing survival may stem from the coordination of multigenic differences. Our data provides a genome-wide basis for understanding how S. sonnei adapted to nutrient-deprived settings, which may be advantageous in the gut lumen and also in hostile environments, potentially contributing to its dominance in high-income countries.