Abstract
Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.
Highlights
The genus Shigella is a major cause of diarrhoeal disease worldwide, and is responsible for around 188 million cases and 600,000 deaths each year [1, 2]
We demonstrate that MvpAT, a member of the VapBC family of toxin:antitoxin systems encoded on pINV, is responsible for both plasmid maintenance through post-segregational killing, and retention of adjacent sequences
We show for the first time that insertion sequences (ISs) on the plasmid and chromosome mediate inter-molecular recombination events, resulting in spontaneous and reversible integration of pINV into the chromosome; following integration, T3SS expression is down-regulated
Summary
The genus Shigella is a major cause of diarrhoeal disease worldwide, and is responsible for around 188 million cases and 600,000 deaths each year [1, 2]. The prevalence of each species depends on the geographic region, S. flexneri remains the leading cause of endemic shigellosis worldwide [2]. The four species of Shigella have emerged from Escherichia coli following the acquisition of a large plasmid, pINV, a 213 kb element that is essential for virulence [4]. PINV is a single copy, non-conjugative element that consists of a patchwork of pathogenesis-associated and plasmid maintenance genes, separated by regions of repeated sequences such as insertion sequence (IS) elements [5]. ISs are highly abundant in S. flexneri, and account for 53% of pINV-encoded genes and 6.7% of all chromosomal sequence [5, 6]
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