Deletion of toxin–antitoxin systems in the evolution of Shigella sonnei as a host-adapted pathogen

Abstract

Pathogenic Shigella spp. are the leading cause of bacterial dysentery, with Shigella flexneri and Shigella sonnei accounting for around 90 % of cases worldwide. While S. flexneri causes most disease in low-income countries (following ingestion of contaminated food and/or water), S. sonnei predominates in wealthy countries and is mainly spread from person-toperson. Although both species contain a large virulence plasmid, pINV, that is essential for the organism to cause disease, little is known about its maintenance. Here, using a counterselectable marker within the virulence-encoding region of pINV, we show that the S. flexneri plasmid is less stable than that of S. sonnei, especially at environmental temperatures. GmvAT, a toxin-antitoxin system, is responsible for the difference in stability, and is present in pINV from S. flexneri but is absent in S. sonnei pINV; GmvT is an acetyltransferase toxin that inhibits protein translation. Loss of GmvAT and a second toxinantitoxin system, CcdAB, from pINV reduces S. sonnei plasmid stability outside the host, reflecting the host-adapted lifestyle and person-to-person transmission of this species, and hence the striking differences in its epidemiology. Many critical functions in bacteria, including antibiotic resistance and virulence, are encoded on plasmids 1 which establish long-standing associations with particular bacterial lineages, even though they can impose fitness costs on their bacterial host. Human pathogens such as Shigella spp., Salmonella enterica and Yersinia spp. harbour plasmids which are essential for pathogenesis 1-4 , although little is known about the mechanisms of plasmid maintenance that lead to the retention of a functional plasmid, and how they are adapted to the lifecycle of these important human pathogens. 3 Shigella spp. are an important cause of bacillary dysentery that have emerged from Escherichia coli on several occasions following the acquisition of a single copy 210 kb plasmid, pINV 5. The plasmid contains a pathogenicity island (PAI) of ~ 30 kb that encodes a Type III Secretion System (T3SS) essential for virulence, as well as effector molecules and regulatory proteins 6,7. The Shigella T3SS acts as a molecular syringe that delivers effectors into host cells 8,9 and mediates entry of the bacterium 3,8. During in vitro growth, Shigella flexneri can lose the PAI or the entire plasmid, which results in increased growth rate 10 , highlighting the fitness cost of pINV in S. flexneri (pINVSf). S. sonnei and S. flexneri cause the overwhelming majority of shigellosis worldwide 11,12. Within less wealthy countries, S. flexneri infections are more common than S. sonnei 11,13 , and are typically acquired from contaminated food or water 14. In contrast, S. sonnei accounts for over 70 % of disease in wealthy countries 11. In this setting, S. sonnei is mainly spread by direct person-to-person contact between individuals in close proximity, such as children in playgroups, men having sex with men (MSM), and those living in institutions 15-19 .

Identification and characterization of acetyltransferase‐type toxin‐antitoxin locus in Klebsiella pneumoniae

“…The GNAT superfamily proteins are widely distributed in eukaryotes and prokaryotes, and have been shown to influence many physiological events by transferring the acetyl group from AcCoA to different substrates (Xie et al ., ). Three examples of GNAT toxins have been described, namely TacT of Salmonella enterica Typhimurium (Cheverton et al ., ), AtaT of enterohemorrhagic Escherichia coli O157:H7 (Jurėnas et al, ) and GmvT of Shigella flexneri pINV plasmids (McVicker and Tang, ). The toxicity of these GNAT toxins is abolished by the co‐expression of their cognate antitoxins that contain the ribbon‐helix‐helix (RHH) conserved domains and form the GNAT‐RHH family of type II TA systems.…”

Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle

“…Another translational inhibitor, the AtaT toxin from Escherichia coli O157:H7, was shown to specifically acetylate the amino group of initiator Met-tRNA fMet preventing its interaction with IF2·GTP (14,15). Toxicity of several other GNAT type II toxins has been demonstrated although their actual targets remain unknown (12,14,16–20). …”

Shigella sonneiO-antigen inhibits internalisation, vacuole escape and inflammasome activation

“…harbour a large virulence plasmid (LVP) that encodes the T3SS, its effectors and additional important virulence factors. The LVP of S. sonnei is less stable than S. flexneri due to the evolution of different toxin:anti-toxin systems (30). We inserted an antibiotic resistance cassette onto the LVP to create a stabilised LVP and used this strain to test if LVP loss was affecting the amount of cell death that was induced.…”