Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that Oantigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries.

Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii, bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii, unlike those of Legionella pneumophila, which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered.IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei, as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections.

42 Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the 43 closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The 44 underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) 45 model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. 46 Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence 47 depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show 48 in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can 49 mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. 50 sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical 51 inhibition or promotion of phagolysosome maturation respectively decreases and increases 52 neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal 3 53 dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-54 dependent manner, indicating that exposure to O-antigen can train the innate immune system 55 against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic 56 target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in 57 developing countries. 58 59 60 Author Summary 61 Shigella sonnei is predominantly responsible for dysentery in developed countries, and is 62 replacing Shigella flexneri in areas undergoing economic development and improvements in 63 water quality. Using Shigella infection of zebrafish (in vivo) and human neutrophils (in vitro), 64 we discover that S. sonnei is more virulent than S. flexneri because of neutrophil tolerance 65 mediated by its O-antigen oligosaccharide acquired from the environmental bacteria 66 Plesiomonas shigelloides. To inspire new approaches for S. sonnei control, we show that 67 increased phagolysosomal acidification or innate immune training can promote S. sonnei 68 clearance by neutrophils in vivo. These findings have major implications for our evolutionary 69 understanding of Shigella, and may explain why exposure to P. shigelloides in low and middle-70 income countries (LMICs) can protect against dysentery incidence. 71 72 73 4 80 has been a valuable discovery tool in the field of innate immunity, helping to illuminate the role 81 of neutrophil extracellular traps (NETs) [5], nucleotide-binding oligomerisation domain (NOD)-82 like receptors (NLRs) [6], bacterial autophagy [7], interferon-inducible guanylate-binding 83 proteins (GBPs) [8,9] and septin-mediated cell-autonomous immunity [10,11] in host 84 defence. 85 86 The genus Shigella comprises four different species (S. flexneri, S. sonnei, S. boydii, S. 87 dysenteriae), although DNA sequencing suggests they evolved from convergent evolution of 88 different founders [12]. The most recent strains of S. flexne...

15Two Shigella species, flexneri and sonnei, cause approximately 90% of bacterial dysentery 16 worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes 17 the majority of infections in middle and high-income countries. S. flexneri is a prototypic 18 cytosolic bacterium; once intracellular it rapidly escapes the phagocytic vacuole and causes 19 pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. By 20 contrast little is known about the invasion, vacuole escape and induction of pyroptosis during 21 S. sonnei infection of macrophages. We demonstrate that S. sonnei causes substantially less 22 pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to 23 reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic 24 S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-25 antigen, which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was 26 responsible for reduced uptake and the T3SS was required for vacuole escape. Our findings 27 suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating additional O-28 antigen into its surface structures compared to other Shigella species. 29 30 in this age group (1). Closely related to Escherichia coli, the genus is made up of four species; 34 S. flexneri, S. sonnei, S. dysenteriae and S.boydii. These are divided into serotypes based on 35 the O-antigen (O-Ag) structure. S. flexneri and S. sonnei are responsible for the majority of 36 infections however, dominance is highly dependent on the socioeconomic status of an area. 37 S. flexneri is associated with poor water sanitation and hygiene in developing countries. In 38 sub-Saharan Africa and Asia, S. flexneri accounts for 66% of cases and S. sonnei 24% of 39 cases (2). However, in areas with good socioeconomic conditions and a high gross domestic 40 product per capita, such as North America and Europe, S. sonnei is responsible for up to 80% 41 of infections (3). Transitional countries that have recently undergone socioeconomic 42 improvements show a shift from S. flexneri to S. sonnei as the dominant species (4-6). As a 43 number of large populous countries undergo this shift (eg. Brazil, India, China), S. sonnei is 44 emerging as an important pathogen. 45The pathogenesis of S. sonnei is poorly understood and generally assumed to be similar to S. 46 flexneri. The growing importance of S. sonnei has led to a re-evaluation of its pathogenesis 47 and has revealed some important differences from S. flexneri. These include a novel adhesin, 48 (7, 8), an antibacterial T6SS (9) and a group 4 capsule (G4C) which protects it from serum-49 mediated killing (10). Both species have a homologous T3SS that promotes secretion of 50 effectors into host cells. 51 Unlike other Shigella species which contain multiple serotypes, there is only one S. sonnei 52 serotype. The genes encoding biosynthesis and export of the O-Ag are encoded...

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