Animal experiments were performed according to the Animals (Scientific Procedures) Act 1986 and approved by the Home Office (Project licences: P4E664E3C and PP5900632). Human neutrophil experiments were conducted under research ethics committee reference 17/LO/1531, and written informed consent was obtained prior to conducting blood draws.

Bacterial strains

Clinical isolates of S. sonnei (Table 1) were obtained in collaboration with the United Kingdom Health and Security Agency (UKHSA), Institut Pasteur, and Oxford University Clinical Research Unit (OUCRU). Strains were originally collected as part of routine public health surveillance in the UK and France^60,61, or as part of a cohort study in Vietnam⁶². Strains were received on agar slants and streaked on plates of Tryptic Soy Agar (TSA; Sigma Aldrich), supplemented with 0.01% Congo Red (CR; Sigma Aldrich) to select for pINV+ isolates. Red colonies, which indicate T3SS and pINV presence, were picked, stored in glycerol at 25% (v/v) and stored at −80 °C. Overnight cultures were prepared by inoculating 5 mL Trypticase Soy Broth (TSB; Sigma Aldrich), where necessary supplemented with 100 μg/mL carbenicillin, with a single red colony and incubating at 37 °C for ~16 h, with shaking at 200 rotations per minute (rpm).

Fluorescent bacterial strains were generated by transformation with pFPV25.1, which encodes a green fluorescent protein (GFP) reporter and resistance to carbenicillin as a selective marker. Electrocompetent cells were generated by growing bacteria to an optical density (OD) of 0.3–0.4, centrifugation at 4 °C and washing with 10% (v/v) ice-cold glycerol. 100 ng of DNA was added, and the suspension was electroporated using the Ec2 setting on a Bio-Rad Micropulser. 1 mL of TSB was immediately added, and bacteria were left to recover at 37 °C for 2 h. Following recovery, the suspension was plated on TSA supplemented with 50 μg/mL carbenicillin to select for positive colonies.

In silico analysis

Complete S. sonnei genomes (accessions available in Table 1, sequencing and assembly described previously¹⁵) were screened for virulence using ABRicate (v.1.0.1)⁶³ with the VFDB option selected (accessed on 18/08/2024)⁶⁴. MAFFT (v.7.526)⁶⁵ was used to perform a core genome alignment with default settings. The resulting alignment was used as an input to FastTree (v.2.1.1)⁶⁶, which was run using the generalised time-reversible model to produce a maximum likelihood phylogenetic tree. Visualisation was performed using Phandango (accessed on 19/12/2024)⁶⁷ and InkScape (v.1.3.2). To generate gene cluster comparison figures, relevant gene clusters were extracted and aligned using Clinker (v.0.0.29)⁶⁸. Fimbrial operons were manually inspected and compared using Mauve (v.2.4.0)⁶⁹.

Zebrafish infection

All zebrafish experiments were performed on larvae up to 5 days post fertilisation (dpf). Zebrafish embryos were obtained from naturally spawning larvae and incubated at 28.5 °C in 0.5 x E2 medium (15 mM NaCl, 1 mM MgSO4, 500 μM KCl, 150 μM KH2PO4, 50 μM Na2HPO4, 0.3 μg/ml methylene blue).

20 mL of TSB was inoculated with 400 μL overnight culture and grown to mid-exponential phase. Bacteria was harvested by centrifugation (4000 x g, 5 min), washed in 1 mL phosphate-buffered saline (PBS; Sigma Aldrich) to remove residual media and pelleted again (1 min, 6000 x g). The desired inoculum concentration was achieved by measuring the OD of bacteria and correction to the desired OD. Injection inoculum was prepared by resuspension of bacteria in inoculum buffer (2% polyvinyl-pyrrolidone (PVP; Sigma Aldrich), PBS and 0.5% phenol red (Sigma Aldrich)) to a final volume of 100 μL. Control groups of larvae were injected with inoculum buffer.

Dexamethasone treatment

Dexamethasone (Sigma Aldrich) was resuspended in dimethyl sulfoxide (DMSO, Sigma Aldrich) at a concentration of 25 mg/mL. Injections were performed as described above, and recovered larvae were split into two groups, treated and control. The treated group were incubated in E2 medium containing 50 µg/mL dexamethasone, and the control group were incubated in E2 medium with the same concentration of DMSO added, as reported previously⁷⁰.

Survival assays and bacterial burden

Larvae were maintained in groups of two or three in 24-well plates at 28.5 °C or 32.5 °C and visualised using a light stereomicroscope to check survival. The precise inoculum was determined retrospectively by the mechanical disruption of a single larva in 200 µL 0.4% Triton-X-100 (Sigma Aldrich) at 0 hpi, and at 6 and 24 hpi for bacterial burden quantifications. For each time point, four different larvae were selected at random as representatives for the infected population. Larvae homogenates were serially diluted in PBS, plated on TSA plates supplemented with CR and colonies were counted manually following overnight incubation at 37 °C.

Eukaryotic cell work

HeLa cells (Human ATCC CCL-2) were used for eukaryotic cell infections. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Aldrich) supplemented with 10% foetal-bovine serum (FBS, ThermoFisher Scientific) and incubated in an incubator supplied with 5% CO₂ at 37 °C. Cells were seeded at a density of 1.5 × 10⁵ in a 6-well plate (VWR) 48 h before infection. Bacterial cultures were grown to mid-exponential phase and then diluted in DMEM (not supplemented with FBS) to reach a multiplicity of infection (MOI) of 100:1. 1 mL of bacterial suspension was added to each well, and cells were centrifuged at 500 x g for 10 min at room temperature. Cells were then incubated for 30 min with 5% CO₂ at 37 °C. Bacterial suspension was removed from the cells, and the cells were washed three times with PBS. Washed cells were treated with 2000 µL of 50 mg/mL gentamycin (Sigma Aldrich) in DMEM + FBS for 1 or 3 h for invasion and replication timepoints, respectively. Following treatment, cells were lysed with 0.1% Triton-X-100 (Sigma Aldrich) in PBS for 5 min at 37 °C. Cell lysates were then serially diluted and plated on TSA, plates were incubated overnight, and CFU counts were determined the following morning.

For imaging, cells previously seeded and infected on coverslips were washed 3 times with PBS and then fixed using 4% (v/v) paraformaldehyde (PFA) in PBS for 15 min at room temperature. Fixed cells were washed 3 times in PBS and permeabilised in blocking (0.1% Triton X-100 and 1% bovine serum albumin (BSA) in PBS). Cells were stained with Hoechst 33342 (1:500, #H3570, ThermoFisher Scientific) and AlexaFluor-647 conjugated Phalloidin (1:500, #10656353, ThermoFisher Scientific) in blocking solution at room temperature in a humid chamber for 1 h. Coverslips were washed 9 times in PBS and then mounted onto glass slides using ProLong Gold antifade reagent with DAPI (#P36935, Thermofisher) mounting media. Fluorescence microscopy was performed using a ZEISS CellDiscover 7 (CD7) microscope using a ZEISS Plan-APOCHROMAT 20× / 0.95 Autocorr Objective coupled to a 0.5x tubelens. Z-stack confocal images of 32 slices over 5 µM were acquired. Confocal images were processed using Airyscan processing (Weinerfilter) using “Auto Filter” and “3D Processing” options.

Human neutrophil infections

Blood was drawn from a healthy donor, using EDTA as an anticoagulant (BD Vacutainer, Becton Dickinson). Neutrophils were isolated using Polymorphprep (Serumwerk Bernburg) solution as per manufacturers guidelines. Briefly, 5–7 mL of blood was slowly layered over an equal quantity of Polymorphprep solution, and the layered solution was centrifuged for 30 min at 500 x g with decreased acceleration and no brakes on deceleration. The neutrophil layer was collected and washed with 10 mL 0.5% (v/v) PBS. Where red blood cells (RBC) were present, a lysis step was incorporated, where 3 mL of 1X RBC lysis buffer (Invitrogen) was added per 5 mL of blood collected and incubated for 10 min. Following RBC lysis, neutrophils were washed twice as previously described and finally resuspended in neutrophil medium (RPMI 1640 Medium, GlutaMAX™ Supplement, Sigma Aldrich). Neutrophils were counted using Trypan Blue Staining and resuspended to the desired concentration.

Infections were performed in a 48-well plate (VWR), with 10⁵ neutrophils added per well. S. sonnei was cultured as previously described, and 10³ bacteria were added per well, with experiments performed in technical duplicates. 20 µL of the initial inoculum was plated to determine precise bacterial input. The 48-well plate was incubated for 1 h at 37 °C with 5% CO₂. For CFU determination, 7.5 µL of 0.1% Triton-X was added, and neutrophils were placed on ice to lyse as previously reported⁵; to prevent excessive neutrophil cell death, cells were not centrifuged prior to lysis. A 10-fold serial dilution was next performed, and 20 µL of lysate at each dilution was plated. CFUs were counted the following morning, and bacterial survival was determined by normalising CFU at 1 hpi to the initial inoculum.

Bacterial RNA extraction and qRT-PCR

Bacterial cultures were grown to mid-exponential phase, as described above. An amount of culture corresponding to 1 × 10⁹ bacterial cells was pelleted by centrifugation at 5000 x g at 4 °C for 10 min, the supernatant was removed, and the pellet was kept at −80 °C overnight to aid with cell lysis. RNA was then extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs) as per the manufacturer’s instructions. RNA concentration was measured using a DeNovix DS-11 spectrophotomer.1000 ng of RNA was then converted to cDNA using a QuantiTect reverse transcription kit (Qiagen). Template cDNA was subjected to quantitative reverse transcription PCR (qRT-PCR) using a 7500 Fast Real-Time PCR System machine and SYBR green master mix (Applied Biosystems), with samples run in technical duplicates. Primers generated for this study were designed using the NCBI Primer Design Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and all primers used can be found in Table 2, rrsA was used as a housekeeping gene, and the delta-delta Ct method was used to quantify gene expression.

Zebrafish RNA extraction and qRT-PCR

For each condition, ~15 embryos were pooled and frozen overnight at −80 °C. RNA was then extracted using the RNAeasy Minikit (Qiagen), converted to cDNA and subjected to qRT-PCR as previously described. Primers used for zebrafish qRT-PCR can be found in Table 3. Zebrafish gene eef1a1a was used as a housekeeping gene, and the delta-delta Ct method was used to quantify changes in gene expression.

Zebrafish microscopy

For imaging, larvae were anaesthetised in 1X tricaine (Sigma Aldrich), placed into a 96-well plate (Perkin Elmer) and embedded in 1 drop of 1% low-melting point agarose (w/v, Thermo Scientific), and wells were topped up with tricaine for the duration of the imaging process. For leucocyte counts, larvae were imaged using a Leica M205FA microscope and counts were performed manually.

A Zeiss Celldiscoverer 7 (CD7) microscope was used for acquiring representative images. For whole embryo imaging, larvae were placed laterally, and a 5 x /0.35 plan-apochromat objective with a 0.5 x tube lens was used to capture widefield z-stacks of 13 slices of 18.46 µM each. For HBV imaging, larvae were placed head-down and widefield imaging was performed using the same objective, but with a 2 x tube lens, to capture z-stacks across the HBV.

Bacterial resistance assay

One fresh red colony was inoculated into 50 µL PBS. Lyophilised baby rabbit complement (Bio-Rad, C12CA) was resuspended in 2 mL ice-cold water, and 150 µL was added to the bacterial suspension. 10 µL was taken for serial dilution and plating to determine the initial inoculum. The bacterial-rabbit complement mixture was incubated at 37 °C, shaking at 200 rpm for 4 h before another 10 µL was taken, serially diluted and plated. CR+ colonies were counted the following morning, and fold change CFU was calculated by normalising colonies counted at 4 h to the initial inoculum. As a control, baby rabbit complement was heat killed at 56 °C for 30 min to inactivate the complement system, as previously reported^71,72.

Protein secretion assay

To analyse the secretion of T3SS effector proteins, bacterial cultures were grown until OD 0.2–0.3 and CR was added at a concentration of 200 µg/mL. Cultures were then grown to maximum OD (2–3). Bacterial cells were pelleted by centrifugation at 10,000 x g, at 4 °C for 10 min. The supernatant was then removed and filtered using 0.2 µm filters to remove cell debris. A volume of filtered supernatant corresponding to OD 2 was taken and trichloroacetic acid (Sigma Aldrich) was added at 10% (v/v) concentration, this mixture was then incubated at −20 °C overnight. The following day, samples were pelleted at 4 °C at maximum speed and washed with 1 mL ice-cold acetone (Sigma Aldrich) 3 times to remove any residual acid. Samples were resuspended in 25 µL 1X Laemmli buffer (10 mM Tris-HCl, pH 6.8, 2% sodium dodecyl 63 sulphate [SDS], 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) prior to SDS-PAGE.

SDS PAGE

Protein samples were boiled at 100 °C, 17.5 µL of sample was loaded onto 12% SDS polyacrylamide gels and gels were run at 100 V in 1X Tris-glycine-SDS running buffer in a Bio-Rad Protean Tetra cell. For secretion experiments, gels were rinsed with distilled water, then stained using Coomassie Brilliant Blue R-250 (Bio-Rad) overnight, before de-staining in a solution of methanol, acetic acid, and water (30%, 5% and 65% (v/v) respectively).

LPS extraction

Crude LPS extraction was carried out as described previously⁷³. Bacteria were grown overnight and then sub-cultured to reach mid-exponential phase as described above. Samples were normalised by OD to ensure an equal density of bacteria in each sample before pelleting at 10,000 x g for 10 min at 4 °C. Pelleted bacteria were resuspended in 200 µL 1X Laemmli buffer and boiled for 15 min. 5 µL DNAse I and RNAse (10 mg/mL) was added, and the mixture was incubated at 37 °C for 30 min. 10 µL of Proteinase K (10 mg/mL) was then added, and the mixture was incubated at 59 °C for a further 3 h. Following this, 200 µL of ice-cold Tris-saturated phenol was added to each sample. Samples were then heated to 65 °C for 15 min, with occasional vortexing and once cool, 1 mL petroleum ether was added. Samples were centrifuged at 14,000 x g for 10 min; the aqueous layer was isolated and added to 150 µL Laemmli buffer. 10 µL was added to a 12% SDS polyacrylamide gel and run as described above.

LPS was then visualised using a modified silver stain, which oxidises LPS, allowing for better visualisation⁷⁴. Briefly, following SDS-PAGE, gels were rinsed with water and then incubated with a fixing solution (40% ethanol, 5% acetic acid) overnight. Fixing solution was then replaced with an oxidising solution (0.7% periodic acid, 40% ethanol, 5% acetic acid) for 20 min. Following the oxidation step, the gel was washed three times in distilled water for 10 min and was then stained using the Pierce Silver Staining Kit according to the manufacturer’s directions. All gels were visualised using a ChemiDoc Touch Gel Imaging System.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.