SPATEs promote the survival of Shigella to the plasma complement system upon local hemorrhage and bacteremia

Abstract

Shigella spp. are the causative agents of shigellosis, which remains a leading cause of death in children under the age of 5. Symptoms of shigellosis include bloody diarrhea, associated to colon hemorrhage; in more severe cases, Shigella bacteremia is induced. These clinical features indicate that Shigella are exposed and survive exposure to plasma, locally and systemically, although this has not yet been studied at a molecular level. In this report, we confirmed in a guinea pig model of shigellosis that both S. flexneri 5a and S. sonnei induced local hemorrhages and we demonstrated that Shigella reached CD31+/CD34+ blood vessels located in the mucosa during the late stages of infection, and further disseminated in the bloodstream. These results confirmed the exposure of Shigella to plasma components during its virulence cycle. We demonstrated that all the tested Shigella strains survived plasma exposure in vitro, and we showed that Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) contribute to Shigella dissemination within the colonic mucosa and in the bloodstream. We have confirmed that SPATEs are expressed and secreted in poorly oxygenated environments encountered by Shigella during late infection stages. We further demonstrated that SPATEs promoted Shigella survival in plasma, by cleaving complement component 3 (C3), thereby impairing the complement system activation. We have shown here that the ability of Shigella to survive plasma exposure is a key factor in its virulence, both within primary foci and systemically.

Keywords: SPATE; Shigella; bacteremia; hemorrhage; plasma.

Fig. 1.

Shigella induces local hemorrhage, reaches blood vessels in vivo and survives to plasma exposure in vitro. (A) Guinea pig colonic mucosa infected for 48 h with S. sonnei et S. flexneri 5a wild-type (WT) strains. Black dotted lines delineate abscesses. Bars, 100 µm. Representative results from n > 3 animals per condition. Hemorrhage is associated with infiltration of RBCs within infected tissues, which were counted (Nb RBCs/mm²) in each condition. (B) CD31+/CD34+ colonic blood vessels were immunodetected in each condition (red/magenta), together with S. sonnei et S. flexneri 5a (green). DNA was stained with Dapi (blue). White boxes indicate individual blood vessels. Capillary lumens are figured with white dashed circles. Bars, 20 µm. Representative results from n > 3 animals per condition. (C, D) Growth of E. coli K12, S. sonnei, and S. flexneri 5a and 2a strains on M9 agar pads in the presence of fresh human plasma at 37 °C for 4 h and 18 h (C). Quantification of bacterial growth (D) by calculating confluence (%) of cultures. Results are expressed as mean ± S.D (n = 3). “ns” indicates P > 0.05, * indicates P < 0,05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001. See SI Appendix, Table S3 for additional information.

Fig. 2.

Shigella SPATEs structure, secretion regulation, and importance during infection. (A) SPATEs (SigA, SepA, Pic) secreted by S. sonnei, S. flexneri 5a, and S. flexneri 2a WT strains were identified by mass spectrometry. (B) The 3D structure of S. sonnei SigA was generated with AlphaFold 2 (ModelArchive 10.5452/ma-s7yqq) and compared to the available structures of SepA (Uniprot: Q8VSL2/PDB: 5J44) and Pic (Uniprot: Q54151) (–27). A rainbow color gradient was used from dark blue (N terminus) to dark red (C terminus). Black arrows show the position of the catalytic triad. (C) SPATE-containing culture supernatants from S. sonnei, S. flexneri 5a, and S. flexneri 2a grown in -O₂ and +O₂ conditions. Samples were separated on a 10% SDS-PAGE gel and stained with Coomassie. Representative result of three independent experiments. ns indicates P > 0.05. (D) The expression of SPATE-encoding genes was quantified by qRT–PCR in S. sonnei, S. flexneri 5a, and S. flexneri 2a grown in −O₂ and +O₂. rrsA mRNA levels were used as a control. Relative gene expression levels are expressed as mean ± S.D (n = 3). ns indicates P > 0.05. SI Appendix, Table S4 for additional information. (E) The presence of indicated Shigella strains (WT, SPATE mutants, and complemented strains) in the colonic lumen, mucosa, and in the blood circulation of guinea pigs was assessed 48 h.p.i. Results are expressed as mean ± S.D (n = 6). One-way ANOVA statistical tests were performed with a 95% CI (ns indicates P > 0.05, ** indicates P < 0.01, *** indicates P < 0.001). SI Appendix, Tables S5 and S6 for additional information.

Fig. 3.

SPATEs are essential for Shigella survival to plasma exposure, by cleaving Complement C3. (A) and (B) S. sonnei WT and ∆sigA strains A) and S. flexneri 5a WT and ∆sepA strains B) were incubated up to 18 h in the presence of fresh human plasma on M9 agar pads and the bacterial confluence was quantified. Results are expressed as mean ± S.D (n = 3). ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.0001. SI Appendix, Tables S7 and S8 for additional information. (C, D) S. sonnei ∆sigA (C) and S. flexneri 5a ∆sepA (D) strains were incubated in the presence of fresh human plasma or decomplemented plasma (dc. plasma) and data were analyzed as in (A) and (B). SI Appendix, Tables S9 and S10 for additional information. (E) Human albumin-free plasma (AF plasma) was incubated overnight at 37 °C with purified SigA or SepA and was analyzed on 10% SDS-PAGE gel, stained with Coomassie. (F) Mass spectrometry analysis of Bands 1 and 2, as indicated (from gel (E). (G, H) Purified human complement C3 (C3, α-chain/β-chain) was incubated with purified SigA and SepA for 18 h at 37 °C, samples were processed like in E). (I) Impact of SigA and SepA heat-inactivation on C3 cleavage. Samples were analyzed by western blot using anti-human C3 antibody (Top) and SigA and SepA were stained with Coomassie (Bottom).

Fig. 4.

SPATEs limit the deposition of C3 on Shigella surface upon plasma exposure in vitro, which is observed in vivo during hemorrhage. (A) S. sonnei, S. sonnei ∆sigA, S. flexneri 5a, and S. flexneri 5a ∆sepA pGFP (green) strains were grown in presence of plasma for 1 h at 37 °C (n = 3). Anti-human C3 (red) and rabbit anti-SigA or rabbit anti-SepA antibodies (magenta) were used to detect C3 and SPATEs at the cell surface of the bacteria. Dapi (blue) was used to stain DNA. Bars, 5 µm. (B) Guinea pig colonic mucosa infected by S. sonnei and S. flexneri 5a WT strains (green) for 48 h. Infected and noninfected tissues were stained with an anti-human complement C3 antibody (red) and DNA was stained with Dapi (blue). Bars, 50 µm. Representative results from n > 3 animals per condition. (C) Tissue hemorrhage was further emphasized using an anti-human albumin antibody (red) upon infection by S. sonnei and S. flexneri 5a (green). DNA was stained with Dapi (blue). Bars, 50 µm. Representative results from n > 3 animals per condition.