Natural avirulentShigella boydiistrain in the Brazilian Amazon lacks major virulence genes and present Type II, Type III and Type VI Secretion Systems

Abstract

24 ABSTRACT:25 Background: Among Shigella species, Shigella boydii has always displayed a smaller role to the overall 26 Shigella burden, frequently placed at third in epidemiological studies and described as restricted to 27 Southeast Asia. Here we characterize an S. boydii isolated from an epidemiological study enrolling 28 1,339 Brazilian children from the Amazon region, in which Shigella species solely was the fourth cause 29 of bacterial diarrhea. S. boydii strain 183 was isolated from rotavirus co-infected children with acute 30 diarrhea. Here we aimed to characterize this strain regarding virulence and, immune response in a 31 pulmonary model. Methods: An in vitro HEp-2 epithelial cell invasion assay was used to compare the Running Title 2 32 invasive phenotype of S. boydii strain 183 with clinical and highly virulent S. flexneri strain, both 33 isolated from Brazilian children. A murine pulmonary model was performed to assess lung damage 34 by histopathological analysis. mRNA expression of immune response key genes was retrieved by 35 multiplex real-time PCR and correlations were obtained by network analysis. Broad genome analysis 36 was performed to confirm S. boydii identity and define its virulence profile. Results: S. boydii strain 37 183 showed fewer invasion rates in vitro and tissue damage in vivo as compared to virulent S. flexneri 38 201. When compared to a survival challenge in mice, S. boydii had 100% survival against 10% of 39 virulent S. flexneri. Overall, mRNA immune gene expression suggests a protective response against S. 40 boydii strains 183, in contrast to the inflammatory response induced by the virulent S. flexneri strain 41 201. Network analysis with S. boydii strain 183 displayed IFN-γ protagonism, contrasting with the 42 correlations centralized on TNF-α by the virulent S. flexneri strain 201. The genome showed a lack of 43 effector proteins and enterotoxins in S. boydii strain 183, and sequencing analysis of Ipa invasins 44 revealed mutations at functional sites. This avirulent S. boydii strain 183 presents the Type II Secretion 45 System, T6SS, in addition to T3SS. Conclusions: In addition to causing no disease, S. boydii strain 183 46 lacks effector proteins and enterotoxins. The presence of T6SS additional secretion system could 47 provide an advantage to establish this strain among commensal bacteria. 48 49 AUTHOR SUMMARY:50 The Shigella genus is a human pathogen responsible to shigellosis and remains one of the significant 51 causes of morbidity and mortality in children under five years old. This genus has four species, Shigella 52 flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae. S. flexneri and S. sonnei are the 53 most common in the worldwide infections; S. dysenteriae is rarely found, and S. boydii is responsible 54 for 1% of the infections and is known to be restricted to Southeast Asia. Once S. boydii have a Running Title 3 55 relatively small role in global Shigella disease, there are few studies regarding its virulence and 56 mechanisms. Here we c...