Natural avirulentShigella boydiistrain in the Brazilian Amazon lacks major virulence genes and present Type II, Type III and Type VI Secretion Systems
Authors: Serra Paula Taquita , Verçosa João Victor , de Souza Ruth Moura , de Souza Dantas Paloma Inessa , de Oliveira Rezende Alan , Barros Ana Paula Miranda , de Souza Aline Rubens , Alves Marcelo Ribeiro , de Souza Fernandes Pereira Marcelo , Balieiro Antônio , Raiol Tainá , Mariúba Luiz André Moraes , de Moraes Milton Ozório , Epiphanio Sabrina , Matos Najla Benevides , da Mota Adolfo José , Pontes Gemilson Soares , de Magalhães Júnior Paulo Franco Cordeiro , de Lacerda Marcus Vinícius Guimarães , Nogueira Paulo Afonso , Orlandi Patrícia Puccinelli 
Published: November 2018

Background

AmongShigellaspecies,Shigella boydiihas always displayed a smaller role to the overallShigellaburden, frequently placed at third in epidemiological studies and described as restricted to Southeast Asia. Here we characterize anS. boydiiisolated from an epidemiological study enrolling 1,339 Brazilian children from the Amazon region, in whichShigellaspecies solely was the fourth cause of bacterial diarrhea.S. boydiistrain 183 was isolated from rotavirus co-infected children with acute diarrhea. Here we aimed to characterize this strain regarding virulence and, immune response in a pulmonary model.

Methods

Anin vitroHEp-2 epithelial cell invasion assay was used to compare the invasive phenotype ofS. boydiistrain 183 with clinical and highly virulentS. flexneristrain, both isolated from Brazilian children. A murine pulmonary model was performed to assess lung damage by histopathological analysis. mRNA expression of immune response key genes was retrieved by multiplex real-time PCR and correlations were obtained by network analysis. Broad genome analysis was performed to confirmS. boydiiidentity and define its virulence profile.

Results

S. boydiistrain 183 showed fewer invasion ratesin vitroand tissue damagein vivoas compared to virulentS. flexneri201. When compared to a survival challenge in mice,S. boydiihad 100% survival against 10% of virulentS. flexneri. Overall, mRNA immune gene expression suggests a protective response againstS. boydiistrains 183, in contrast to the inflammatory response induced by the virulentS. flexneristrain 201. Network analysis withS. boydiistrain 183 displayed IFN-γ protagonism, contrasting with the correlations centralized on TNF-α by the virulentS. flexneristrain 201. The genome showed a lack of effector proteins and enterotoxins inS. boydiistrain 183, and sequencing analysis ofIpainvasins revealed mutations at functional sites. This avirulentS. boydiistrain 183 presents the Type II Secretion System, T6SS, in addition to T3SS.

Conclusions

In addition to causing no disease,S. boydiistrain 183 lacks effector proteins and enterotoxins. The presence of T6SS additional secretion system could provide an advantage to establish this strain among commensal bacteria.

AUTHOR SUMMARY

TheShigellagenus is a human pathogen responsible to shigellosis and remains one of the significant causes of morbidity and mortality in children under five years old. This genus has four species,Shigella flexneri,Shigella sonnei,Shigella boydii, andShigella dysenteriae.S. flexneriandS. sonneiare the most common in the worldwide infections;S. dysenteriaeis rarely found, andS. boydiiis responsible for 1% of the infections and is known to be restricted to Southeast Asia. OnceS. boydiihave a relatively small role in globalShigelladisease, there are few studies regarding its virulence and mechanisms. Here we characterize anS. boydiiisolated from Brazilian children from the Amazon region, and aimed to describe this strain regarding virulence. It is known thatShigellaspecies use the Type 3 Secretion System (T3SS) to invade and colonize the human intestine. We found inS. boydiithe presence of Type 2 Secretion System (T2SS), Type 6 Secretion System (T6SS), in addition to the T3SS. The T6SS have been described inS. sonneionly, granting a competitive advantage againstS. flexnerimixed cultures. The presence of T6SS additional secretion system could provide a benefit to establish this strain among commensal bacteria.