Shigeaki MatsudaRyu OkadaSarunporn TandhavanantHirotaka HiyoshiKazuyoshi GotohTetsuya IidaToshio KodamaThe Research Foundation for Microbial Diseases of Osaka UniversityOsaka UniversityOkayama UniversityUniversity of California, DavisMahidol University2020-01-272020-01-272019-05-01Nature Microbiology. Vol.4, No.5 (2019), 781-788205852762-s2.0-85061729397https://repository.li.mahidol.ac.th/handle/20.500.14594/50191© 2019, The Author(s), under exclusive licence to Springer Nature Limited. Many Gram-negative pathogens utilize dedicated secretion systems to export virulence factors such as exotoxins and effectors 1–4 . Several exotoxins are synthesized as precursors containing amino-terminal Sec signal peptides and are exported through the inner-membrane-bound Sec machinery to the periplasm, followed by secretion across the outer membrane to the exterior using a type II secretion system (T2SS) 3,5 . Here, we report that thermostable direct haemolysin (TDH), an exotoxin of the food-borne pathogen Vibrio parahaemolyticus, can be exported through the type III secretion system (T3SS), which engages in one-step secretion of effectors 4 , despite possessing a Sec signal peptide and being mainly secreted via the T2SS. Although the precursor of TDH is targeted to the Sec pathway, a fraction of mature TDH was observed to re-enter the bacterial cytoplasm. The N terminus of mature TDH comprises a T3SS signal sequence, allowing it to be loaded into the T3SS. We also show that T3SS-delivered TDH as an effector contributes to intestinal fluid accumulation in a rabbit diarrhoeal model of V. parahaemolyticus infection. Thus, our results show that an unconventional export mechanism for a bacterial toxin via the T3SS in tandem with the Sec machinery facilitates the virulence trait of V. parahaemolyticus.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyMedicineLetterSCOPUS10.1038/s41564-019-0368-y