title: Piericidin A1 BlocksYersiniaYsc Type III Secretion System Needle Assembly.
abstract: The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS inYersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alterYersiniamembrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action againstYersiniatype III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components inYersiniaare positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited theY. enterocoliticaYsc T3SS, it did not inhibit the SPI-1 family Ysa T3SS inY. enterocoliticaor the Ysc family T3SS inPseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibitsYersiniaYsc T3SS needle assembly.IMPORTANCEThe bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenicYersiniabut did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to theYersiniaYsc T3SS.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Morgan
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Miles C
authors.initials: MC
authors.lastname: Duncan
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Kevin S
authors.initials: KS
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Andreas
authors.initials: A
authors.lastname: Diepold
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Hanh
authors.initials: H
authors.lastname: Lam
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Dupzyk
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Lexi R
authors.initials: LR
authors.lastname: Martin
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Weng Ruh
authors.initials: WR
authors.lastname: Wong
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
authors.forename: Judith P
authors.identifier: 0000-0003-4983-9731
authors.initials: JP
authors.lastname: Armitage
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Roger G
authors.initials: RG
authors.lastname: Linington
authors.affiliation: Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Auerbuch
keywords: T3SS; Yersinia; piericidin; type III secretion system; virulence blocker