Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

Abstract

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

Energy source of flagellar type III secretion

“…Our conclusions are consistent with previous observations of a PMF requirement for flagellar growth from more than 25 years ago 19 , and extend the earlier findings in showing that export can be energized by PMF alone in the absence of any type III secretion ATPase 7 . Use of the proton gradient is perhaps not surprising given the speed of type III secretion and the likely advantage of tapping a proximal energy source.…”

“…The maintenance of normal ATP levels in the presence of CCCP was noted previously in experiments with Y. enterocolitica 7 and is thought to be due to a regulatory mechanism that inhibits the hydrolytic activity of the ATP synthase when the membrane is deenergized 10 . Because this protective mechanism may not act instantaneously, cellular ATP levels might undergo a transient drop following CCCP treatment that would escape detection in our measurements.…”

“…Homologues of FliI also occur in the type III secretion apparatus of injectisomes and are usually assumed to energize export in those systems as well. Some evidence for a different view has also been reported: it was observed that type III secretion in Yersinia enterocolitica was prevented by the protonophore CCCP 7 , and it was shown that the secretion ATPase InvC of Salmonella functions to dissociate export substrate from the chaperone 8 , a role distinct from transport itself. The energy source for type III secretion thus remains uncertain.…”

Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export

“…As it has been shown that Yersinia enterocolitica type III secretion is inhibited considerably by treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) 22 , and that flagellar formation is also dependent on proton motive force (PMF) 23 , we investigated whether flagellar protein export is still dependent on PMF in the gain-of-function mutant as well as wild-type cells. The rate of cell growth decreased when CCCP concentration increased, Fig.…”

The type III secretion injectisome, a complex nanomachine for intracellular ‘toxin’ delivery

“…InvC from S. enterica SPI-1 has been shown to unfold the exported proteins in an ATP-dependent manner and to detach some T3S substrates from their cytoplasmic chaperones before their export (Akeda and Galan, 2005). It is also probable that the ATPase energizes export but the proton motive force is also involved (Wilharm et al, 2004;Minamino and Namba, 2008;Paul et al, 2008). HrcN, the ATPase from P. syringae, forms hexamers and dodecamers that are activated by oligomerization and peripherally associated with the cytoplasmic side of the inner membrane (Pozidis et al, 2003).…”