Requirements for assembly of PtlH with the pertussis toxin transporter apparatus of Bordetella pertussis.

PtlH is an essential component of the Ptl system, the type IV transporter responsible for secretion of pertussis toxin (PT) across the outer membrane of Bordetella pertussis. The nine Ptl proteins are believed to interact to form a membrane-spanning apparatus through which the toxin is secreted. In this study, we monitored the subcellular localization of PtlH in strains of B. pertussis lacking PT, lacking other Ptl proteins, or from which ATP has been depleted in order to gain insight into the requirements for assembly of PtlH with the remainder of the Ptl transporter complex that is thought to be tightly embedded in the membrane. We found that PtlH is exclusively localized to the inner membrane fraction of the cell in a wild-type strain of B. pertussis. In contrast, PtlH localized to both the cytoplasmic and inner membrane fractions of a mutant strain of B. pertussis that does not produce PT. In comparison to how it localized in wild-type strains of B. pertussis, PtlH exhibited aberrant localization in strains lacking PtlD, PtlE, PtlF, and PtlG. We also found that localization of PtlH was perturbed in B. pertussis strains that were treated with carbonyl cyanide m-chlorophenylhydrazone and sodium arsenate, which are capable of depleting cellular ATP levels, and in strains of B. pertussis that produce an altered form of PtlH that lacks ATPase activity. When taken together, these results indicate that tight association of PtlH with the membrane, likely through interactions with components of the transporter-PT complex, requires the toxin substrate, a specific subset of the Ptl proteins, and ATP. Based on these data, a model for the assembly of the Ptl transporter-PT complex is presented.