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

Pertussis toxin (PT) is an essential virulence factor that is secreted by Bordetella pertussis (40), the causative agent of pertussis (whooping cough). PT is a large, complex, oligomeric protein that interferes with signaling pathways in host cells by ADP-ribosylating a family of GTP-binding regulatory proteins (16, 18). The toxin, which belongs to the AB class of bacterial toxins, is composed of five different subunits, S1, S2, S3, S4, and S5, found in a ratio of 1:1:1:2:1 (24, 25, 37). The enzymatically active S1 subunit (the A component) sits atop a ring formed by the remaining subunits that make up the binding component, or the B oligomer, of the toxin (33). Secretion of PT from the bacterium requires a transport system that comprises nine accessory proteins known as the Ptl proteins (41). The genes encoding the transport system are located directly downstream from the genes encoding the toxin structural subunits (Fig. ​(Fig.1A).1A). The Ptl transport apparatus is a member of the type IV family of secretion systems that are involved in the transport of proteins and/or DNA from bacterial cells (8, 42). The Ptl system displays considerable homology to other type IV transporters, including the prototypic type IV transporter, the VirB/D4 system of Agrobacterium tumefaciens, which has been studied extensively. Because of the similarities between these systems, much can be surmised concerning the general architecture of the Ptl transporter based on what is known about the VirB/D4 system. However, extrapolations from the VirB/D4 system to the Ptl system have certain limitations because distinct differences exist between the Ptl and VirB/VirD4 systems. In particular, these two systems differ dramatically in regards to their substrates and the locations within the bacteria at which that the substrates are believed to initially interact with the transporters. The substrates for the VirB/D4 transporter are cytoplasmic proteins, certain of which are covalently bound to DNA. These substrates are thought to interact with the VirB/D4 transporter at the cytoplasmic surface of the inner membrane through initial interaction with VirD4, a protein that is lacking in the Ptl system (2, 7). In contrast, the substrate for the Ptl system, PT, is an oligomeric protein that is transported via a Sec-like system to the bacterial periplasm independent of the Ptl transporter (41). Thus, PT is believed to interact initially with the Ptl transporter in the periplasm rather than the cytoplasm and therefore must interact with the Ptl transporter in a manner distinctly different from the manner in which the substrates of the VirB/D4 transporter initially interact with that assembly. Currently, little is known about how and when PT initially interacts with its transporter. FIG. 1. The ptx-ptl region and conserved motifs in transfer ATPases. (A) Schematic representation of the structure and nucleotide numbering of the ptx-ptl operon. Pr represents the promoter region of ptx-ptl operon. (B) Schematic representation of the conserved ... In our previous studies, we examined mutants of B. pertussis that lack specific Ptl proteins or the toxin itself in order to gain a better understanding of the transporter and its interaction with PT. In the course of these studies, we noted that subcellular localization of PtlH, a critical component of the Ptl transporter, is altered in certain of these mutants. PtlH is a member of a family of type IV transporter proteins that possess ATPase activity and which have been demonstrated to be peripheral inner membrane proteins (9, 27). Because tight association of peripheral membrane proteins with the membrane can depend on their interaction with other integral membrane proteins (19, 29), we monitored the subcellular localization of PtlH in specific mutant strains of B. pertussis in order to gain insight into the requirements for assembly of PtlH with the remainder of the membrane-embedded Ptl transporter. In this study, we examined the subcellular localization of PtlH in mutants of B. pertussis lacking PT and/or specific Ptl proteins. We also examined localization of PtlH in the absence of ATP binding. We found that tight association of PtlH with the membrane is dependent on the toxin substrate, certain other Ptl proteins, and ATP binding and/or hydrolysis, information that sheds light on the requirements for transporter assembly, interactions of PT with the transporter, and the sequence of events that take place during secretion of PT from B. pertussis.