In this study, we found that localization of PtlH in the B. pertussis
bacterium is dependent both on Ptl proteins and on the toxin substrate. Our finding that both the toxin and other Ptl proteins are required for proper localization of PtlH suggests that PtlH may interact either directly or indirectly with one or more of these proteins. Given the homologies that exist between the Ptl system and other type IV transporters, it is expected that PtlH would interact directly with other Ptl transporter proteins (9
), possibly PtlF, since data from yeast two-hybrid studies of the VirB proteins have demonstrated a direct interaction between VirB11 (PtlH homolog) and VirB9 (PtlF homolog) (39
). While direct interactions of PtlH with other Ptl proteins would be predicted, our finding that PT is required for proper localization of PtlH was unexpected and provides important clues as to when and how PT initially interacts with its transporter.
The finding that PT is required for proper localization of PtlH suggests that PtlH tightly engages with the transport apparatus only when PT is present. We found that neither the S1 subunit nor the B oligomer, composed of subunits S2, S3, S4, and S5, can substitute for the holotoxin. These results are consistent with and extend our previous finding, that secretion of PT from the bacterium requires assembly of the S1 subunit with subunits of the B oligomer (15
). Importantly, these findings suggest that final assembly of the transporter does not occur in the absence of the holotoxin substrate.
Localization of PtlH in specific ptl
mutants provides further clues as to the events that may occur during Ptl transporter morphogenesis and the interaction of the transporter with PT. Mutants of B. pertussis
lacking PtlD, PtlE, PtlF, and PtlG displayed the greatest abnormalities in PtlH localization, suggesting that these proteins may interact either directly with PtlH or indirectly, perhaps by stabilizing the protein(s) that interact with PtlH. PtlD, PtlE, PtlF, and PtlG proteins are homologs of VirB6, VirB8, VirB9, and VirB10, respectively, which are part of the membrane-associated core complex of VirB/D4 transporter proteins, the formation of which is believed to represent the first step in the assembly of that transporter (9
). Our results suggest that, likewise, association of PtlD, PtlE, PtlF, and PtlG may represent an initial step in Ptl transporter assembly and that assembly of this complex would occur before PtlH tightly engages with the transporter assembly. Subcellular localization of PtlH in the ptlI
mutant did not differ significantly from that of the wild-type strain, suggesting that PtlI, which is homologous to another VirB/D4 core-complex protein (VirB7), has only minimal effects on the interaction of PtlH with other proteins of the transporter complex. PtlA, which is homologous to the pilin subunit VirB2, has essentially no effect on PtlH localization. The finding that PtlA is not required for correct localization of PtlH indicates that the full complement of Ptl transporter proteins is not required for PtlH to tightly engage with the transporter. Importantly, the finding that PT but not PtlA is required for correct localization of PtlH suggests that PT can interact with an incomplete form of the transporter that lacks PtlA.
In this study, we also examined the requirement for ATP binding to PtlH and/or hydrolysis of ATP by PtlH for wild-type localization of PtlH. We believe that our result demonstrating a dependence of PtlH localization on ATP helps to shed light on the dynamics of the interaction of PtlH with the transporter-PT complex. In cells from which ATP has been deleted by the energy poisons CCCP or sodium arsenate, PtlH associates only loosely with the membrane, suggesting that normal interactions of PtlH with the membrane likely occur only if ATP is bound to the protein. These results are consistent with our finding that a mutant form of PtlH with alterations in its Walker A box also showed aberrant localization. Since the Walker A box of PtlH is essential for the ATPase activity of the protein and it is thought that this region is involved in nucleotide binding (38
), this form of PtlH is predicted to be defective in ATP binding.
Savvides et al. (30
) have demonstrated that HP0525, the Helicobacter pylori
type IV transporter homolog of PtlH, can exist in three forms, an ATP-bound form, an ADP-bound form, and an apo form devoid of any nucleotides. These three forms differ in conformation with the largest differences observed between the apo form and the two nucleotide-bound forms. These workers suggest that, because of the similarity in conformation between the ADP- and ATP-bound forms, nucleotide binding is likely the critical step for biological activity and that ATP hydrolysis would serve only to accelerate nucleotide release, regenerating the apo form for another cycle of activity. Our results are consistent with a model in which binding of ATP to PtlH results in tighter association of PtlH with the membrane, likely through interactions with other protein members of the transport apparatus and/or PT. Hydrolysis of ATP to ADP and subsequent release of the nucleotide would convert PtlH back to the form that is only loosely associated with the membrane because it exhibits a lower affinity for the transport apparatus.
When taken together, our results on the effects of PT, Ptl proteins, and ATP on the localization of PtlH allow us to significantly extend our model of PT secretion (Fig. ). In this model, toxin subunits are first individually secreted across the inner membrane into the periplasm. The periplasmic space contains the signal peptidase that would then cleave the signal sequences of each of the toxin subunits (26
), followed by assembly of the toxin. At the same time, portions of the Ptl apparatus would assemble, in particular, a core complex consisting, at least in part, of PtlD, PtlE, PtlF, and PtlG. PT would then interact with the Ptl subassembly followed by tight association of the ATP-bound form of PtlH with the complex. The pilus-like protein PtlA, which has been shown to be essential for toxin secretion (10
), may not be recruited to the transporter complex until the final stage of transporter assembly. Only after assembly of the transporter-PT complex is completed would secretion of the toxin proceed. Because we were unable to obtain mutants of B. pertussis
from which ptlB
were completely deleted, we cannot, as yet, say when these proteins might interact with the transporter apparatus during the assembly process.
FIG. 9. Model for secretion of PT. After synthesis, pertussis toxin subunits are individually transported across the inner membrane, likely by a Sec-like system. The signal sequences of the individual polypeptide chains are then cleaved, followed by assembly (more ...)
The mechanism by which PT, which is located in the periplasm, interacts with its secretion apparatus has remained an enigma. Because of its homology with other type IV transporters, the Ptl apparatus is believed to span both the inner and outer membranes of the bacterium, forming a translocation channel through which the substrate would pass (9
). The substrates for other type IV transporters are cytoplasmic and are thought to interact with the transporter at the cytoplasmic face of the inner membrane through initial interactions with VirD4 (9
), a protein that is absent in the Ptl system. Thus, current models of type IV transport do not easily accommodate a periplasmic substrate such as PT. The model shown in Fig. , which is based on the data presented in this study, depicts PT interacting with a partially assembled Ptl apparatus. Only after PT interacts with this subassembly would complete assembly of the apparatus occur. Such a model would explain how the periplasmic toxin is secreted by a transport apparatus that is believed to span the inner and outer membranes.