|Abstract:||The bacterium Pasteurella multocida is a gram-negative, non-spore- forming, capsulated coccobacillus that lives an aerobic to facultative anaerobic lifestyle. P. multocida is a zoonotic pathogen that causes a myriad of diseases, including atrophic rhinitis, pasteurellosis, and dermonecrosis in a wide gamut of different host types. A major virulence factor produced by serotype D and some serotype A strains of P. multocida is P. multocida toxin (PMT), a 146-kDa modular protein deamidase that enters host cells via receptor-mediated endocytosis and manipulates intracellular signaling by constitutively activating the a-subunit of heterotrimeric G-proteins. This dissertation will focus primarily on two major unanswered questions regarding PMT and its action on host cells. First major question: During translocation of PMT from late endosomes into the cytosol of host cells, does the N-terminus of PMT (PMT-N) deliver its C-terminal cargo as an intact moiety, or as separate functional subdomains? Second major question: During cellular intoxication, by what molecular mechanism does PMT uncouple and subsequently downregulate G-protein-coupled receptor- heterotrimeric Gaq-protein phospholipase C-b1 (GPCR-Gaq-PLCb1) signaling? These two questions are the basis for the generated hypotheses tested, experimental approaches taken, and sub-questions developed in this thesis. Here, I present data that contribute to answering these two major questions regarding delivery of C-terminal cargo by PMT-N into the host cytosol and its subsequent downregulation of Gaq-protein signaling in intoxicated cells.
To address the first question, we hypothesized that PMT-N delivers the C- terminal cargo into the cytosol of host cells as an intact polypeptide comprised of amino acid residues 569-1285. Previous studies have shown that PMT-N facilitates host cell entry and cytosolic delivery of the C-terminal catalytic cargo from late endocytic vesicles. The putative cargo of PMT is a 78-kDa polypeptide of three discrete modular subdomains C1-C2-C3, for which there is a crystal structure available. Subdomain C1 harbors the membrane localization domain (MLD); C2 has an unknown function; and C3 contains the enzymatic deamidase activity that converts an active-site glutamine residue into glutamic acid in specific Gα-proteins. What is not clearly understood is whether the three C- terminal cargo subdomains are delivered intact or undergo proteolytic cleavage between each discrete subdomain once translocated into the cytosol from late endosomes. In this dissertation, I present evidence that demonstrates that PMT- N mediates the cytosolic delivery of its C-terminal cargo as a single-chain polypeptide, corresponding to C1-C2-C3 with the MLD. Additionally, the delivered cargo showed no indication of being cleaved between subdomains. I present evidence that shows that PMT-N can deliver C1-C2 alone, as well as C1-C2 and a truncated C3, which is in line with our previous work that PMT-N can deliver non-native cargo into the cytosol of host cells. This further supports the notion that receptor-binding and translocation modules are encompassed within PMT-N. Furthermore, I show that C1-C2 may facilitate the cytosolic delivery of the catalytic C3 subdomain in coordination with PMT-N. In addition, I present data that refines the minimum C3 domain required for intracellular signaling activity as containing residues 1105-1278.
To address the second question, we hypothesized that PMT uncouples and downregulates GPCR-Gaq-PLCb1 signaling by inducing, subsequent to activation, the redistribution and removal of Gaq subunits from the host plasma membrane. Previous studies have demonstrated that once inside the host cytosol, the initial activation of Gaq-PLCb1 signaling by PMT is subsequently followed by an uncoupling between of the GPCR and the a subunit of the Gaq protein, causing a significant decrease in downstream calcium and mitogenic signaling. What is not fully understood is the mechanism by which this uncoupling occurs. Preliminary studies in our laboratory indicated that Gaq/11, but neither Gas nor Gb were redistributed in the plasma membrane. Due to their high homology, it had not been discerned whether both Gaq and Ga11 or if only one of them is being redistributed and depleted from plasma membranes. In this thesis, I present data that PMT decreases over-expressed Gaq proteins in host plasma membranes and in detergent resistant membrane (DRM) fractions. I show that this membrane depletion of Gaq protein, but not Ga11, was dependent on PMT catalytic activity. The results from this study also indicate that in PMT-treated cells, Gaq is redistributed within the host cell plasma membrane, as evidenced in subcellular fractionation experiments where by Gaq moved from DRM fractions into more soluble membrane and cytosolic fractions. Conversely, PMT did not appear to change the levels of Ga11 levels, although Ga11 has been shown to be a deamidation substrate for PMT. This strongly suggests that membrane depletion of Ga protein may be specific to certain Ga-proteins.
The findings from these two studies reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo subdomains are delivered intact as C1-C2-C3 including the MLD. The findings in this study offer insights into the determinants that define the delivered cargo subdomains and the intrinsic nature of the cargo subdomains required for delivery, which furthers our understanding of the cytosolic cargo delivery process and may be applicable to other related A-B type toxins. Additionally, these findings show that PMT treatment induces the depletion of Gaq protein from the plasma membrane as a mechanism of downregulating GPCR-Gaq-PLCb1 signaling, which may be a phenomenon that applies to other heterotrimeric Ga- protein targets of PMT and other protein toxins that target Ga-proteins; thereby offering insights to the intoxication effects of PMT and related toxins in various cells types.
Taken together both sets of findings have broader implications on how we understand the structural packaging and functioning of A-B type protein toxins; both in terms of their endosome-to-cytosol translocation and use as vehicles to deliver exogenous cytosolic cargos into host cells. These findings also offer insights on the inherent dual functions of toxins to affect signaling and alter cell fate to promote infection by their cognate organisms. Exogenous cargo delivery by different A-B type toxins opens doors for their potential use as pharmacological tools to deliver powerful novel therapeutics into unhealthy cells as a way of treating or curing human illnesses. Understanding and defining the signaling dynamics and intracellular activity modulation of the activity cargos of these A-B toxins gives way for their potential use as the novel toxin-based cytosolic therapeutics, especially in cases where specific abnormal cell types may require targeted killing or inhibition of proliferation. In any case, such work as presented in this thesis may provide the information necessary to help fellow research scientists and physicians obtain the molecular tools needed to have a positive impact on human health.