Investigation of the intracellular trafficking pathways of the dermonecrotic toxin family

Pasteurella multocida toxin;Cytotoxic necrotizing factor 1;Cytotoxic necrotizing factor 2;Cytotoxic necrotizing factor y;serum response element (SRE);translocation;endocytosis;Arf6

Repella, Tana

关键词: Pasteurella multocida toxin;    Cytotoxic necrotizing factor 1;    Cytotoxic necrotizing factor 2;    Cytotoxic necrotizing factor y;    serum response element (SRE);    translocation;    endocytosis;    Arf6;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/29558/Repella_Tana.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The aim of this thesis is to further define the entry and trafficking pathwaysof the dermonecrotic toxin family composed of related AB toxins whose membersinduce dermonecrosis upon injection into animal skin. The dermonecrotic toxinsPasteurella multocida toxin (PMT) from Pasteurella multocida (P. multocida),cytotoxic necrotizing factors 1, 2, and 3 (CNF1, 2 and 3) from Escherichia coli (E.coli), the cytotoxic necrotizing factor Y (CNFY) from Yersinia pseudotuberculosis(Y. pseudotuberculosis), and the dermonecrotic toxin (DNT) from Bordetellaspecies are related by sequence similarity and share similar intracellular GTPaseproteintargets. PMT, CNF1, CNF2, CNFY, and DNT are bacterial exotoxins thatare responsible for a wide range of human and zoonotic diseases.The potent mitogenic toxin from Pasteurella multocida (PMT) is the majorvirulence factor associated with a number of epizootic and zoonotic diseases causedby infection with this respiratory pathogen. PMT is a glutamine-specific proteindeamidase that acts on its intracellular G-protein targets to increase intracellularcalcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptormediatedendocytosis and then translocates into the cytosol through a pH-dependentprocess that is inhibited by ammonium chloride (NH4Cl) or bafilomycin A1(BafA1). However, the detailed mechanisms that govern cellular entry, trafficking,and translocation of PMT remain unclear. Co-localization studies described hereinrevealed that while PMT shares an initial entry pathway with transferrin (Tfn) andcholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequentlydiverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde tothe ER, and PMT is trafficked to late endosomes. This study implicates the smallregulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMTfrom the endocytic vesicle occurs through a pH-dependent process that is alsodependent on both microtubule and actin dynamics, as evidenced by inhibition ofPMT activity in our SRE-based reporter assay, with nocodazole and cytochalasinD, respectively, suggesting that membrane translocation and cytotoxicity of PMT isdependent on its transfer to late endosomal compartments. In contrast, disruption ofGolgi-endoplasmic reticulum (ER) trafficking with brefeldin A (BFA) increasediiiPMT activity, suggesting that inhibiting PMT trafficking to non-productivecompartments that do not lead to translocation, while promoting formation of anacidic tubulovesicle system more conducive to translocation, enhances PMTtranslocation and activity.CNF1, CNF2, and CNF3 are virulence factors of pathogenic E. coli.Pathogenic E. coli are responsible for a wide range of diseases including intestinalinfections, urinary tract infections, septicemia, neonatal meningitis, pneumonia, andhemolytic-uremic syndrome. CNFY is an exotoxin produced by pathogenicYersinia pseudotuberculosis. DNT is an exotoxin produced by Bordetella speciesthat induces the lesions characteristic of atrophic rhinitis. The CNFs and DNTmodify and activate Rho proteins with CNF1 preferentially modifying RhoA andCdc42, CNF2 preferentially deamidating RhoA and Rac1, and CNFY acting as aselective activator of RhoA. The work reported in this thesis uses the SRE assay tocompare activation of SRE signaling pathways among the CNFs and DNT. Theseresults show that CNF2 and CNFY are the strongest activators of SRE signalingpathways. SRE activity peaks at a concentration of 100 ng/mL for CNF1 andCNF2, while concentrations of 1 μg/mL CNFY elicited the highest SRE activation.DNT elicited minimal SRE response, presumably due to paucity of DNT cellsurfacereceptors on HEK 293T/17.It has been previously demonstrated that the CNFs are dependent uponendosomal acidification for translocation into the cytosol. The results reportedherein demonstrate that while high concentrations of agents of endosomalacidification (BafA1 and NH4Cl) inhibit translocation of CNFs, low concentrationsof these inhibitors actually enhance the activity of CNF1 and CNF2. Furthermore,a region of the N-terminus, residues 199-267 of CNF1, was identified in which thepI and charge of CNF1/2 differ from that of CNF3/Y, DNT, and PMT and tertiarystructural changes that occur in this region with changing pH may be responsiblefor the increase in CNF1/2 activity. These results also demonstrate that the CNFstranslocate from the late endosomes as treatment with nocodazole inhibits theiractivity. Treatment with nocodazole, which inhibits the progression from early tolate endosome, was able to inhibit the NH4Cl-induced increase in CNF1 activity.ivTaken together these results support a model in which the CNFs translocate from anacidified late endosome. In the case of CNF1 and CNF2, small concentrations ofinhibitors of this acidification may be able to increase CNF1/2 activity by alteringthe pH and thereby altering tertiary structure and folding of the toxin proteins tomake translocation more favorable.