title: Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.
abstract: Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS) toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN) Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.
authors.affiliation: Department of Microbiology and Immunology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.
authors.forename: Coreen
authors.initials: C
authors.lastname: Johnson
authors.forename: T R
authors.initials: TR
authors.lastname: Kannan
authors.forename: Joel B
authors.initials: JB
authors.lastname: Baseman
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D016253:Microscopy, Immunoelectron; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects* / Q000378:metabolism*; D014709:Vero Cells; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015415:Biomarkers; C546120:CARDS toxin, Mycoplasma pneumoniae; D018942:Macrolides; D011994:Recombinant Proteins; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases; D020691:rab GTP-Binding Proteins