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.

Mycoplasma penetrans is a urogenital tract pathogen implicated in the deterioration of the immune system in human immunodeficiency virus-infected AIDS patients. Here, we describe a 78-kDa protein from M. penetrans, designated MYPE9110, that exhibits sequence similarity to known ADP-ribosyltransferases (ADPRTs) such as Bordetella pertussis pertussis toxin and Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin. MYPE9110 possesses key amino acid residues found in all ADPRTs that are essential for ADPRT activity. Several mammalian cell proteins are ADP-ribosylated by MYPE9110, and the full-length recombinant protein exhibits a strong auto-ADP-ribosylating activity. In the absence of target proteins, MYPE9110 demonstrates a NAD-glycohydrolase activity by hydrolyzing NAD. Furthermore, this toxin elicits cytopathology in HeLa cells by inducing cytoplasmic vacuolization in the presence of ammonium chloride. The deletion of the C-terminal region of MYPE9110 significantly diminishes its binding to host cells while still exhibiting an ADPRT activity, suggesting that MYPE9110 is a member of the family of A-B ADPRT toxins.

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