The following text describes a protein:
Function:
The main virulence factor for this bacteria, a mono-ADP-ribosylating toxin (mART), that transfers the ADP-ribosyl group from NAD(+) to multiple target proteins in vitro. Also elicits cytopathic effects in mammalian cells, such as disorganization and disruption of respiratory epithelial integrity in tracheal epithelium and vacuolization in the cytoplasm of CHO and HeLa cells as well as in mice and baboons. Treatment of mice or baboons with CARDS elicits a response that is consistent with human M.pneumoniae infections and mouse models of both infection and intoxication, suggesting that CARDS toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction. Treatment of baboons with CARDS induces a number of cytokines; G-CSF (40 fold), IL-1Ra (10 fold), IL-6 and IL-8 (333 and 100 fold, respectively), MIP-1a (5 fold), and RANTES (9 fold). Treatment of mice gives a similar response. Binds phosphatidyl choline, dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin via domains D2 plus D3; Has at least 2 host receptors SFTPA1 and ANXA2. Internalized by a clathrin-mediated process; protein is rapidly taken up at 37 degrees Celsius. Clathrin-independent or caveolin-dependent endocytosis were not detected. In HeLa cells internalized CARDS trafficks toward the nucleus by retrograde transport from early to late endosomes, then the Golgi apparatus; at 16 hours most toxin is concentrated in the perinuclear region in the host endoplasmic reticulum (ER). Failure to localize to the host ER prevents ADP-ribosylation and vacuolization. An acidic compartment is required to mediate retrotransport and processing of toxin into an N-terminal fragment with mART activity and a C-terminal fragment that is able to induce vacuolization; Induces the host NLRP3 inflammasome to release interleukin-1 beta (IL-1 beta); IL-1 beta release requires ADP-ribosylation activity and uptake by host macrophages. In the host colocalizes with the NLRP3 inflammasome; ADP-ribosylates NLRP3 in vitro. ADP-ribosylation of NLRP3 may lead to hyperinflammation (Probable).
Miscellaneous:
MISCELLANEOUS: Provokes a strong immune reaction in infected patients; MISCELLANEOUS: In a mouse infection model, pulmonary disease severity correlates with the expression level of CARDS toxin; strain S1 produces the highest amount of protein among the tested strains (S1 / Subtype 2, M129-B7 / Subtype 1 and M129-B9 / Subtype 1) and the most severe infection. Other differences between the strains were not explored in this study.
Subcellular Localization:
Cell membrane. Cytoplasm. Cell surface. Cell projection, attachment organelle. Host cytoplasm, host cytosol. Host endoplasmic reticulum. Note=In bacteria most protein is cytoplasmic, 7-10% is on cell surface, including in the tip (attachment) organelle. In host cells is found in the cytosol and in a punctate pattern in the perinuclear region in the host ER. In host cells, an isolated C-terminal fragment (residues 273-591) is detected in a larger punctate pattern. In 10% of host cells colocalizes with the speck NLRP3 inflammasome complex (purified protein incubated with mouse macrophages).
Domain:
The N-terminus (up to about residue 249) has mART activity, as well as NADase activity. The C-terminal 41 residues are required for internalization by HeLa cells and for vacuolization. The C-terminus (residues 266-591) is required for interaction with annexin A2. Has 3 structural domains, the N-terminal mART domain (mono-ADP ribosyltransferase, residues 1-205), D2 (residues 273-439) and D3 (residues 440-591). mART is joined to D2 by a linker (residues 257-272) which has a disulfide bond. The 3 domains assemble into an isosceles triangle, where the probable active site and NAD(+)-binding site are occulded. Enzyme activity will require either a conformation change or proteolytic processing. Domain D2 and D3 together bind lipids, domain D3 is at least partially responsible for binding to and internalization by host cells. A C-terminal fragment (residues 273-591) is capable of inducing vacuolization upon incubation with HeLa cells.
Sequence:
MPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF