title: Deletion analysis identifies key functional domains of the cytadherence-associated protein HMW2 of Mycoplasma pneumoniae.
abstract: Mycoplasma pneumoniae attachment to host cells requires biogenesis of a functional attachment organelle, including proper localization of the adhesion protein P1 to this structure. Mutations in the hmw2 gene result in the inability to cytadhere, failure to localize P1 to the attachment organelle, altered cell morphology and accelerated turnover of the cytadherence-associated proteins HMW1, HMW3 and P65. The hmw2 gene encodes HMW2 (190 kDa) and P28 (28 kDa), the latter apparently the product of internal translation initiation near the 3' end of the hmw2 coding region. Transformation of hmw2 mutant I-2 with recombinant wild-type hmw2 restores a wild-type phenotype. In the current study, a severely truncated hmw2 gene with an in frame internal deletion of 80% of the HMW2 coding region that leaves the P28-encoding region intact restored cytadherence to mutant I-2. Transformants produced the expected 38 kDa HMW2 derivative (HMW2Deltamid) at levels comparable to that of HMW2 in wild-type cells; like HMW2, HMW2Deltamid exhibited marked Triton X-100 insolubility. HMW3, P65 and P28 were fully restored, but not HMW1. These transformants were morphologically similar to wild-type M. pneumoniae but failed to localize P1 to the attachment organelle. Finally, a C-terminally truncated HMW2 derivative was partly Triton X-100 soluble and incapable of restoring HMW1, HMW3 and P65 to wild-type levels. These data are consistent with a model in which the C-terminal domain of HMW2 imparts normal localization to the protein, and this localization itself is required for productive interactions with downstream cytadherence-associated proteins. Furthermore, these results emphasize the association of HMW1 with P1 clustering.
authors.affiliation: Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Mitchell F
authors.initials: MF
authors.lastname: Balish
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Ross
authors.forename: Makda
authors.initials: M
authors.lastname: Fisseha
authors.forename: Duncan C
authors.initials: DC
authors.lastname: Krause
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017353:Gene Deletion; D008855:Microscopy, Electron, Scanning; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; C117432:HMW2 protein, bacteria; C035471:adhesin, Mycoplasma pneumoniae