Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally.

The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in M. pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria. M. pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in hmw2 result in the loss of HMW1 and HMW3. However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW- mutants.

Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence-associated protein HMW1 and identification of HMW4 as a product.

Mycoplasma pneumoniae is a major cause of tracheobronchitis and pneumonia in older children and young adults. The lack of adequate tools for genetic analysis has hindered the elucidation of function and regulation of mycoplasma virulence determinants. We describe here the use of a transposon vector to deliver the cloned gene for the cytadherence-associated protein HMW1 in M. pneumoniae. A 4.95 kbp BamHI fragment encoding all but the C-terminal end of HMW1 was cloned into a modified Tn4001 and transformed into wild-type M. pneumoniae and into a non-cytadhering mutant lacking HMW1-HMW5. Southern blot hybridizations confirmed insertion of the transposon and the presence of both the resident and recombinant hmw1 alleles. Analysis by Western immunoblotting revealed a truncated HMW1 (HMW1') in the transformants, the level of HMW1' being dependent upon the orientation of the hmw1 gene in the transposon and the site of insertion. Similar expression patterns were noted in wild-type and mutant backgrounds. However, expression of wild-type levels of HMW1' in the mutant did not restore adherence. Finally, HMW4 and HMW1 were shown to be products of the same gene, HMW4 being a heat-modified derivative of HMW1.

Phosphorylation of Mycoplasma pneumoniae cytadherence-accessory proteins in cell extracts.

A cell-free system was used to characterize the phosphorylation of Mycoplasma pneumoniae proteins HMW1 and HMW2, which are involved in the adherence of this organism to human tracheal epithelium during infection. The pH and cation requirements for phosphorylation of HMW1 and HMW2 were determined, and the effects of glycolytic intermediates, cyclic AMP, and eukaryotic kinase-phosphatase inhibitors and stimulators on this process were examined. Phosphoamino acid analysis identified serine as the major phosphate acceptor for both HMW1 and HMW2 in this system.

Phosphorylation of cytadherence-accessory proteins in Mycoplasma pneumoniae.

Attachment to host cells of the respiratory epithelium by Mycoplasma pneumoniae is a complex, multicomponent process, requiring a number of accessory proteins in addition to adhesins directly involved in receptor binding. In this study, protein phosphorylation of the cytadherence-accessory proteins HMW1, HMW2, and HMW4 of M. pneumoniae was examined using biochemical and immunological techniques. The initial indication of protein modification came from Western immunoblot analysis of the two-dimensional polyacrylamide gel electrophoresis (PAGE) profile of M. pneumoniae proteins, revealing multiple spots for both HMW1 and HMW4 that varied in pI but not in size. M. pneumoniae cultured in the presence of H3(32)PO4 exhibited numerous phosphorylated proteins as detected by sodium dodecyl sulfate-PAGE and autoradiography. These included proteins corresponding to HMW1, HMW2, and HMW4 in electrophoretic mobility. The Triton X-100 partitioning characteristics of these phosphorylated proteins was identical to that described previously for HMW1, -2, and -4. Furthermore, these protein bands were absent when a noncytadhering variant deficient in HMW1-5 was examined in the same manner. Finally, the availability of antiserum to HMW1 and -4 enabled us to confirm by radioimmunoprecipitation that HMW1 and HMW4 are phosphoproteins. Phosphoamino acid analysis of acid-hydrolyzed HMW1 and HMW2 identified primarily phosphothreonine and, to a lesser extent, phosphoserine in HMW1 and predominantly phosphoserine, with a trace of phosphothreonine, in HMW2. Neither protein contained phosphotyrosine. HMW1-HMW5 are components of a cytoskeleton-like structure in M. pneumoniae that is thought to function in cell division, changes in cell morphology, gliding motility, and the localization of adhesins in the mycoplasma membrane. Phosphorylation may regulate cytoskeleton dynamics involving these cytadherence-accessory proteins.

Peripheral blood mononuclear cells express antigens associated with multidrug resistance in a small cell lung cancer cell line.

H69AR is a multidrug resistant small cell lung cancer (SCLC) cell line that does not overexpress P-glycoprotein, the plasma membrane drug efflux pump usually associated with this type of resistance. Monoclonal antibodies (MAbs) were previously raised against H69AR cells, and three of these MAbs, 2.54, 3.50, and 3.186, cross-reacted with peripheral blood mononuclear cells. T cells (CD3+), B cells (CD19+), NK cells (CD16+) and monocytes (CD14+) expressed each of the three antigens, but to differing degrees. Immunoprecipitation and partial proteolytic mapping experiments demonstrated that the antigens detected by MAbs 2.54 and 3.186 are identical in H69AR cells and PBMCs. SCLC cells are known to express many hematopoietic antigens; however, this is the first report of SCLC multidrug resistance-associated antigens being expressed on hematopoietic cells.