Mycoplasma pneumoniae J-domain protein required for terminal organelle function

Abstract

The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Colonization of the respiratory epithelium requires proper assembly of a complex, multifunctional, polar terminal organelle. Loss of a predicted J-domain protein also having domains unique to mycoplasma terminal organelle proteins (TopJ) resulted in a non-motile, adherence-deficient phenotype. J-domain proteins typically stimulate ATPase activity of Hsp70 chaperones to bind nascent peptides for proper folding, translocation or macromolecular assembly, or to resolve stress-induced protein aggregates. By Western immunoblotting all defined terminal organelle proteins examined except protein P24 remained at wild-type levels in the topJ mutant; previous studies established that P24 is required for normal initiation of terminal organelle formation. Nevertheless, terminal organelle proteins P1, P30, HMW1 and P41 failed to localize to a cell pole, and when evaluated quantitatively, P30 and HMW1 foci were undetectable in >40% of cells. Complementation of the topJ mutant with the recombinant wild-type topJ allele largely restored terminal organelle development, gliding motility and cytadherence. We propose that this J-domain protein, which localizes to the base of the terminal organelle in wild-type M. pneumoniae, functions in the late stages of assembly, positioning, or both, of nascent terminal organelles.

Fig. 1

Schematic alignment of acidic- and proline-rich (APR) and enriched in aromatic and glycine residue (EAGR) domains of M. pneumoniae proteins (modified from Balish and Krause, 2002). Scale bar, number of amino acid residues.

Fig. 2

Comparison of the topJ operon in M. pneumoniae with orthologues from the closely related M. genitalium (Peterson et al., 1993) identified by basic local alignment search tool (BLAST). The black arrow preceding MG200 indicates the promoter for the operon as identified by primer extension (Musatovova et al., 2006). The inverted triangle indicates the transposon insertion in MPN119 in the topJ mutant. The dotted line represents reading frames not included in the schematic. Open reading frames are not to scale. Primer pairs corresponding to a, b and a, c were used to amplify MPN119 or MPN119 + MPN120, respectively, for complementation studies. The underlined sequence corresponds to an engineered SmaI restriction site. Hyp, hypothetical protein. a. 5′-AAAGCTATCACCCGGGCCAGATTCATCACT-3′ b. 5′-CACCATGTTAAACCCGGGGGTATAGT-3′ c. 5′-CCTTCAAGGCTTCCCGGGCTTTTTCTAAATCC-3′.

Fig. 3

Satellite growth by wild-type M. pneumoniae (A), topJ mutant (B), and topJ transformants with MPN119 alone (C) or MPN119 + MPN120 (D). Mycoplasmas were incubated in SP4 medium with 3% gelatin for 72 h. Bar, 15 μm. Inset, enlarged view of the indicated region.

Fig. 4

Western immunoblot analysis of wild type, topJ mutant and transformant M. pneumoniae strains. Proteins were separated by 4–12% gradient SDS-PAGE and analysed by immunoblotting with the indicated antisera.

Fig. 5

Cytadherence capacity of wild type, topJ mutant and transformant M. pneumoniae strains. A. Qualitative assessment of HA. WT, wild type; II-3, negative control. Bar, 30 μm. B and C. Quantitative assessment of HA and attachment to A549 cells, respectively, normalized to wild-type binding. III-4, negative control. Error bars, standard deviation.

Fig. 6

Immunolocalization of terminal organelle proteins in wild-type M. pneumoniae and the topJ mutant. A. Localization of P1 and TopJ: panel 1, phase-contrast; panel 2, merged-phase and fluorescence images; panel 3, schematic representation; panel 4, TopJ false-coloured yellow; panel 5, P1 false-coloured red; panel 6 (wild type), merge of wild type panels 4 and 5; panel 6 (topJ), DAPI staining. B. Localization of P30 and P41: panel 1, phase-contrast; panel 2, merged-phase and fluorescence images; panel 3, schematic representation; panel 4, P41 false-coloured yellow; panel 5, P30 false-coloured red; panel 6, merge of panels 4 and 5. Yellow arrow, P41 focus unpaired with P30. Bar, 1 μm.

Fig. 7

Immunolocalization of P30 and HMW1 in wild-type M. pneumoniae and the topJ mutant. Panel 1, phase-contrast; panel 2, merged-phase and fluorescence images; panel 3, schematic representation; panel 4, HMW1 false-coloured yellow; panel 5, P30 false-coloured red; panel 6, merge of panels 4 and 5. Wild type: white arrow, fluorescent polar foci. topJ: white arrow, pole of terminal cell in chain with no fluorescent focus; white arrowhead, HMW1 and P30 pair within cell chain having no clearly polar focus in terminal cell; yellow arrowhead, unpaired HMW1 focus within cell chain; red arrowhead, unpaired P30 focus within cell chain. Bar, 2 μm.