Summary 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 macro-molecular 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.

Summary Mycoplasma pneumoniae is a wall-less human respiratory tract pathogen that colonizes mucosal epithelium via a polar terminal organelle having a central electron-dense core and adhesin-related proteins clustered at a terminal button. A mutant lacking J-domain co-chaperone TopJ is noncytadherent and nonmotile, despite having a core and normal levels of the major cytadherence-associated proteins. J-domain co-chaperones work with DnaK to catalyze polypeptide binding and subsequent protein folding. Here we compared features of the topJ mutant with other cytadherence mutants to elucidate the contribution of TopJ to cytadherence function. The topJ mutant was similar ultrastructurally to a non-cytadherent mutant lacking terminal organelle proteins B/C, including aberrant core positioning and cell morphology in thin sections, but exhibited a hybrid satellite growth pattern with features of mutants both having and lacking a core. Time-lapse images of mycoplasmas expressing a YFP fusion with terminal organelle protein P41 suggested that terminal organelle formation/positioning was delayed or poorly coordinated with cell growth in the absence of TopJ. TopJ required a core for localization, perhaps involving HMW1. P1 trypsin accessibility on other non-cytadherent mutants was significantly enhanced over wild-type but unexpectedly was reduced with topJ mutant cells, suggesting impaired processing, translocation, and / or folding of this adhesin.

Summary Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J-domain co-chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J-domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J-domain, central acidic and proline-rich (APR) domain, and C-terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J-domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J-domain co-chaperone function. In contrast, TopJ derivatives having APR or C-terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild-type cells. Results from immunofluorescence microscopy suggest that both the APR and C-terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.

The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.