Protein Kinase/Phosphatase Function Correlates with Gliding Motility in Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae exhibits a novel form of gliding motility that is mediated by the terminal organelle, a differentiated polar structure. Given that genes known to be involved in gliding in other organisms are absent in M. pneumoniae, random transposon mutagenesis was employed to generate mutants with gliding-deficient phenotypes. Transposon insertions in the only annotated Ser/Thr protein kinase gene (prkC; MPN248) and its cognate phosphatase gene (prpC; MPN247) in M. pneumoniae resulted in significant and contrasting effects on gliding frequencies. prkC mutant cells glided at approximately half the frequency of wild-type cells, while prpC mutant cells glided more than twice as frequently as wild-type cells. Phosphoprotein staining confirmed the association between phosphorylation of the cytoskeletal proteins HMW1 and HMW2 and membrane protein P1 and the gliding phenotype. When the prpC mutant was complemented by transposon delivery of a wild-type copy of the prpC allele, gliding frequencies and phosphorylation levels returned to the wild-type standard. Surprisingly, delivery of the recombinant wild-type prkC allele dramatically increased gliding frequency to a level approximately 3-fold greater than that of wild-type in the prkC mutant. Collectively, these data suggest that PrkC and PrpC work in opposition in M. pneumoniae to influence gliding frequency.M ycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract causing primary atypical pneumonia and tracheobronchitis (1). Mycoplasmas lack major biosynthetic pathways, classical transcriptional regulators, chemotactic and other two-component systems, and the prototypical prokaryotic cell division apparatus (2, 3). The limited biosynthetic capabilities of mycoplasmas are generally explained by their evolution as obligate parasites of diverse eukaryotic hosts (4). Colonization of the host respiratory epithelium by M. pneumoniae requires gliding motility (5), which might facilitate access to receptors on the host cell surface and subsequent lateral spread.The gliding apparatus of M. pneumoniae is a polar terminal structure (6) that also functions in cell division (7) and adhesion to host receptors (2, 5). While the cytoskeletal protein HMW1 (MPN447) and membrane proteins P1, B/C, and P30 (MPN141, MPN142, and MPN453, respectively) localize to the terminal organelle and are required for gliding (5,(8)(9)(10), these proteins are also essential for cytadherence and attachment to surfaces. Accordingly, their distinct functions in gliding motility are difficult to define by mutagenesis alone. Given that the M. pneumoniae genome exhibits no homology to elements of defined gliding mechanisms (2, 3), including those of other gliding mycoplasmas (11-14), it was necessary to perform saturating transposon mutagenesis in order to identify the components specific to gliding (15). Transposon insertions in the genes encoding the cytoskeletal proteins P41 and P65 (MPN311 and MPN309, respectively) (16), which are known to localize to the termina...

Electron cryotomography of Mycoplasma pneumoniae mutants correlates terminal organelle architectural features and function

“…wild-type levels in both mutants (Page and Krause, 2013), consistent with identification of all core substructures in tomograms here. As likely regulatory rather than structural proteins, both may be present in low abundance, and attempts at their detection using antibodies raised against multiple antigenic peptides for each were unsuccessful (Page and Krause, 2013).…”

Insights into the pathogenesis of Mycoplasma pneumoniae

“…Electron microscopy has demonstrated that the adhesion of a M. pneumoniae variant is concentrated in the adherend in the following order: HMW1, HMW3, Pl, P30, P90, P40 and P65, which indicates that these proteins have formed an interrelated adhesion network (12). Specifically, HMW1, HMW2 and HMW3 function as stable adherends and allow other adhesions to locate onto the adherend, and, they are involved in the adhesion onto the respiratory tract epithelia (13). As reported, the M. pneumoniae mutant strains, HMW1 and HMW2, can prevent the P1 protein from correctly locating onto the apical structure, which leads to irregular cell morphology, loss of toxicity and sliding ability, and loss of adhering function (14).…”

In Vitro Spatial and Temporal Analysis of Mycoplasma pneumoniae Colonization of Human Airway Epithelium

“…Mycoplasmas were grown at 37°C in SP-4 medium (30) until the phenol red pH indicator turned orange. Antibiotic selection was included for the prpC mutant (25). For radiolabeling studies, mycoplasmas were cultured in the presence of 100 Ci [methyl- 3 H]thymidine (6.7 Ci/mmol; PerkinElmer), harvested and collected by centrifugation, and washed 3 times in fresh SP-4 medium as described previously (31).…”

“…Wild-type M. pneumoniae (strain M129, 17th broth passage) (15); P30 mutants II-3, II-7, and II-3R (7,24); the P200 mutant (22); and the prpC mutant (25) were included in the current study. P30 is a terminal organelle protein required for adherence to host cells and gliding motility (5,6,26).…”