Domain analysis of protein P30 in Mycoplasma pneumoniae cytadherence and gliding motility

Abstract

The cell wall-less prokaryote Mycoplasma pneumoniae causes bronchitis and atypical pneumonia in humans. Mycoplasma attachment and gliding motility are required for colonization of the respiratory epithelium and are mediated largely by a differentiated terminal organelle. P30 is a membrane protein at the distal end of the terminal organelle and is required for cytadherence and gliding motility, but little is known about the functional role of its specific domains. In the current study, domain deletion and substitution derivatives of P30 were engineered and introduced into a P30 null mutant by transposon delivery to assess their ability to rescue P30 function. Domain deletions involving the extracellular region of P30 severely impacted protein stability and adherence and gliding function, as well as the capacity to stabilize terminal organelle protein P65. Amino acid substitutions in the transmembrane domain revealed specific residues uniquely required for P30 stability and function, perhaps to establish correct topography in the membrane for effective alignment with binding partners. Deletions within the predicted cytoplasmic domain did not affect P30 localization or its capacity to stabilize P65 but markedly impaired gliding motility and cytadherence. The larger of two cytoplasmic domain deletions also appeared to remove the P30 signal peptide processing site, suggesting a larger leader peptide than expected. We propose that the P30 cytoplasmic domain may be required to link P30 to the terminal organelle core, to enable the P30 extracellular domain to achieve a functional conformation, or perhaps both.

FIG. 1.

(A) Schematic of wild-type protein P30. Brackets designate the predicted signal peptide (SP), intracellular, transmembrane (TM), and extracellular regions of P30. Domains I, II, and III are indicated by black horizontal lines, while horizontal gray bars marked a and b correspond to antigens used to prepare antisera to the cytoplasmic and extracellular regions of P30, respectively. Numbers indicate amino acid residues. Vertical black and gray bars within domain III correspond to P-rich repeat motifs PGMAPR (black), PGMPPH (dark gray), and PGFPPQ (light gray). (B) Schematic of P30 domain deletion derivatives. Numbers indicate the deleted residues for each. (C) Amino acid residues of the P30 TM domain. Underlining, alanine substitutions generated; X, identical residues in M. pneumoniae, M. genitalium, and M. gallisepticum; *, identical residues in M. pneumoniae and M. genitalium only.

FIG. 2.

Boxshade of M. pneumoniae P30 (MPN P30), M. genitalium P32 (MGN P32), and M. gallisepticum MGC2 (MGA MGC2). Black and gray blocks indicate identical and similar amino acids, respectively. The solid bar is above the predicted P30 TM domain, and the double line is above the conserved region between P30 and P32. The alignment of sequences was analyzed by the “ClustalW2” program on http://www.ebi.ac.uk/ and boxshaded by BOXSHADE 3.21 on http://www.ch.embnet.org.

FIG. 3.

Western immunoblot analysis of P30 and P65 in domain deletion mutants (A and B) and A-scanning TM domain substitution derivatives (C). Total mycoplasma protein loaded per lane for polyacrylamide gel electrophoresis was 100 μg (A) or 40 μg (B and C). P30 C-terminus-specific antibodies were used for results shown in panels A and B, and N-terminus-specific antibodies were used for results shown in panel C, but comparable results were obtained with both antibody specificities for all P30 derivatives tested here (data not shown). P65 and P30 or derivatives thereof are indicated on the right (all panels), and size standards in kilodaltons are indicated on the left (A). WT, wild-type M. pneumoniae; rP30, recombinant P30.

FIG. 4.

(A) Immunofluorescence localization of P30 and P65 in wild-type M. pneumoniae (WT), P30 mutants II-3 and II-7, and mutant II-3 containing the indicated domain deletion derivatives. The panels in rows 3 and 4 correspond to the same fields. (B) Immunofluorescence localization of P30 and HMW1 in the P30ΔI domain deletion mutant. Merged fluorescence and phase-contrast images of individual cells are shown. Antibody specificity was established previously (20, 34, 39). Bar, 1 μm.

FIG. 5.

Qualitative hemadsorption analysis of wild-type M. pneumoniae (WT), P30 mutant II-3, and the indicated P30 domain deletion or amino acid substitution derivatives. Bar, 65 μm.

FIG. 6.

Satellite growth by wild-type M. pneumoniae (WT), P30 mutant II-3, and the indicated P30 domain deletion or amino acid substitution derivatives. Mycoplasmas were incubated in SP4 medium with 3% gelatin for 72 h. Bar, 15 μm.