Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis

Abstract

The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal beta-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N-acetylglucosamine.

Fig. 1

Representative electron micrograph of M. pulmonis cell with surrounding capsular material.

Fig. 2

Polyacrylamide gel electrophoresis of M. pulmonis lysates stained with PAS (panel A), destained, and restained with Coomassie (panel B). Numbers on left refer to protein standards in kilodaltons. The EPS does not enter the gel as noted by the arrows.

Fig. 3

Schematic of the overlapping MYPU_7410 and 7420 genes and flanking regions. Arrows refer to direction of transcription. The location of the transposon in the mutants are as shown. The nucleotide position of the transposon within the M. pulmonis genome in CTG1701, CTG1516, CTG2028, and CTG1291 is 911295, 911453, 911543, and 913643, respectively. In CTG1701, CTG1516, CTG2028, the 542-amino acid MYPU_7410 gene product would be truncated to 176, 228, 258 amino acids, respectively. In CTG1291, the 538-amino acid MYPU_7420 gene product would be truncated to 423 amino acids. Small arrows refer to the binding sites of the forward (For) and reverse (Rev) primers used to amplify the MYPU_7410 and 7420 genes for complementation of EPS mutants.

Fig. 4

M. pulmonis strains reacted with FITC-conjugated lectins (green) and stained with Hoechst 33342 (blue) to label DNA. Panels A, B, C, and G are wild-type CT, the EPS-I mutant CTG2028, the EPS-I mutant CTG1291, and the complemented mutant CTG1701-C, respectively, reacted with GS-I lectin. Panels D, E, and F are CT, CTG2028, and CTG1291, respectively, reacted with GS-II lectin.

Fig. 5

Gas chromatogram of methyl glycosides of EPS. Panels A–D, wild-type M. pulmonis strain CT, melibiose standard (a disaccharide composed of glucose and galactose), EPS-I mutant CTG2028, and EPS-I mutant CTG1291, respectively.

Fig. 6

Gas chromatogram of methyl glycosides of EPS. Panels A–C, wild-type M. pulmonis strain CT, EPS-I mutant CTG1516, and GlcNAc standard, respectively.

Fig. 7

Gas chromatogram of methyl glycosides of EPS. Panels A–C, strains CTG1701, CTG1701-C and CTG38, respectively.

Fig. 8

Western blot of M. pulmonis proteins reacted with Vsa-specific monoclonal antibody. Numbers on left refer to protein standards in kilodaltons. Arrows indicate the upper most Vsa band which corresponds to a Vsa protein with about 40 tandem repeats in CTG38 and a smaller protein with fewer tandem repeats in CTG2028.

Fig. 9

Biofilms formed on glass by M. pulmonis strains. Shown in panels A–F are biofilms for strains CTG1701, CTG1516, CTG1291, CTG-R40, CTG-R5, and CTG38, respectively. Top or overhead view of each biofilm is shown on the left and side view shown on the right.

Fig. 10

Association of M. pulmonis with A549 cells. A549 cells (1 × 10⁶) were incubated with the indicated strain of M. pulmonis, incubated for 2.5 hours, washed, and assayed for mycoplasma CFU. The data represents a combination of three separate experiments that were performed in triplicate. The data are represented as the mean (+ standard error of the mean). Asterisk refers to the statistical difference between CTG38 and the other two groups.