Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis

Abstract

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

FIG. 1.

Identification of Tn4001 insertions within the genomes of M. gallisepticum HA⁻ transformants. Lanes: 1, wild-type M. gallisepticum R-low; 2 to 4, M. gallisepticum mutants E117, E345, and E325, respectively. In each lane, 2 to 4 μg of chromosomal DNA was digested with EcoRI (A) or XbaI (B and C) restriction enzymes, subjected to Southern blot hybridization, and probed with the ³²P-labeled gentamicin gene (A), gp-1 fragment (B), or cm-1 fragment (C). The sizes of the hybridization bands are shown on the left.

FIG. 2.

Identification of Tn4001 insertion sites within the M. gallisepticum R-low gapA-crmA operon. The solid line represents an 8-kb genomic fragment of M. gallisepticum R-low that was cloned and sequenced. The locations and directions of the mgc-2, gapA, and crmA genes are indicated by large labeled arrows. The positions of HaeIII (Ha), HindIII (H), XbaI (X), and EcoRI (E) restriction sites are indicated. The positions of Tn4001 insertions in mutants E117, E345, and E325 are indicated by labeled arrows. The locations of the gp-1 and cm-1 genomic fragments used as probes are indicated by broken lines. Two XbaI genomic fragments (shown in Fig. 1) are indicated by dotted brackets along with their corresponding sizes.

FIG. 3.

Western blot analysis of M. gallisepticum R-low Tn4001 mutants. Total cell proteins from wild-type M. gallisepticum R-low (lane 1), mutants E117, E345, and 325 (lanes 2 to 4, respectively), and M. gallisepticum R-high (lane 5) were subjected to SDS-PAGE and immunoblotted with monospecific anti-GapA (A) or anti-CrmA (B) antibodies. The 105-kDa GapA and 116-kDa CrmA protein bands are indicated.

FIG. 4.

In vitro attachment of M. gallisepticum to MRC-5 cells. Hatched and stippled bars indicate the attachment of M. gallisepticum mutants and strains relative to that of wild-type M. gallisepticum R-low (data are presented relative to the R-low cytadherence value of 100%). The Tn4001 transposon mutants and M. gallisepticum strains used are indicated below the bars. Division of the attachment values for the strains by multiple comparisons of the LSMeans into three significantly different clusters is indicated by letters above the bars. Error bars represent standard deviations.

FIG. 5.

Western blot analysis of M. gallisepticum strains. (A and B) Total cell proteins from M. gallisepticum strains were subjected to SDS-PAGE and immunoblotted with monospecific anti-GapA (A) or anti-CrmA (B) antibodies. The GapA and CrmA protein bands are indicated. M. gallisepticum strains included ts-11, F, and 6/85 (lanes 1 to 3, respectively). M. imitans is shown in lane 4. (C) A base substitution at position 139 (boxed) in the gapA structural gene of M. gallisepticum ts-11, generating a TAA termination codon, is shown in comparison to the corresponding region in strain R-low.

FIG. 6.

Monitoring the presence and genomic location of the Tn4001 transposon in M. gallisepticum R-low mutants. (A) Tn-position-PCR of various mutants. Primers ISb1 and GAf4 were used to amplify the junction between the IS256 arm of the Tn4001 transposon and the gapA gene in M. gallisepticum R-low mutants E117, E345, and E325 (lanes 1 to 3, respectively). The sizes of the PCR products corresponding to the mutants are shown on the left. (B) Tn-position-PCR of mutant E117 isolated directly from the trachea at 3, 7, 11, 21, and 28 days p.i. (lanes 1 to 5, respectively) with primers ISb1 and GAf4. The 0.25-kb PCR product is indicated. (C). Western blot analysis of total cell proteins from mutant E117 (depicted in panel B) with anti-CrmA antibodies. The 116-kDa CrmA protein band is indicated. The HA phenotype of colonies from each isolate on agar plates (positive or negative, + or −, respectively) is shown at the bottom.