Comparative genomic analyses of attenuated strains of Mycoplasma gallisepticum

Abstract

Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage M. gallisepticum strain R (R(low)) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of R(low), R(high), indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R(low) isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum R(low). Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter- and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.

FIG. 1.

Major genomic differences between F and R strain genomes. (A) Dot plot of F and R_low genome sequences. vlhA loci are indicated with dotted lines and expanded to demonstrate gene structure at vlhA5 loci (B), vlhA1 loci (C), and vlhA4/3 loci (D). vlhA genes are indicated with gray arrows and also noted with vlhA numbers in R strain; genes present only in F strain adjacent to the vlhA5 locus are indicated with black arrows. Genes similar and potentially orthologous between the two genomes are indicated with blocks or diagonals.

FIG. 2.

Attenuation of MGA_1107 mutant of R strain in chickens. (A) Histopathological lesion scores in tracheas of chickens infected with Hayflick's medium (Medium), mutant (MGA_1107) organism, and virulent organism (R_low). Horizontal bars indicate 25th percentile (bottom), median, and 75th percentile (top). Lowercase letters indicate statistically similar groups. (B) Tracheal mucosal thickness. Bar and letters are as described for panel A. (C) Microbiological recovery from tracheas of chickens. Dots indicate titers of samples recovered from individual birds.