Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein Gene vlhA Expression during In Vivo Infection of the Natural Chicken Host

Abstract

Mycoplasma gallisepticum is the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms of M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomic vlhA gene expression directly from M. gallisepticum populations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minor vlhA gene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specific vlhA genes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominant vlhA gene expression in the colonizing bacterial population. The dominant expression of a given vlhA gene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominate vlhA gene when no longer faced with host pressures. Overall, these data indicate that vlhA phase variation is dynamic throughout the earliest stages of infection and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms.

FIG 1

The vlhA expression profile of the broth-grown M. gallisepticum input cultures as determined from RNA sequencing.

FIG 2

The vlhA expression profile of M. gallisepticum extracted directly from tracheas of experimentally infected birds over the course of the 7-day infection as determined through RNA sequencing. Each data point represents an average RPKM value from the results determined for five animals (with the exception of the broth sample). Error bars show standard errors of the means (SEM). Key statistically significant changes between two time points are indicated by paired upper- and lowercase letters for the following genes: vlhA 3.03 (A/a and A′/a′) and vlhA 4.07.1/4.08 (B/b).

FIG 3

(A) vlhA expression of M. gallisepticum recovered and cultured at five (5′) or 10 (10′) passages from the tracheas of the two different infected birds at 7 days postinfection. Only data from vlhA genes with an average RPKM value of >100 are displayed. (B) The sequences of the region upstream of the highly expressed vlhA 2.02 gene in the cultures recovered from the infected birds. nt, nucleotide.