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.

The Variable Internal Structure of the Mycoplasma penetrans Attachment Organelle Revealed by Biochemical and Microscopic Analyses: Implications for Attachment Organelle Mechanism and Evolution

“…Although another group reported changes in the expression of a specific set of phase-variable genes in response to infection of a host (52), this study is the first, to our knowledge, to report on the expression of an entire mycoplasma transcriptome upon exposure to host cells. Particularly striking was the absence of major differences in the transcript profiles of M. penetrans cells grown independently and grown in the presence of HeLa cells for 6 h, which is enough time for interactions to occur between the two cell types, including invasion (31,33).…”

“…Briefly, the fastq files were assembled, mapped, and analyzed for differential gene expression using Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper) (68), with the M. penetrans HF-2 genome as the reference genome. Parameters were as described previously (52). For comparison of gene expression values, the data were first normalized by determining the ratio of reads per kilobase per million (RPKM) for mycoplasmas grown with HeLa cells to those for mycoplasmas grown axenically and then the fold change was calculated by taking the log 2 transformation of the RPKM difference between the two biological samples.…”

“…Data from the RNA-Seq experiment were analyzed as described previously for Mycoplasma gallisepticum data (52). Briefly, the fastq files were assembled, mapped, and analyzed for differential gene expression using Rockhopper (http://cs.wellesley.edu/~btjaden/Rockhopper) (68), with the M. penetrans HF-2 genome as the reference genome.…”

“…cDNA libraries were created using the Illumina TruSeq stranded mRNA library preparation kit (Illumina), following the manufacturer's protocol, as described previously (52). The cDNA libraries were examined for quality using the Agilent 2100 Bioanalyzer and then were quantified using a library quantification kit (Kapa Biosystems).…”

“…Purified bacterial RNA was enriched by performing poly(A) depletion using the NEBNext poly(A) mRNA magnetic isolation module (New England BioLabs) on all samples, as described previously (52). The RNA was then depleted of both prokaryotic and eukaryotic rRNA by using the Ribo-Zero Gold rRNA removal kit (Illumina), as directed by the manufacturer's instructions, and was cleaned and concentrated by using the Zymo RNA Clean and Concentrator-25 kit (Zymo Research), as described previously (52). The RNA was eluted in 25 l RNase-free water, examined for quality using an Agilent 2100 Bioanalyzer (Agilent Technologies), and used for cDNA synthesis.…”