pMGA phenotypic variation in Mycoplasma gallisepticum occurs in vivo and is mediated by trinucleotide repeat length variation

Abstract

Chickens were infected with a pathogenic strain of Mycoplasma gallisepticum, and the expression of pMGA, the major surface protein, was inferred by examination of colonies from ex vivo cells. Within 2 days postinfection, 40% of cells had ceased the expression of the original pMGA surface protein (pMGA1.1), and by day 6, the majority of recovered cells were in this category. The switch in pMGA phenotype which had occurred in vivo was reversible, since most colonies produced from ex vivo progenitors exhibited frequent pMGA1. 1(+) sectors. After prolonged in vivo habitation, increasing proportions of recovered cells gave rise to variant pMGA colonies which had switched from the expression of pMGA1.1 to another gene, pMGA1.2, concomitant with the acquisition of a (GAA)(12) motif 5' to its promoter. Collectively, the results suggest that changes in M. gallisepticum pMGA gene expression in vivo are normal, common, and possibly obligate events for successful colonization of the host. Surprisingly, the initial cessation of pMGA1.1 expression occurred in the absence of detectable pMGA antibodies and seemed to precede the adaptive immune response.

FIG. 1

Kinetics of in vivo S6J growth and pMGA antibody production in chickens p.i. (A) Semilogarithmic plot of the average number of S6J organisms (CFU) recovered from the tracheal washes from two birds at each time point p.i. (days 1, 2, 4, 6, 8, 10, 13, 14, 28, and 42 p.i.). (B) Semilogarithmic plot of the average pMGA antibody levels in serum and tracheal wash samples at all time points p.i. and from air sac samples on days 14, 28, and 42 p.i. Levels of pMGA-specific antibodies were calculated relative to a selected high-titer serum. Tracheal wash levels have been multiplied by 100, and air sac levels have been multiplied by 10,000. Error bars represent the range between the two samples tested for each point rather than the standard deviation.

FIG. 2

S6J ex vivo colony pMGA phenotype categories. Typical S6J colony pMGA phenotypes obtained before infection and after reisolation from infected chickens were identified by sequential staining using the double-staining protocol. Colony phenotypes after immunostaining were grouped into four categories. Category 1 colonies were predominantly brown (MAb66⁺) and were assumed to be derived from pMGA1.1⁺ cells. Category 2 colonies were predominantly white with only brown sectors and were assumed to be derived from pMGA1.1⁻ cells. Category 3 colonies, also called mixed colonies, contained both brown (MAb66⁺) and red (rabbit anti-pMGA1.1⁺) sectors and were assumed to be derived from pMGA1.1⁻ cells. Category 4 colonies stained red only and were derived either from pMGA1.1⁻ cells or from pMGA variant cells.

FIG. 3

Changes in S6J colony pMGA1.1 and variant pMGA phenotypes with time p.i. (A) Bar graph comparing the average proportion of colonies in categories 1 and 2 (see Fig. 2) in the starting inoculum and from tracheal wash samples at various time points p.i. The total number of colonies counted for each time point ranged from 119 to 663. The mean proportions of category 1 and 2 colonies on days 2, 4, 6, 8, 13, and 28 were found to be significantly different from those on day 0 (P < 0.05). (B) Bar graph comparing the average proportion of colonies in categories 3 and 4 (see Fig. 2) in the starting inoculum and from tracheal wash samples at various time points p.i. The total number of colonies counted for each time point ranged from 119 to 663. The mean proportions of category 3 colonies on days 10, 13, and 28 were significantly different from the proportion seen on day 0 (P < 0.05). Only the mean proportion of category 4 colonies on day 28 was significantly different from that on day 0 (P < 0.05).

FIG. 4

Detection of pMGA1.2 expression. (A) Northern analysis of 3 μg of total RNA from M. gallisepticum S6 cells and clones, described in the text, using a probe specific for the pMGA1.1 or pMGA1.2 gene as indicated. (B) Alignment of the DNA sequences of the 5′ region of the pMGA1.2 genes amplified from S6J and C20.2A cells. Dots represent identities to the S6J sequence, and dashes represent deletions. Arrows above the S6J sequence indicate the orientations and locations of the oligonucleotides [1.2(GAA)F and 1.2(GAA)R] used to amplify the PCR products.

FIG. 4

Detection of pMGA1.2 expression. (A) Northern analysis of 3 μg of total RNA from M. gallisepticum S6 cells and clones, described in the text, using a probe specific for the pMGA1.1 or pMGA1.2 gene as indicated. (B) Alignment of the DNA sequences of the 5′ region of the pMGA1.2 genes amplified from S6J and C20.2A cells. Dots represent identities to the S6J sequence, and dashes represent deletions. Arrows above the S6J sequence indicate the orientations and locations of the oligonucleotides [1.2(GAA)F and 1.2(GAA)R] used to amplify the PCR products.