Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

Abstract

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation

“…Sun et al demonstrated that surface expressed PDHB of Mycoplasma bovis exhibits excellent antibodies‐induced immunogenicity in naturally infected animals. The presence of surface displaced PDHB had been also observed in Mycoplasma agalactiae , where it might be an important pathogenicity determinant . To our knowledge, the present report is the first to show an interaction PDHB/collagen and the involvement of this subunit in development of biofilms.…”

Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep

“…A total of 45 mutants, which exhibited stable transposon insertions and normal growth profiles except pdhB mutant [ 9 , 19 ], were tested in the initial round of screening by intramammary infection of lactating ewes in three different groups, as described in Table 1 . Two weeks post-infection (pi), samples of the right and left kidneys, lungs, uterus, liver, spleen and synovial fluids from stifle joints were checked for the presence of M. agalactiae by culture and PCR [ 20 ], and if positive then for the presence or absence of specific mutants via SSM PCR.…”

“…This gene is responsible for the metabolism of pyruvate, which is the major energy yielding process in M. agalactiae [ 38 ]. The pdhB mutant showed reduced growth in axenic medium during logarithmic phase, but was recovered in vivo from the udder and LNs of infected animals in the confirmatory screening experiment [ 9 , 19 ], indicating its relative survival in local sites of infection. Additionally, this mutant also showed reduced invasion into HeLa cells [ 19 ], indicating its role in host cell invasion and the significance of the latter in the systemic spread of M. agalactiae .…”

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection

“…However, mutants 6-29 (pdhB) and 6-14 (uhpT) exhibited smaller colony size than the PG2 strain. Interestingly, only 6-29 (pdhB) showed slower log-phase growth (40). Nevertheless, this mutant also was included in the current in vivo study, because pyruvate dehydrogenase mutants in other bacteria also are known to exhibit in vitro slow-growth phenotypes, yet they have been used in STM screens and are accepted as playing important roles in the pathogenicity of these bacteria (41,42).…”