Extracellular protease: Potential virulence factor of Mycoplasma mycoides subspecies mycoides

Mycoplasmas are prokaryotes that lack a cell wall and infect a variety of hosts, including humans, mammals, reptiles, fish, arthropods, and plants. Unlike most mycoplasmal infections (which cause clinically silent, chronic disease), infections caused by the Mycoplasma mycoides cluster have an acute progression of pneumonia, polyarthritis, mastitis, and septicemia that frequently results in disseminated intravascular coagulopathy and death. Five of six organisms that comprise the mycoides cluster produce an extracellular caseinolytic protease. The extracellular role of this caseinolytic protease is important in substrate utilization and microenvironment. Proteases are known to have a broad spectrum of activities, including extracellular and intracellular functions. The intracellular role of this caseinolytic protease is still unknown.; We investigated this caseinolytic protease in Mycoplasma mycoides subspecies mycoides LC type (Mmm) GM12 as a potential virulence factor. The wild-type Mmm GM12 was mutagenized with transposon Tn916 and selected for tetracycline resistance and the inability to degrade casein. Two mutants (Mmm GM12-Tn916#8 and GM12-Tn916#286) were isolated. The genomic DNA from Mmm GM 12-Tn916#8 mutant was isolated; and the regions flanking the Tn916 insertion were sequenced. Primer walking the wild-type Mmm GM 12 genomic DNA completed the protease gene sequence. Results from the putative gene sequence BLAST revealed homology to a family of carboxy-terminal processing proteases (CtpA) of seven microorganisms. The PCR amplified protease gene ( ctpA) was cloned into pCR 2.1 (Invitrogen, Carlsbad, CA), which restored caseinolytic activity in the Lon- strain BL21 (lambda DE3) of Escherichia coli.; The partially purified CtpA is capable of stimulating humoral immunity in the host. The ctpA gene products may play a role in the virulence of many pathogens, including Bartonella bacilliformis, Staphylococcus aureus, Synechocystis, E. coli, and Salmonella typhimurium . However, an Mmm ctpA- mutant is more virulent than wild-type Mmm, has higher numbers of organisms recovered from tissue, has greater dissemination, and causes more severe lung histopathology. Transcription of the ctpA gene was greatly reduced, as was that of at least one additional gene 3' to the ctpA, suggesting that this chromosomal locus, and perhaps the protease activity, is associated with control of virulence.