Attenuation of Corynebacterium pseudotuberculosis by targeted mutagenesis of the phospholipase D gene.

A toxic phospholipase D (PLD), is thought to be an essential virulence determinant of Corynebacterium pseudotuberculosis, an animal pathogen which causes chronic, suppurative infections. In this study, PLD was purified from C. pseudotuberculosis biovar equi isolate 155 and the gene encoding PLD (pld) was cloned, pld from 155 and C. pseudotuberculosis biovar ovis isolate Whetten 1 were sequenced, Pld⁻ mutants of Whetten 1 were constructed, and one mutant was tested for reduced virulence for goats. PLD was purified by size exclusion chromatography and isoelectric focusing. In the purest fraction, sphingomyelinase, synergistic hemolysis, and staphylococcal β-hemolysin inhibition activities were associated with the presence of 31 and 21 kDa proteins. Only the 31 kDa protein was identified by Western blot analysis with PLD-neutralizing antibodies. Similar 1.6 kb inserts with pld from 155 and Whetten 1, in plasmids pCpE13s and pCpO51s respectively, conferred all three PLD-associated activities on E. coli. DNA sequence analysis revealed that both inserts encode a 307 amino acid protein which, in secreted form, has a predicted a molecular weight of 31.1 kDa and a pI of 8.84. The primary structure of the corynebacterial PLDs were compared to those produced by Corynebacterium ulcerans and Arcanobacterium haemolyticum. The four PLDs were found to shared 64% to 97% identity, and except for small regions of amino acid homology which includes two catalytic domains from glyceraldehyde-3-phosphate dehydrogenases, the sequences were unique when compared to other sequences found in GenBank. Two Pld⁻ strains of Whetten 1 were engineered by allele replacement. In strain W1.23r1, pld was deleted and in strain W1.31r1, pld contains a nonsense mutation which is predicted to truncate PLD after 65 amino acids. The virulence of W1.31r1 was compared to that of Whetten 1 by inoculation of goats. Whetten 1 caused abscessation at the site of infection and spread to the regional lymph node, while W1.31r1 had reduced ability to establish a primary infection and was incapable of dissemination. These results confirm that PLD is a virulence determinant of C. pseudotuberculosis which allows the persistence and spread of the bacteria within the host