SEQUENCE-ANALYSIS OF THE MIP GENE OF THE SOILBORNE PATHOGEN LEGIONELLA-LONGBEACHAE

To understand the basis of pathogenesis by Legionella longbeachae sero group 1, the importance of the Mip protein in this species was examine d, Amino-terminal analysis of the purified, cloned L. longbeachae sero group 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coil background, DNA seq uence analysis of plasmid pIMVS27, containing the entire L, longbeacha e serogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophila serogroup 1, 76% homology at the DNA le vel and 87% identity at the amino acid level, Primer extension analysi s determined that the start site of transcription was the same for bot h species, with some differences observed for the -10 and -35 promoter regions, Primers designed from the mip gene sequence obtained for L. longbeachae serogroup 1 ATCC 33462 were used to amplify the mip genes from L. longbeachae serogroup 2 ATCC 33484 and an Australian clinical isolate of L. longbeachae serogroup 1 A5H5, The mip gene from A5H5 was 100% identical to the type strain sequence, The serogroup 2 strain of t. longbeachae differed by 2 base pairs in third-codon positions, Alle lic exchange mutagenesis was used to generate an isogenic mip mutant i n ATCC 33462 and strain A5H5. The ATCC mip mutant was unable to infect a strain of Acanthamoebae sp. both in liquid and in a potting mix coc ulture system, while the A5H5 mip mutant behaved in a manner siilar to that of L. pneumophila serogroup 1, i.e., it displayed a reduced capa city to infect and multiply within Acanthamoebae. To determine if this mutation resulted in reduced virulence in the guinea pig animal model , the A5H5 mip mutant and its parent strain were assessed for their ab ilities to establish an infection after aerosol exposure, Unlike the v irulent parent strain, the mutant strain did not kill any animals unde r two different dose regimes. The data indicate that the Mip protein p lays an important role in the intracellular life cycle oft. longbeacha e serogroup 1 species and is required for full virulence.