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Sequence analysis of the mip gene of the soilborne pathogen Legionella longbeachae
R M Doyle1, T W Steele, A M McLennan
1Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, South Australia. Robyn.Doyle@imvs.sa.gov.au
Abstract:
To understand the basis of pathogenesis by Legionella longbeachae serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned L. longbeachae serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an Escherichia coli background. DNA sequence analysis of plasmid pIMVS27, containing the entire L. longbeachae serogroup 1 mip gene, revealed a high degree of homology to the mip gene of Legionella pneumophila serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both 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 L. longbeachae differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic mip mutant in 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 coculture system, while the A5H5 mip mutant behaved in a manner siilar to that of L. pneumophila serogroup 1, i.e., it displayed a reduced capacity 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 abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle of L. longbeachae serogroup 1 species and is required for full virulence.
Insights
The Mip protein is crucial for Legionella longbeachae serogroup 1 pathogenesis, enabling infection of Acanthamoebae and full virulence in animal models. Its absence significantly impairs the bacterium's ability to cause disease.
Area of Science:
- Microbiology
- Pathogenesis
- Bacterial Virulence Factors
Background:
- Legionella longbeachae is a significant cause of pneumonia.
- The Mip protein is known to be important for Legionella pneumophila virulence.
- Understanding L. longbeachae pathogenesis requires investigating key virulence factors.
Purpose of the Study:
- To investigate the role of the Mip protein in the pathogenesis of Legionella longbeachae serogroup 1.
- To characterize the mip gene in L. longbeachae and compare it to L. pneumophila.
- To assess the contribution of the Mip protein to L. longbeachae's ability to infect host cells and cause disease.
Main Methods:
- Cloning and sequencing of the L. longbeachae mip gene.
- Construction of isogenic mip mutants using allelic exchange mutagenesis.
- In vitro infection assays with Acanthamoebae.
- In vivo virulence studies in a guinea pig aerosol model.
Main Results:
- The L. longbeachae mip gene shows high homology to that of L. pneumophila.
- Mip mutants of L. longbeachae serogroup 1 were unable to infect Acanthamoebae or showed reduced infectivity and multiplication.
- The mip mutant strain of L. longbeachae did not cause mortality in guinea pigs, unlike the wild-type strain.
Conclusions:
- The Mip protein is essential for the intracellular lifestyle of L. longbeachae serogroup 1 within Acanthamoebae.
- The Mip protein is required for the full virulence of L. longbeachae serogroup 1 in a mammalian host.
- Targeting the Mip protein could be a strategy to control L. longbeachae infections.