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DNA sequence of mip, a Legionella pneumophila gene associated with macrophage infectivity
N C Engleberg1, C Carter, D R Weber
1Ann Arbor Veterans Administration Hospital, Michigan 48109.
Abstract:
In a previous study, a 24-kilodalton (kDa) protein surface antigen of Legionella pneumophila was cloned into Escherichia coli and found to be expressed on the host cell surface. Subsequently, a site-directed mutation in this gene (designated mip) in L. pneumophila was found to impair the capacity of this bacterium to initiate intracellular infection in human macrophages. The work presented here indicates that the antigenic gene product is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila. In addition, the antigen was identified as a highly basic protein on two-dimensional nonequilibrium polyacrylamide gels and on two-dimensional monoclonal antibody immunoblots. When the DNA fragment encoding this protein was sequenced, a long open reading frame of 699 base pairs was identified within a region to which antigen expression was previously mapped. mip mRNA isolated from both L. pneumophila and transformed E. coli had the same 5' end, as determined by primer extension analysis, indicating that the same promoter sequences are used in both species. A likely factor-independent transcriptional terminator was found 20 residues downstream of the stop codon, suggesting that mip is encoded on a monocistronic message. The inferred polypeptide began with a possible 20- to 24-residue signal sequence, and, as predicted by two-dimensional electrophoresis, had a molecular weight of 24,868 and was a potent polycation with an estimated pI of 9.8.
Insights
The Legionella pneumophila mip gene encodes a 24.8 kDa surface antigen, distinct from other major outer membrane proteins. This antigen is crucial for bacterial infection of human macrophages.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- A previous study cloned a 24-kilodalton (kDa) surface antigen gene (mip) from Legionella pneumophila into Escherichia coli, observing its expression.
- A mutation in the mip gene impaired L. pneumophila's ability to infect human macrophages, highlighting its role in pathogenesis.
Purpose of the Study:
- To further characterize the mip gene product and its role in Legionella pneumophila infection.
- To determine if the mip antigen is distinct from other major outer membrane proteins.
- To elucidate the genetic and molecular properties of the mip gene and its product.
Main Methods:
- Two-dimensional nonequilibrium polyacrylamide gel electrophoresis and monoclonal antibody immunoblots to analyze the antigen's properties.
- DNA sequencing of the mip gene to identify open reading frames and regulatory elements.
- Primer extension analysis to determine mRNA 5' ends and promoter usage.
- Analysis of predicted polypeptide sequence for signal peptides and physicochemical properties.
Main Results:
- The mip antigen is distinct from the 24- to 29-kDa major outer membrane protein of L. pneumophila.
- The antigen was identified as a highly basic protein (pI 9.8) with a molecular weight of 24,868.
- A 699-base pair open reading frame was identified, encoding the mip protein.
- Identical promoter sequences are used in both L. pneumophila and E. coli, and the gene is likely monocistronic.
Conclusions:
- The mip gene product is a distinct, highly basic, surface-exposed protein of Legionella pneumophila.
- The mip antigen plays a significant role in the bacterium's capacity to initiate intracellular infection in macrophages.
- Understanding the mip gene and its product provides insights into Legionella pathogenesis and potential therapeutic targets.