|CULLEN, P - MONASH UNIVERSITY
|HAAKE, D - UCLA SCHOOL OF MEDICINE
|BULACH, D - MONASH UNIVERSITY
|ADLER, B - MONASH UNIVERSITY
Submitted to: International Leptospirosis Society Meeting Abstracts and Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 11/28/2002
Publication Date: 11/28/2002
Citation: Cullen, P.A., Haake, D., Bulach, D.M., Zuerner, R.L., Adler, B. 2002. Identification and characterization of a novel leptospiral 'mini-momp' [abstract]. International Leptospirosis Society Meeting Abstracts and Proceedings. pg. 27.
Technical Abstract: A global analysis of Leptospira interrogans serovar lai outer membrane proteins identified pL21, a new protein that was the second most abundant constituent of the L. interrogans serovar lai outer membrane proteome. In this investigation, a pL21 peptide sequence generated from the above study was used to search the partial genomic sequences of L. interrogans serovar pomona and L. borgpetersenii serovar hardjo. A match to a gene encoding a 20-kDa protein with a signal peptidase II cleavage site characteristic of bacterial lipoproteins was obtained from both data sets. Using Southern blot analysis, a pL21 probe was found to hybridize to genomic DNA from all the pathogens examined and none of the nonpathogens examined. pL21 was amplified from 5 additional strains of Leptospira by PCR and the DNA sequences of these PCR products were determined. Alignment of the deduced amino acid sequences encoded by each of the seven pL21 homologues revealed 96.3-100% amino acid identity. A recombinant pL21 protein was produced and used to generate specific polyclonal antisera, which, in conjunction with antisera to known membrane markers, showed that pL21 was extracted alongside other known leptospiral OMPs using both TritonX-114 extraction and a newly developed sucrose gradient method. Immunoprecipitated pL21 from leptospiral culture containing tritiated palmitic acid was shown to incorporate palmitic acid, consistent with its prediction as a lipoprotein. Hence, in accordance with the current nomenclature of leptospiral outer membrane proteins, the name of pL21 was changed to LipL21. Among 8 strains investigated by immunoblotting, including pathogenic strains that were culture attenuated or virulent, all expressed LipL21 in relatively equal amounts. Both recombinant LipL21 and LipL32 reacted with the infected hamster sera while LipL36, which is known to be down regulated during infection, did not. In conclusion, LipL21, which appears to be one of the major constituents of the leptospiral outer membrane, was confirmed as a lipidated outer membrane protein. It was shown to be highly conserved among leptospiral pathogens and to be recognized by infected animal sera.