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Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

ABSTRACT: Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.

SUBMITTER: Haake DA

PROVIDER: S-EPMC204853 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

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Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Haake D A DA Champion C I CI Martinich C C Shang E S ES Blanco D R DR Miller J N JN Lovett M A MA

Journal of bacteriology 19930701 13

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 19 ...[more]

PMID: 8320237

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Project description:Shedding of DNA of pathogenic Leptospira spp. has been documented in naturally infected cats in several countries, but urinary shedding of infectious Leptospira spp. has only recently been proven. The climate in Southern Chile is temperate rainy with high annual precipitations which represents ideal preconditions for survival of Leptospira spp., especially during spring and summer. The aims of this study were to investigate shedding of pathogenic Leptospira spp. in outdoor cats in Southern Chile, to perform molecular characterization of isolates growing in culture, and to assess potential risk factors associated with shedding. Urine samples of 231 outdoor cats from rural and urban areas in southern Chile were collected. Urine samples were investigated for pathogenic Leptospira spp. by 4 techniques: qPCR targeting the lipL32 gene, immunomagnetic separation (IMS)-coupled qPCR (IMS-qPCR), direct culture and IMS-coupled culture. Positive urine cultures were additionally confirmed by PCR. Multilocus sequence typing (MLST) was used to molecularly characterize isolates obtained from positive cultures. Overall, 36 urine samples (15.6%, 95% confidence interval (CI) 11.4-20.9) showed positive results. Eighteen (7.8%, 95% CI 4.9-12.1), 30 (13%, 95% CI 9.2-18), 3 (1.3%, 0.3-3.9) and 4 cats (1.7%; 95% CI 0.5-4.5) were positive in qPCR, IMS-qPCR, conventional culture, and IMS-coupled culture, respectively. MLST results of 7 culture-positive cats revealed sequences that could be assigned to sequence type 17 (6 cats) and sequence type 27 (1 cat) corresponding to L. interrogans (Pathogenic Leptospira Subgroup 1). Shedding of pathogenic Leptospira spp. by cats might be an underestimated source of infection for other species including humans. The present study is the first one reporting growth of leptospires from feline urine in culture in naturally infected cats in South-America and characterisation of culture-derived isolates. So far, very few cases of successful attempts to culture leptospires from naturally infected cats are described worldwide.

Dataset Information

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Publications

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

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