Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity.
Abstract: We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted alpha-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.
Publication Date: 1997-06-01 PubMed ID: 9218769DOI: 10.1046/j.1365-2958.1997.4291786.xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research involves cloning the gene of a protein in Borrelia burgdorferi, the bacteria that causes Lyme disease, which interacts with cell membranes and has haemolytic activity, or the ability to lyse red blood cells.
Cloning the Membrane-Interactive Protein
- The research cloned the gene of a protein which interacts with cell membranes.
- This ability was discovered through its haemolytic activity, which refers to the capability of breaking down red blood cells.
- This action is erythrocyte-species specific, meaning it functions with varying degrees of activity on red blood cells from different species, such as horses, sheep, and rabbits.
Analysis of Haemolytic Determinant
- Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, Central to the study, the authors found two specific genes, blyA and blyB, with haemolytic properties.
- They are part of a predicted four-gene operon, a functionally united group of genes that are transcribed together, which is found in multiple copies on certain plasmids – small, circular, double-stranded DNA molecules – in B. burgdorferi B31 strain.
Detailed findings about blyA and blyB
- Gene blyA codes for a small alpha-helical protein that structurally resembles a broad group of toxins known to form pores in cell membranes and exhibit cytolytic activity.
- By contrast, gene blyB codes for a soluble protein that stabilizes the function of BlyA and enhances its haemolytic activity.
- The haemolytic activity was observed to be sensitive to protease enzymes, unstable under heat, independent of divalent cations, and highly dependent on protein concentration. This suggests that the protein works through oligomerization, a process in which identical molecules join together to perform a biological function.
Experimental Methods and Further Analyses
- The protein BlyA was purified from E. coli using a procedure called Triton X-114 phase partitioning.
- Analyses of blyA and blyB gene mutants showed that the membrane association and activity of BlyA relied on minor changes in its sequence and on the protein BlyB.
- Finally, the study reported that the bly genes are expressed at very low levels in B. burgdorferi cultured under laboratory conditions.
Cite This Article
APA
Guina T, Oliver DB.
(1997).
Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity.
Mol Microbiol, 24(6), 1201-1213.
https://doi.org/10.1046/j.1365-2958.1997.4291786.x Publication
Researcher Affiliations
- Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
MeSH Terms
- Amino Acid Sequence
- Animals
- Bacterial Proteins / genetics
- Bacterial Proteins / metabolism
- Base Sequence
- Binding Sites
- Borrelia burgdorferi Group / genetics
- Borrelia burgdorferi Group / growth & development
- Borrelia burgdorferi Group / metabolism
- Chromosome Mapping
- Cloning, Molecular
- DNA, Bacterial
- Hemolysin Proteins / genetics
- Hemolysin Proteins / metabolism
- Horses
- Molecular Sequence Data
- Nucleic Acid Conformation
- Plasmids
- Rabbits
- Sequence Analysis, DNA
- Sheep
Citations
This article has been cited 11 times.- Stevenson B, Brissette CA. Erp and Rev Adhesins of the Lyme Disease Spirochete's Ubiquitous cp32 Prophages Assist the Bacterium during Vertebrate Infection.. Infect Immun 2023 Mar 15;91(3):e0025022.
- Anderton JM, Tokarz R, Thill CD, Kuhlow CJ, Brooks CS, Akins DR, Katona LI, Benach JL. Whole-genome DNA array analysis of the response of Borrelia burgdorferi to a bactericidal monoclonal antibody.. Infect Immun 2004 Apr;72(4):2035-44.
- Damman CJ, Eggers CH, Samuels DS, Oliver DB. Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system.. J Bacteriol 2000 Dec;182(23):6791-7.
- Porcella SF, Fitzpatrick CA, Bono JL. Expression and immunological analysis of the plasmid-borne mlp genes of Borrelia burgdorferi strain B31.. Infect Immun 2000 Sep;68(9):4992-5001.
- Stevenson B, Porcella SF, Oie KL, Fitzpatrick CA, Raffel SJ, Lubke L, Schrumpf ME, Schwan TG. The relapsing fever spirochete Borrelia hermsii contains multiple, antigen-encoding circular plasmids that are homologous to the cp32 plasmids of Lyme disease spirochetes.. Infect Immun 2000 Jul;68(7):3900-8.
- Caimano MJ, Yang X, Popova TG, Clawson ML, Akins DR, Norgard MV, Radolf JD. Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297.. Infect Immun 2000 Mar;68(3):1574-86.
- Eggers CH, Samuels DS. Molecular evidence for a new bacteriophage of Borrelia burgdorferi.. J Bacteriol 1999 Dec;181(23):7308-13.
- Yang X, Popova TG, Hagman KE, Wikel SK, Schoeler GB, Caimano MJ, Radolf JD, Norgard MV. Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family.. Infect Immun 1999 Nov;67(11):6008-18.
- Zückert WR, Meyer J, Barbour AG. Comparative analysis and immunological characterization of the Borrelia Bdr protein family.. Infect Immun 1999 Jul;67(7):3257-66.
- Kornacki JA, Oliver DB. Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters.. Infect Immun 1998 Sep;66(9):4115-22.
- Gilmore RD Jr, Mbow ML. A monoclonal antibody generated by antigen inoculation via tick bite is reactive to the Borrelia burgdorferi Rev protein, a member of the 2.9 gene family locus.. Infect Immun 1998 Mar;66(3):980-6.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
