title: Production and applications of an N-terminally-truncated recombinant beta-haemolysin from Staphylococcus aureus.
abstract: The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a β-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.
authors.affiliation: ICAR Centre of Advanced Faculty Training, Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 125004 Haryana, India. Electronic address: drmahaviryadav@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Singh
authors.affiliation: ICAR Centre of Advanced Faculty Training, Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 125004 Haryana, India. Electronic address: adjitprinik@msn.com.
authors.forename: A
authors.initials: A
authors.lastname: Singh
authors.affiliation: ICAR Centre of Advanced Faculty Training, Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar, 125004 Haryana, India. Electronic address: drarchanasharma62@gmail.com.
authors.forename: A
authors.initials: A
authors.lastname: Sharma
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D001688:Biological Products / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000627:therapeutic use; D002020:Buffaloes; D002417:Cattle; D003001:Cloning, Molecular; D018389:Codon, Nonsense; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004912:Erythrocytes / Q000187:drug effects; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006461:Hemolysis / Q000187:drug effects; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D012756:Sheep; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D001688:Biological Products; D018389:Codon, Nonsense; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D013108:Sphingomyelin Phosphodiesterase; C029878:hlb protein, Staphylococcus aureus
keywords: CAMP test; N-terminal deletion; Recombinant β-haemolysin; Staphylococcus aureus