Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.
Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.
Wren
Brendan W
BW
Department of Medical Microbiology, St Bartholomew's and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK.
Stabler
Richard A
RA
Department of Medical Microbiology, St Bartholomew's and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK.
Das
Satya S
SS
Department of Medical Microbiology, St Bartholomew's and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, UK.
Butcher
Philip D
PD
Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
Mangan
Joseph A
JA
Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
Clarke
Jonathan D
JD
Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
Casali
Nicola
N
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
Parish
Tanya
T
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
Stoker
Neil G
NG
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D017401:Brachyspira hyodysenteriae / Q000737:chemistry / Q000235:genetics* / Q000472:pathogenicity; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000737:chemistry / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; C112817:TlyA protein, Mycobacterium tuberculosis