Identification and characterization of an in vivo regulated D15/Oma87 homologue in Shigella flexneri using differential display polymerase chain reaction.
Shigella genes expressed during infection likely contribute to adaptation and virulence in the host. Using differential display PCR (DDPCR), a cDNA fragment from Shigella flexneri serotype 5 that showed enhanced expression in a murine model was identified, cloned and sequenced. Enhanced expression was verified by RNA dot blot. The full-length gene was cloned using PCR and sequenced. The complete gene sequence was BLAST searched against GenBank, and exhibited strong homology to genes encoding Haemophilus influenzae D15 and Pasteurella multocida Oma87 protective outer membrane antigens. The S. flexneri gene putatively encodes a approximately 90-kDa protein and was termed oma90. The deduced amino acid sequence from oma90 was analyzed and compared to the D15/Oma87 antigens. Additionally, oma90 mapped to a cluster of orthologous groups, and probably contains an ancient conserved domain. The chromosomal organization of oma90 was similar to that for H. influenzae and P. multocida as well as for other known homologues. Northern blot revealed that the oma90 transcript encoded only oma90. This report represents the first description of a S. flexneri gene identified based on enhanced expression in the host. Furthermore, we report the first evidence demonstrating in vivo regulation of a member of the d15/oma87 gene family.
Robb
C W
CW
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA. dniesel@utmb.edu
Orihuela
C J
CJ
Ekkelenkamp
M B
MB
Niesel
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D005260:Female; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009876:Operon; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012763:Shigella flexneri / Q000235:genetics*
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C093356:D15 antigen, bacteria; C101027:Oma87 protein, bacteria; C429750:Oma90 protein, Shigella flexneri