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Infection and Immunity, May 2000, p. 2441-2448, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of an Isogenic Mutant of
Streptococcus pyogenes Manfredo Lacking the Ability To Make
Streptococcal Acid Glycoprotein
B. A.
Degnan,1,2,*
M. C.
Fontaine,2
A. H.
Doebereiner,2
J. J.
Lee,2
P.
Mastroeni,3,4
G.
Dougan,3
J. A.
Goodacre,1,5 and
M. A.
Kehoe2
School of Clinical Medical Sciences
(Rheumatology)1 and Department of
Microbiology and Immunology,2 Medical
School, University of Newcastle, Newcastle upon Tyne NE2 4HH,
Department of Biochemistry, Imperial College of Science, Technology and
Medicine, South Kensington, London SW7 2AZ,3
Centre for Veterinary Science, University of Cambridge,
Cambridge CB3 0ES,4 and Lancashire
Postgraduate School of Medicine and Health, University of Central
Lancashire, Preston PR1 2HE,5 United Kingdom
Received 29 October 1999/Returned for modification 22 November
1999/Accepted 25 January 2000
An isogenic mutant of Streptococcus pyogenes Manfredo
that lacks the ability to make streptococcal acid glycoprotein (SAGP) has been constructed by inserting a deletion in the sagp
gene using the method of allelic exchange. An assay of cell extracts (CE) prepared from the wild-type and mutant Manfredo strains for the
enzyme arginine deiminase (AD) showed that significant activity was
present in wild-type CE but none could be detected in mutant CE. These
findings confirm our earlier conclusion that SAGP has AD activity
(B. A. Degnan, J. M. Palmer, T. Robson, C. E. D. Jones, M. Fischer, M. Glanville, G. D. Mellor, A. G. Diamond,
M. A. Kehoe, and J. A. Goodacre, Infect. Immun.
66:3050-3058, 1998). Wild-type CE but not mutant CE potently inhibited
human peripheral blood mononuclear cell proliferation in response to
phytohemagglutinin, and this inhibition was overcome by the addition of
L-arginine to proliferation assay mixtures. Invasion assays
showed that the isogenic mutant organisms lacking SAGP, and thus AD
activity, were between three and five times less able to enter
epithelial cells (Hep-2C and A549) than were the wild-type
streptococci. Both wild-type and mutant S. pyogenes
bacteria were extremely sensitive to low pH. However,
L-arginine (1 mM or above) significantly increased the
viability of the wild type but not the isogenic mutant organisms under
acidic conditions. The difference in acid susceptibility between
wild-type and mutant bacteria may explain the reduced capacity of the
isogenic mutant bacteria to invade and survive intracellularly.
*
Corresponding author. Mailing address: c/o Dr. P. Mastroeni, Centre for Veterinary Science, Madingley Rd., Cambridge CB3
0ES, United Kingdom. Phone: 44 1223 766233. Fax: 44 1223 337610. E-mail: pm274{at}cam.ac.uk.
Infection and Immunity, May 2000, p. 2441-2448, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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