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Infect Immun, February 1998, p. 549-557, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Intracellular Multiplication and Virulence of
Shigella flexneri Auxotrophic Mutants
Antonella
Cersini,
Anna Maria
Salvia, and
Maria Lina
Bernardini*
Dipartimento di Biologia Cellulare e dello
Sviluppo, Fondazione Istituto Pasteur-Cenci Bolognetti,
Università di Roma "La Sapienza," 00185 Rome, Italy
Received 8 July 1997/Returned for modification 2 September
1997/Accepted 11 November 1997
We have constructed and analyzed a group of Shigella
flexneri 5 auxotrophic mutants. The wild-type strain M90T was
mutagenized in genes encoding enzymes involved in the synthesis of (i)
aromatic amino acids, (ii) nucleotides, and (iii) diaminopimelic acid. In this way, strains with single (aroB, aroC,
aroD, purE, thyA, and
dapB) and double (purE aroB, purE
aroC, purE aroD, purE thyA) mutations
were obtained. Although the Aro mutants had the same nutritional
requirements when grown in laboratory media, they showed different
degrees of virulence in vitro and in vivo. The aroB mutant
was not significantly attenuated, whereas both the aroC and
aroD strains were severely attenuated.
p-Aminobenzoic acid (PABA) appeared to be the main
requirement for the Aro mutants' growth in tissue culture. Concerning
nucleotides, thymine reduced the pathogenicity, whereas adenine did
not. However, when combined with another virulence-affecting mutation,
adenine auxotrophy appeared to potentiate that mutation's effects.
Consequently, the association of either the purE and
aroC or the purE and aroD mutations
had a great effect on virulence as measured by the Sereny test, whereas
the purE aroB double mutation appeared to have only a small
effect. All mutants except the dapB strain seemed to move within a Caco-2 cell monolayer after 3 h of infection.
Nevertheless, the auxotrophs showing a high intracellular generation
time were negative in the plaque assay. Knowledge of each mutation's
role in attenuating Shigella strains will provide useful
tools in designing vaccine candidates.
*
Corresponding author. Mailing address: Dipartimento di
Biologia Cellulare e dello Sviluppo, Fondazione Istituto Pasteur-Cenci Bolognetti, Università di Roma "La Sapienza," via degli Apuli 1, 00185 Rome, Italy. Phone: (39-6) 49917579. Fax: (39-6) 49917594. E-mail: bernardini{at}axcasp.caspur.it.
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