Attenuation, Persistence, and Ability to Induce Protective Immunity in Mice of an aroA Staphylococcus aureus Mutant

BUZZOLA FERNANDA R.; BARBAGELATA, MARIA SOL; CACCURI, ROBERTO L.; SORDELLI, DANIEL O.

INFECTION AND IMMUNITY

ASM Press

Lugar: Washington DC, USA; Año: 2006 vol. 74 p. 3498 - 3506

0019-9567

Staphylococcus aureus is the most important etiological agent of bovine mastitis, a disease that  causes significant economic losses to the dairy industry.  Several vaccines to prevent the disease have been tested, with limited success.  This aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as live vaccine to induce protective immunity in a murine model of intramammary infection.  To this purpose we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917.  After screening of 3493 erythromycin-resistant colonies, one mutant incapable to grow onto plates lacking phenylalanine, tryptophane and tyrosine was isolated and characterized.  Molecular characterization of the mutant showed that the affected gene was aroA and that the insertion occurred 756 bp downstream of the aroA start codon.  Complementation of the aroA mutant with a plasmid carrying aroA recovered the wild type phenotype.  The mutant exhibited an LD50 (1x106 CFU/mouse) higher than that of the parental strain (4.3x104 CFU/mouse).  The aroA mutant showed decreased ability to persist in the lungs, spleens and mammary glands of mice.  Intramammary immunization with the aroA mutant stimulated both Th1 and Th2 responses in the mammary gland, as ascertained by RT-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a bovine with mastitis.