Attenuation and Persistence of and Ability To Induce Protective Immunity to a Staphylococcus aureus aroA Mutant in Mice

Abstract

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. The aim of this study was to obtain a suitable attenuated aro mutant of S. aureus by transposon mutagenesis and to demonstrate its efficacy as a live vaccine to induce protective immunity in a murine model of intramammary infection. To do this, we transformed S. aureus RN6390 with plasmid pTV1ts carrying Tn917. After screening of 3,493 erythromycin-resistant colonies, one mutant incapable of growing on plates lacking phenylalanine, tryptophan, 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 a 50% lethal dose (1 ؋ 10 6 CFU/mouse) higher than that of the parental strain (4.3 ؋ 10 4 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 reverse transcription-PCR, and induced significant protection from challenge with either the parental wild-type or a heterologous strain isolated from a cow with mastitis.

Staphylococcus aureus Fatty Acid Auxotrophs Do Not Proliferate in Mice

“…Its ⌬accD derivative, strain JP103, was created using a TargeTron gene knockout system (16) to insert a 0.9-kb group II intron at 164 bp into the accD gene using the plasmid and primers as described previously (3). The first step was to introduce the plasmid containing the group II intron flanked by accD sequences into strain RN6930 by phage-mediated transfer (15). After selection for insertions and curing the plasmid, the insertional inacti- vation of the accD gene was confirmed by PCR, which showed a 0.9-kb insert in the accD gene in strain JP103 (Fig.…”

“…The laboratory experiments used the genetically malleable strain RN4220; however, for the mouse experiments, we used strain S. aureus RN6930 (13) because this strain has a track record in mouse infection models (14,15). Its ⌬accD derivative, strain JP103, was created using a TargeTron gene knockout system (16) to insert a 0.9-kb group II intron at 164 bp into the accD gene using the plasmid and primers as described previously (3).…”

Network analysis of S. aureus response to ramoplanin reveals modules for virulence factors and resistance mechanisms and characteristic novel genes

“…The literature has that strains of virulent organisms are attenuated when the genes of aromatic amino acid biosynthesis are turned off. AroA mutant of S. aureus generated by transposon mutagenesis had reduced virulence in mice (Buzzola et al, 2006). …”

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

“…We chose two different mutagenesis targets: aromatic amino acid metabolism and the QS system. Auxotrophic strains (notably auxotrophic for aromatic amino acids) have been generated to obtain attenuated vaccine strains in mammals (1,4,10) and fish (26) to trigger a protective antibacterial response and in immunotherapy studies to obtain safer vectors (16). Our ⌬aroA mutants (CHA-OA and CHA-OAL) have reduced growth rates in LB and VB media, and the dramatic mortality reduction observed when injecting high amounts of these strains into C57BL/6 mice is likely to be related to a low multiplication in the host, allowing elimination of the bacteria.…”