Growth, Virulence, and Immunogenicity ofListeria monocytogenes aroMutants

Abstract

Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K 2 , suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10 4 -fold for the aroA, aroB, and aroA/B mutants and >10 5 -fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 ؋ 10 6 aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 ؋ 10 8 aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis

“…Auxotrophs generated by mutations in the shikimate pathway cannot produce chorismate, the precursor for the biosynthesis of aromatic compounds such as PABA, dehydroxybenzoic acid, tryptophan, tyrosine, and phenylalanine. Mutants in the shikimate pathway are attenuated and protective as vaccines in a number of pathogens including S. enterica serovar Typhimurium (17), L. monocytogenes (40), and P. aeruginosa (32). By contrast, in Y. pestis strain GB, an aroA mutant was found to be virulent in BALB/c mice, suggesting that a functional shikimate pathway does not diminish virulence in this pathogen.…”

“…Given that aroA and aroB mutants are attenuated in other bacteria (17,20,32,40), we chose B. pseudomallei mutant strain 13B11, with a transposon insertion in aroB, as a possible vaccine. aroB mutant strain 13B11 was unable to grow in minimal medium.…”

“…Primer P6M3 was then used to sequence the PCR product. An insertion in the aroB gene of B. pseudomallei K96243 was predicted to render the mutant unable to grow on M9 minimal medium (40). To confirm this predicted phenotype, a single colony of mutant 13B11 or B. pseudomallei K96243 was used to inoculate 10 ml LB broth and incubated for 24 h statically at 37°C.…”

The use ofListeria monocytogenesas a DNA delivery vector for cancer gene therapy

“…Attenuation through mutation of classical virulence factors would therefore affect the efficacy of our system. It is likely that other approaches, such as the creation of auxotrophic mutants, as described by others, 15,16 will have to be implemented when developing L. monocytogenes bactofection vehicles so as to retain intracellular growth and cell spread but limit virulence for the host. It will be a challenge to select the correct mutant strain to provide an adequate level of vector amplification in vivo whilst maintaining a significant degree of attenuation to ensure safety.…”

Single-agent combinatorial cancer therapy

“…As discussed by Quispe-Tintaya et al (6), L. monocytogenes could be coupled to (radiolabeled) tumor-specific antibodies to enhance tumor selectivity of the bacteria, thus positively influencing the therapeutic activity. Virulence-attenuated auxotrophic mutants that still possess all of their virulence genes (12,13) 188 Re has a short half-life relative to other β-emitting isotopes, its relatively high mean emission path length of about 2.5 mm may have limited utility treating smaller metastases and may result in increased bystander damage to adjacent healthy tissues. Finally, combining the radiotherapy and biotherapy approach with the expression of radiosensitizing molecules and proteins or the delivery of prodrug-converting enzymes (14) to enhance the antitumor effect could be of value.…”