The ability to adapt to adverse environmental conditions encountered in food and during host infection is a sine qua non for a successful Listeria monocytogenes infection. This ability is likely to depend on complex regulatory pathways controlled by a number of key regulators. We utilized the pORI19 plasmid integration system to analyze the role of six putative regulatory loci in growth under suboptimal environmental conditions and during murine infection. Disruption of loci encoding a topoisomerase III (lmo2756), a putative methyltransferase (lmo0581), and a regulator of the MarR family (lmo1618) revealed roles for the methyltransferase and the MarR regulator in growth under environmental stress conditions. However, plasmid integration into these loci had no impact on virulence potential in the murine model of infection. Disruption of the alternative sigma factor Sigma-H resulted in a mutant that demonstrated reduced growth potential in minimal medium. Murine studies indicated a minor role for this sigma factor in the infectious process. Strikingly, disruption of both perR and fur loci resulted in mutants that are significantly affected in virulence for mice, with the fur mutant demonstrating the greatest reduction in virulence potential. Both perR and fur mutants demonstrated increased resistance to hydrogen peroxide and the fur mutant was sensitive to low-iron conditions. The virulence defect of both fur and perR mutants could be rescued by iron-overload after esculetin treatment of mice, suggesting that the in vivo role of these gene products is to procure iron for bacterial growth.

Deletion of perR in Listeria monocytogenes results in a small-colony phenotype (⌬perR sm ) that is slow growing and exhibits increased sensitivity to H 2 O 2 . At a relatively high frequency, large-colony variants (⌬perR lg ) arise, which are more resistant to H 2 O 2 than the wild-type and ultimately dominate the culture. Transcriptional analysis revealed that the kat gene (catalase) is up-regulated in both types of mutants and that the highest level is apparent in ⌬perR sm mutants, demonstrating PerR regulation of this gene. Overexpression of the catalase gene in the wild-type background resulted in a slower-growing strain with a smaller colony size similar to that of ⌬perR sm . By combining a bioinformatic approach with experimental evidence, other PerR-regulated genes were identified, including fur, lmo0641, fri, lmo1604, hemA, and trxB. The transcriptional profile of these genes in both mutant backgrounds was similar to that of catalase in that a higher level of expression was observed in ⌬perR sm than in the wild type or ⌬perR lg . Murine studies revealed that the virulence potential of the ⌬perR sm mutant is substantially reduced compared to that of the wild-type and ⌬perR lg strains. Collectively, the data demonstrate that the ⌬perR sm mutant represents the true phenotype associated with the absence of PerR, which is linked to overexpression of regulated genes that negatively affect bacterial homeostasis both in vitro and in vivo. A subsequent secondary mutation occurred at a high frequency, which resulted in phenotypic reversion to a large-colony phenotype with increased fitness that may have obstructed the analysis of the role of PerR in the physiology of the bacterial cell.

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.