Phosphatase-negative mutants of Legionella pneumophila and their behavior in mammalian cell infection

Microbial phosphatases are known or suspected to play a role in the pathogenesis of several intracellular pathogens, including Legionella micdadei. Legionella pneumophila also possess phosphatase activites, but their possible roles in cellular infection are unknown. We generated mutants of a serogroup 1 isolate of L. pneumophila that lack the major phosphatase. Isolation of a Pho mutant after random mutagenesis with transposon MudII4041 allowed us to dissociate the major alkaline phosphatase (pH optimum ∼8) from a minor acid phosphatase activity. Both activities were concentrated in the bacterial periplasm. The gene encoding the major alkaline phosphatase (pho) was cloned by expression in E. coli and used to generate a site directed mutation in two L. pneumophila strains. Each parent-mutant pair was compared in a U937 cell tissue culture assay for capacity to infect, lyse, and grow within mammalian cells. Although the parental stains differed in their U937 cell cytopathicity, neither was significantly more infective than its Pho derivative, suggesting that the alkaline phosphatase activity is not essential for cellular infection. Because they are not attenuated, Pho mutants can be used to generate gene fusions with E. coli alkaline phosphatase to study and secretion and cellular infectivity in L. pneumophila.