Characterization of a Legionella micdadei mip mutant

Abstract

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip ؊ organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.

Microbial Peptidyl-Prolyl cis / trans Isomerases (PPIases): Virulence Factors and Potential Alternative Drug Targets

“…In addition, Mip was analyzed under the aspect of virulence only in the two other Legionella species relevant to human disease. For Legionella micdadei, deleting mip decreased the viability of bacteria in the initial hours of infection in the amoeba Hartmanella vermiformis and in U937 macrophage-like cells, though an attachment defect cannot be excluded in the case of U937 cells (207). In Legionella longbeachae, the dominant causative agent of Legionnaires' disease in Australia, similar effects were observed in the natural host amoeba Acanthamoeba castellanii and in the macrophage cell line HL-60.…”

The response regulator LetA regulates the stationary-phase stress response in Legionella pneumophila and is required for efficient infection of Acanthamoeba castellanii

“…Plasmid pDL500 was restricted with XhoI and a Km r cassette was inserted generating pDL500-Kana. Plasmid pDL500-Kana was then restricted with BamHI and XbaI and the fragment, consisting of L. pneumophila JR32 DNA containing the Km r cassette, was cloned into pBOC20 [23] generating pDLboc500-Kana. The plasmid pDLboc500-Kana was electroporated into L. pneumophila JR32 using methods described previously [24].…”

Antigenic and genetic characterization of Leaionella Proteins: Contribution to taxonomy, diagnosis and pathogenesis

“…pneumophila and the Mip-L. micdadei, once inside the macrophage, exhibit a growth rate similar to that of the wild type. The L. micdadei Mip-mutant, however, displayed a prolonged lag phase compared to the mutated L. pneumophila strain, for reasons yet unknown (O'Connell et al 1995).…”

“…These investigators isolated and characterized a Mip analog in C. truchomatis, as determined by immunologic cross-reactivity and DNA sequence homology. Recently, Mip-like proteins have been detected in two other intracellular pathogens, Coxiella burnetii and Rochalimaea quintana (Cianciotto et al 1995). Investigation of all 14 serogroups of L. pneumophila and 29 other Legionella species, ofwhich 18 have been implicated in disease, disclosed the production of a 24-31 kD protein reactive with a monospecific anti-Mip antiserum (Cianciotto et al 1990a) in all but one species (L. brunensis).…”

“…Furthermore, hybridization experiments showed that all species (including L. brunensis) and serogroups contained me-like genes, indicating a role for infectivity of all Legionella species. A L. micdadei mutant specifically lacking expression of Mip has been constructed (O'Connell et al 1995). This mutant was con-structed by allelic exhange in a fashion similar to the L. pneumophila mip mutant and represents the first genetic manipulation of a L. micdadei strain.…”