Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5188-5191, June 1992 Microbiology Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages (intracellular parastism/evolution/Hwimannela/Tebwhymena/FK506-bnidng proteins) NICHOLAS P. CIANCIOTTO*t AND BARRY S. FIELDSt *Department of Microbiology and Immunology, Northwestern University, Chicago, IL 60611; and *Respiratory Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333 Communicated by John R. Preer, March 9, 1992 ABSTRACT Legioneila pneumophia is an intracellular Valkampfia, as well as within the ciliate protozoa Tetrahy- parasite of freshwater protozoa and human. mena (5-16). These protozoa are present in water samples Recent studies determined that the macrophage Infectiity that have been implicated as the sources for cases oflegionel- potentiator (Mip) surface protein, a prokaryotic ole ofthe losis and, more important, the capacity ofsuch water samples FK506-blnding proteins, is required for optimal infection of to support the growth of L. pneumophila is dependent upon macrophages. To determine whether Mip is also Involved in L. the presence of the protozoa (7, 14, 15, 17). We share the pnewnophila infection of protozoa, we examind the ability of belief that adaptation to intracellular niches within protozoa a strain lacking Mip to parasitize Hrmannel amebae and engendered in L. pneumophila the ability to infect mamma- Tethraymena ciliates. After 3 days of incubation, 10W0-fold lian cells. However, support for this hypothesis requires the fewer bacteria were recovered from protozoan cocultures demonstration that Legionella employs similar mechanisms infected with the Mip- strain than from those cocultures (genes) to infect its various host cells. infected with an isogenic Mip+ strain. The mi mutant was, Recent studies demonstrated that aL. pneumophila mutant however, not inpaired in its ability to bind to amoehee ell lacking the 24-kDa macrophage infectivity potentiator (Mip) surfaces, indicating that Mip is involved in bacterial resistance surface protein is impaired in its ability to infect human to intracellular killing and/or intraceflular multiplction. alveolar macrophages in vitro and to cause disease in exper- These data suggest that L. pneumophila employs similar genes imental animals following intratracheal inoculation (18-20). and mechanisms to infect human cells and protozoa. Further- With the identification of mip, a recently described geneti- more, they support the hypothesis that the ability of L. cally defined virulence factor ofLegionella (21), we can begin pneumophia to parasitize macrophages and hence to cause to ascertain whether infection of macrophages and infection human disease is a consequence of-its prior adaptation to of protozoa have a common molecular basis. This report intracellular growth within protozoa. demonstrates that a mip mutant is also defective in its ability to parasitize two different types of freshwater protozoa, Legionella pneumophila, the etiologic agent ofLegionnaires' Hartmannella vermiformis and Tetrahymena pyriformis, disease, represents a major environmental pathogen (1, 2). suggesting that intracellular parasites of mammalian cells Strains of this bacterium are ubiquitous within aquatic envi- may have evolved from free-living forms that first adapted to ronments and can infect humans following the inhalation of growth within lower eukaryotes. contaminated aerosols generated by air-conditioners, foun- tains, and other man-made devices (1, 3). Within the lower respiratory tract, L. pneumophila invades and proliferates to MATERIALS AND METHODS large numbers within alveolar macrophages (1). In the ab- Bacterial Strains and Medium. The Mip+ L. pneumophila sence of an adequate cell-mediated immune response, the strain derived for this study, NU201, is a spontaneous rapid intracellular replication oflegionellae and the release of tissue-destructive substances from either the bacteria, the streptomycin-resistant derivative of the virulent, clinical host, or both result in acute bronchopneumonia (2, 4). L. isolate strain 130b (18). Strain NU203 is an isogenic Mip- pneumophila infection ofmacrophages is characterized by an derivative of strain NU201. To construct NU203, a DNA evasion of the bactericidal oxidative burst, an inhibition of insertion was introduced into the mip gene of strain NU201 phagosome-lysosome fusion, and lysis ofthe host cell (2, 4). by allelic exchange-i.e., mip was replaced with a mutated, Despite our increased understanding of the pathogenesis of kanamycin-resistance (Kmr)-tagged gene from a counterse- legionellosis, it is still not clear how L. pneumophila, an lectable plasmid vector (22). The mutagenesis procedure was organism that neither possesses a mammalian reservoir nor performed as before (18) but with one significant modifica- exhibits a "natural" route ofinfection, evolved the facility to tion; electroporation (see below), rather than conjugation, parasitize human professional phagocytes. The answers to was used to introduce the mutated mip gene into L. pneu- this paradox may lie within studies that identify commonal- mophila. As a result of this modification, we achieved the ities between the human host and the seemingly disparate insertional inactivation ofmip within a virulent strain that had aquatic environment. been passaged less than 10 times on artificial medium. Strain Although the legionellae are capable of extracellular NU203 exhibited the same defect in macrophage infection as growth, much evidence supports the notion that L. pneu- the original mip mutant AA1O5 (18), indicating that mip's role mophila flourishes within aquatic environments as an intra- in intracellular infection is not strain dependent. Although cellular parasite of protozoa (5). L. pneumophila can grow they still differ in their ability to infect macrophages, strain within a variety of amoebae, including strains of Acan- thamoeba, Echinamoeba, Hartmannella, Naegleria, and Abbreviations: Mip (or mip), macrophage infectivity potentiator; Kmr, kanamycin-resistance; BCYE, buffered charcoal yeast extract; cfu, colony forming unit(s). The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Microbiology and Immunology, Northwestern University, 303 East in accordance with 18 U.S.C. §1734 solely to indicate this fact. Chicago Avenue, Searle Building, Room 6-541, Chicago, IL 60611. 5188 Downloaded by guest on October 1, 2021 Microbiology: Cianciotto and Fields Proc. Natl. Acad. Sci. USA 89 (1992) 5189 AA105 and its Mip+ parent AA103 have diminished infec- isogenic parent NU201 to replicate within the amoeba H. tivity due to prolonged passage on artificial medium. vermiformis. Strains ofH. vermiformis have been associated Legionella strains were grown on buffered charcoal yeast with L. pneumophila in water samples implicated in cases of extract (BCYE) medium for 48-72 hr at 370C (23). legionnaires' disease (25, 28). Within Hartmannella cultures, Electroporation of L. pneumophia. Electroporation was the numbers ofMip- bacteria did not increase until 48 hr after performed using the "Cell-Porator electroporation system inoculation, indicating that the mutant is defective in its and voltage booster" according to the manufacturer's spec- ability to initiate intracellular multiplication within amoebae ifications (BRL). To prepare L. pneumophila for electropo- (Fig. 1). Following this prolonged lag period, the numbers of ration, bacteria from fresh BCYE plates were suspended in NU203 bacteria increased at a slower rate than did the 20 ml of double-distilled water to an ODw0 of "'1.8 and numbers of NU201 bacteria such that by 72 hr after inocu- centrifuged. The bacterial pellet was dispersed in 500 ml of lation there was a 1000-fold difference in recovery between sterile, ice-cold 10%o glycerol/90%o double-distilled water, the Mip+ and Mip- strains. These data confirm that mip is and the cell suspension was centrifuged for 25 min at 4300 x required for optimal intracellular infection ofamoebae as well g (6000 rpm, Beckman JA-20 rotor). The bacteria were then as human macrophages. Interestingly, the mutant's growth subjected to a second glycerol wash and recentrifuged. kinetics within amoeba cultures were similar to those ob- Finally, the bacteria were resuspended in 500 1.L of 10o served within alveolar macrophage cultures (18), suggesting glycerol, resulting in a suspension of .1011 colony-forming that mip has a common role in infection in both cell types. units (cfu)/ml. The cells either were used immediately or To begin to understand the function ofMip in the initiation were stored as 100-,ul samples at -700C. ofamoeba we the relative rate at which In the presence ofplasmid DNA, glycerol-treated legionel- infection, quantitated lae were exposed to an electric pulse of 2.4 kV. The pulsed Mip- bacteria associate with Hartmannella cells (Fig. 2). cells were inoculated into 1 ml of buffered yeast extract Over the first 12 hr of incubation, comparable numbers of broth, incubated at 370C for 60-90 min, and then plated on the Mip+ and Mip- bacteria became associated with amoebae, appropriate antibiotic-containing BCYE medium. The 15- suggesting that Mip is not critical for the initial interactions kilobase (kb) pNC31.3 plasmid (18) used for allelic exchange