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Infection and Immunity, November 2002, p. 6273-6283, Vol. 70, No. 11
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.11.6273-6283.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Intracellular Growth of Legionella pneumophila Gives Rise to a Differentiated Form Dissimilar to Stationary-Phase Forms

Rafael A. Garduño,1,2 Elizabeth Garduño,1 Margot Hiltz,1 and Paul S. Hoffman1,2*

Department of Microbiology and Immunology,1 Division of Infectious Diseases, Department of Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H-4H72

Received 26 February 2002/ Returned for modification 8 May 2002/ Accepted 9 July 2002

When Legionella pneumophila grows in HeLa cells, it alternates between a replicative form and a morphologically distinct "cyst-like" form termed MIF (mature intracellular form). MIFs are also formed in natural amoebic hosts and to a lesser extent in macrophages, but they do not develop in vitro. Since MIFs accumulate at the end of each growth cycle, we investigated the possibility that they are in vivo equivalents of stationary-phase (SP) bacteria, which are enriched for virulence traits. By electron microscopy, MIFs appeared as short, stubby rods with an electron-dense, laminar outer membrane layer and a cytoplasm largely occupied by inclusions of poly-ß-hydroxybutyrate and laminations of internal membranes originating from the cytoplasmic membrane. These features may be responsible for the bright red appearance of MIFs by light microscopy following staining with the phenolic Giménez stain. In contrast, SP bacteria appeared as dull red rods after Giménez staining and displayed a typical gram-negative cell wall ultrastructure. Outer membranes from MIFs and SP bacteria were equivalent in terms of the content of the peptidoglycan-bound and disulfide bond cross-linked OmpS porin, although additional proteins, including Hsp60 (which acts as an invasin for HeLa cells), were detected only in preparations from MIFs. Proteomic analysis revealed differences between MIFs and SP forms; in particular, MIFs were enriched for an ~20-kDa protein, a potential marker of development. Compared with SP bacteria, MIFs were 10-fold more infectious by plaque assay, displayed increased resistance to rifampin (3- to 5-fold) and gentamicin (10- to 1,000-fold), resisted detergent-mediated lysis, and tolerated high pH. Finally, MIFs had a very low respiration rate, consistent with a decreased metabolic activity. Collectively, these results suggest that intracellular L. pneumophila differentiates into a cyst-like, environmentally resilient, highly infectious, post-SP form that is distinct from in vitro SP bacteria. Therefore, MIFs may represent the transmissible environmental forms associated with Legionnaires' disease.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, 7th Floor, Dalhousie University, 5859 University Ave., Halifax, Nova Scotia, Canada B3H-4H7. Phone: (902) 494-3889. Fax: (902) 494-5125. E-mail: phoffman{at}tupdean2.med.dal.ca.

Editor: B. B. Finlay

Infection and Immunity, November 2002, p. 6273-6283, Vol. 70, No. 11
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.11.6273-6283.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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