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

Abstract

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 stationaryphase (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-␤-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 ϳ20-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.The genus Legionella is one of the most successful of all aquatic bacteria, consisting of over 40 named species, their numerous serogroups (7), and a collection of Legionella-like amoebal pathogens that usually exhibit an obligate intracellular lifestyle requiring a particular protozoan host (3). An obligate requirement for the amino acid cysteine (38), which cannot be substituted for by cystine (the oxidized form most commonly found in aerobic environments), conceivably limits members of the Legionella genus to an intracellular lifestyle (25,46) or to life in association with other microorganisms (46,56,63,66) that may constitute a source of cysteine. However, in a biofilm coculture model, persistence but not multiplication of legionellae could be strictly demonstrated, suggesting that natural growth may indeed require the intracellular environment of a protozoan host (52). Thus, the natural life cycle of Legionella most likely...

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…Proteomes-To comprehensively visualize proteomic data of E and PE, and transition between the phases, we generated Voronoi treemaps (65). Voronoi treemaps have previously been used to combine quantitative proteomic data and functional relatedness of proteins for S. aureus and B. subtilis (48,66).…”

Growth-related Metabolism of the Carbon Storage Poly-3-hydroxybutyrate in Legionella pneumophila

“…The authors declare that they have no conflicts of interest with the contents of this article. 1 Both authors contributed equally to this work. 2 To whom correspondence may be addressed.…”

“…When nutrients become limiting, L. pneumophila differentiates into the mature intracellular form. This phase corresponds to the transmissive phase in which L. pneumophila becomes flagellated, expresses its virulence factors, and seems to be metabolically dormant (1)(2)(3)(4). This biphasic life cycle is also observed during growth in liquid medium, and therefore, in vitro experiments are considered as valid models to analyze the specific features encountered during both phases (3).…”

“…In the transmissive phase of L. pneumophila, high amounts of cytoplasmic granules of poly-3-hydroxybutyrate (PHB) 4 are observed in L. pneumophila. Generally, this polymer is known as an important energy and carbon storage for some bacteria (1,(5)(6)(7)(8). Indeed, PHB is also essential for the survival of L. pneumophila in the environment where it is catabolized during the viable but non-culturable state of L. pneumophila (7)(8)(9)(10).…”

Effects of Oakmoss and its Components on <i>Acanthamoeba</i><i> castellanii</i> ATCC 30234 and the Uptake of <i>Legionella pneu</i><i>mophila</i> JCM 7571 (ATCC 33152) into <i><i>A. castellanii</i></i>

“…A total of 50 µL of A. castellanii suspension 4 10 6 cells/mL was seeded in duplicate on a 96 well microtiter plate Greiner Bio-One Co. Ltd., Frickenhausen, Germany and the amoebae cells were allowed to adhere to the wells at 37 for 3 h. Then, 50 µL of the often isolated from the same location from where the amoebae are isolated Rowbotham, 1980Sasaki et al, 2003Thomas et al, 2006 . Intracellular L. pneumophila is protected from adverse conditions Thomas et al, 2004 , and shows decreased sensitivity to disinfectants not only because of their intracellular location but also as a result of phenotypic modifications Bandyopadhyay et al, 2004Garduño et al, 2002 It is very important to control the number of L. pneumophila in natural and manmade water systems to prevent outbreaks of legionellosis. Various disinfectants have been used to control L. pneumophila, but control is very difficult because L. pneumophila grows in biofilm or in amoeba cells making it resistant to a variety of disinfectants Barker et al, 1992Cooper and Hanlon, 2010Kim et al, 2002Wright et al, 1991 .…”