Ultrastructural Analysis of Differentiation in Legionella pneumophila

Abstract

Legionella pneumophila is an adaptive pathogen that replicates in the intracellular environment of fundamentally divergent hosts (freshwater protozoa and mammalian cells) and is capable of surviving long periods of starvation in water when between hosts. Physiological adaptation to these quite diverse environments seems to be accompanied by morphological changes (Garduño et al., p. 82-85, in Marre et al., ed., Legionella, 2001) and conceivably involves developmental differentiation. In following the fine-structural pathway of L. pneumophila through both in vitro and in vivo growth cycles, we have now discovered that this bacterium displays an unprecedented number of morphological forms, as revealed in ultrathin sections and freeze-fracture replicas for transmission electron microscopy. Many of the forms were identified by the obvious ultrastructural properties of their cell envelope, which included changes in the relative opaqueness of membrane leaflets, vesiculation, and/or profuse invagination of the inner membrane. These changes were best documented with image analysis software to obtain intensity tracings of the envelope in cross sections. Also prominent were changes in the distribution of intramembranous particles (clearly revealed in replicas of freeze-fractured specimens) and the formation of cytoplasmic inclusions. Our results confirm that L. pneumophila is a highly pleomorphic bacterium and clarify some early observations suggesting sporogenic differentiation in L. pneumophila. Since morphological changes occurred in a conserved sequence within the growth cycle, our results also provide strong evidence for the existence of a developmental cycle in L. pneumophila that is likely accompanied by profound physiological alterations and stage-specific patterns of gene expression.Legionella pneumophila is a gram-negative bacterial pathogen that has evolved to replicate in the intracellular compartment of freshwater amoebae (3, 9, 21). Accidentally, L. pneumophila infects the alveolar macrophages of susceptible humans and causes the atypical pneumonia known as Legionnaires' disease. The intracellular environment not only represents a survival haven for L. pneumophila but also seems to be essential for replication, implying that, in spite of its ability to grow in artificial media in the laboratory, L. pneumophila is a natural obligate intracellular pathogen (3,20,21). After egressing from a wasted host, extracellular L. pneumophila survives extended periods of starvation in fresh water (45,58,60), perhaps in a nonculturable form (61), until it finds a new protozoan host.Central to the pathogenesis and ecology of obligate intracellular bacterial pathogens with an extracellular phase (wellstudied examples being Chlamydia and Coxiella spp.) is the ability to differentiate into various forms within a developmental cycle (35,36,46,48,49,57). Typically, after or during their intracellular replication, these pathogens differentiate into a highly infectious and environmentally resilient form that survives extracellularl...

The Selective Value of Bacterial Shape

“…Cells relying entirely on passive dispersal can adapt their shapes to optimize the rates at VOL. 70,2006 WHY BACTERIA HAVE SHAPE 677 on May 9, 2018 by guest http://mmbr.asm.org/ which they move and to choose the locales in which they will accumulate. Although no one rule can be applied to all geological situations, a general guideline seems to be that the smallest cells may be retained in fine crevices, larger cells are retained by preferential attachment, and cells with dimensions in between may be more apt to flow through.…”

“…70,2006 WHY BACTERIA HAVE SHAPE 683 on May 9, 2018 by guest http://mmbr.asm.org/ prokaryotic and eukaryotic worlds" (246) and "an exciting frontier for microbial ecology and evolution" (206). The relationship has been heralded as a critical force that "promoted major transitions in bacterial evolution" (206), shaped "the phenotypic and taxonomic composition of bacterial communities" (150), is "at least partly responsible for the diversity of present bacteria" (23), and may have "shaped microbial evolution as profoundly as oxygenic photosynthesis" (246).…”

“…Epibiotic predators (e.g., Vampirococcus) attach to and remain at the surface of their targets and suck the VOL. 70,2006 WHY BACTERIA HAVE SHAPE 687 on May 9, 2018 by guest http://mmbr.asm.org/ cytoplasm from the host; periplasmic predators (e.g., Bdellovibrio) burrow into the periplasm of gram-negative bacteria, extract the intracellular nutrients, and leave after lysing the cells; and cytoplasmic predators (e.g., Daptobacter) penetrate all layers of the gram-negative envelope to grow and divide in the host's cytoplasm (110). The effect of these predators on bacterial size or shape is unknown, with little or no work being done on the subject.…”

“…VOL. 70,2006 WHY BACTERIA HAVE SHAPE 671 on May 9, 2018 by guest http://mmbr.asm.org/ direct atomic contact with an adjoining surface (assuming that each surface is perfectly smooth and without projections) (335). This circumstance derives from the geometry of the bacterial surface (highly curved) coupled with the ϳ5-nm range over which the secondary Gibbs free energy minimum allows reversible binding (269,335,336) (Fig.…”

Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines

“…Electron microscopy was done in the Electron Microscope Facility of the Faculty of Medicine, Dalhousie University, using standard protocols (37). A Philips EM300 transmission electron microscope operating at 60 kV was used to visualize Jurkat T leukemia cells that had been cultured in the absence or presence of 200 Ag/mL LfcinB for 2 hours.…”