Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

Abstract

There has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.

Keywords: Biofilm ecology; Biofilm physiology; Cell functionality; Legionella pneumophila; Spatial organization.

Fig. 1

Legionella pneumophila within intracellular and extracellular environments. (A) Freshwater ecosystems. 1. Invasion of protozoan host by a free living planktonic cell. 2. After internalization, the bacterium evades the phagosome-lysosome fusion and starts the intracellular multiplication within the Legionella-containing vacuole (LCV) (replicative phase). 3. After the nutrient's conditions become limited, the bacteria switch to a transmissive phase. 4. New cells can lyse the vacuolar membrane and are released in the extracellular environment. Free-living transmissive bacteria can start a new cycle (1), associate with biofilms (5) or persist in the extracellular environment as viable but non-culturable (VBNC). (B). In nutrient-rich medium, L. pneumophila presents a biphasic life cycle, that resembles the replicative (exponential phase (EP) and transmissive intracellular forms (stationary phase (SP)). (C) Release of the mature infectious form (MIF) from the protozoan host can cause Legionnaire's disease through inhalation of aerosols in humans. Created with BioRender.com.

Fig. 2

- (A)Legionella pneumophila within multispecies biofilms. I.L. pneumophila adheres to a pre-existing biofilm. II. The cells find a favourable environment which allows them to repress some virulence genes. III. When favourable conditions are depleted, L. pneumophila starts expressing virulence genes and detaches from the biofilm. (B) Intracellular regulation during host protozoan infection. 1. The bacteria adhere to the host cell through phagocytosis mechanisms. 2. The T4BSS effector proteins and other factors allow the establishment of the LCV, and the bacteria initiate the multiplication (replicative phase). 3. When nutrients become limiting, the bacteria switch to a transmissive phase. 4. Bacteria are released into the cytosol, as mature infectious form (MIF) or enter the viable-but-not-culturable (VBNC) form. N: nucleus; GC: Golgi complex; ER: endoplasmic reticulum; M: mitochondria; (+) High concentrations; (−) Low concentrations. Created with BioRender.com.

Fig. 3

Multispecies biofilm visualization using (A) Fluorescent protein labeling (FP) or (B) Fluorescence in situ hybridization (FISH). Brief description of the protocol for each technique. Created with BioRender.com.