Use of a Novel DNA-Loaded Alginate-Calcium Carbonate Biopolymer Surrogate to Study the Engulfment of Legionella pneumophila by Acanthamoeba polyphaga in Water Systems

Abstract

The engulfment of Legionella pneumophila by free-living amoebae (FLA) in engineered water systems (EWS) enhances L. pneumophila persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between L. pneumophila and FLA in EWS are lacking. To address this, we have developed a biopolymer surrogate with a similar size, shape, surface charge, and hydrophobicity to those of stationary-phase L. pneumophila. Parallel experiments were conducted to observe the engulfment of L. pneumophila and the surrogate by Acanthamoeba polyphaga in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and L. pneumophila, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from A. polyphaga was also faster compared to that of L. pneumophila. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. L. pneumophila and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between L. pneumophila and FLA in EWS. IMPORTANCE The internalization of L. pneumophila within amoebae is a critical component of their life cycle in EWS, as it protects the bacteria from commonly used water disinfectants and provides a niche for their replication. Intracellularly replicated forms of L. pneumophila are also more virulent and resistant to sanitizers. Most importantly, the bacteria's adaptation to the intracellular environments of amoebae primes them for the infection of human macrophages, posing a significant public health risk in EWS. The significance of our study is that a newly developed L. pneumophila biopolymer surrogate can mimic the L. pneumophila engulfment process in A. polyphaga, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated L. pneumophila persistence in EWS and the associated public health risk management.

Keywords: Legionella pneumophila; amoeba; public health; surrogate; waterborne pathogens.

FIG 1

CLSM 45× bright-field (A), fluorescent (B), and composite (C) time-lapse images of fluorescent surrogate coincubated with A. polyphaga trophozoites in dechlorinated, filter sterilised tap water at 30°C at 0 (top) and 3.5 (bottom) h of incubation.

FIG 2

CLSM bright-field (A), fluorescent (B), and composite (C) images of surrogate exocytosis by A. polyphaga trophozoites at 12 h at 100× (top) and at 24 h at 60× (bottom). The white arrow points to a clump of surrogate microparticles being exocytosed by a trophozoite, and the blue arrows point to retained surrogate microparticles that are concentrated at the periphery of the trophozoite membranes.

FIG 3

CLSM 100× bright-field (A), fluorescent (B), and composite (C) images of an A. polyphaga trophozoite releasing a surrogate clump at 48 h of coincubation.

FIG 4

The log-transformed qPCR concentrations of engulfed surrogate tracer DNA, L. pneumophila DNA, and nonstained extracellular (nonengulfed) surrogate tracer DNA (A ●, B ■, and D ♦, respectively) in surrogate-A. polyphaga and L. pneumophila-A. polyphaga cocultures at 30°C. The counts of live L. pneumophila engulfed by A. polyphaga are shown in panel C (▴). The averages of 6 sample replicates are reported, and the error bars represent the standard deviation at each sampling point.

FIG 5

Normalized concentrations (C/C0) of the surrogate (●) and L. pneumophila (■) engulfment kinetics of A. polyphaga trophozoites in dechlorinated, filter-sterilised tap water at 30°C over a period of 72 h. Error bars represent the normalized standard deviation of 6 sample replicates.