Use of a novel DNA-loaded alginate-calcium carbonate biopolymer surrogate to study the engulfment of Legionella pneumophila by Acanthamoeba polyphaga in water systems

<p> </p> <p><strong>ABSTRACT:</strong> The engulfment of <em>Legionella pneumophila</em> by free-living amoebae (FLA) in engineered water systems (EWS) enhances <em>L. pneumophila</em> persistence and provides a vehicle for rapid replication and increased public health risk. Despite numerous legionellosis outbreaks worldwide, effective tools for studying interactions between <em>L. pneumophila</em> 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 <em>L. pneumophila</em>. Parallel experiments were conducted to observe the engulfment of <em>L. pneumophila</em> and the surrogate by <em>Acanthamoeba polyphaga</em> in dechlorinated, filter-sterilised tap water at 30°C for 72 h. Trophozoites engulfed both the surrogate and <em>L. pneumophila</em>, reaching maximum uptake after 2 and 6 h, respectively, but the peak surrogate uptake was ~2-log lower. Expulsion of the engulfed surrogate from <em>A. polyphaga</em> was also faster compared to that of <em>L. pneumophila</em>. Confocal laser scanning microscopy confirmed that the surrogate was actively engulfed and maintained within vacuoles for several hours before being expelled. <em>L. pneumophila</em> and surrogate phagocytosis appear to follow similar pathways, suggesting that the surrogate can be developed as a useful tool for studying interactions between <em>L. pneumophila</em> and FLA in EWS.</p> <p><br></p> <p><strong>IMPORTANCE:</strong> The internalization of <em>L. pneumophila</em> 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 <em>L. pneumophila</em> 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 <em>L. pneumophila</em> biopolymer surrogate can mimic the <em>L. pneumophila</em> engulfment process in <em>A. polyphaga</em>, a free-living amoeba. With further development, the surrogate has the potential to improve the understanding of amoeba-mediated <em>L. pneumophila</em> persistence in EWS and the associated public health risk management.</p>