<it>Listeria monocytogenes </it>virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate <it>Tetrahymena pyriformis</it>, causes protozoan encystment and promotes bacterial survival inside cysts

<p>Abstract</p> <p>Background</p> <p>The gram-positive pathogenic bacterium <it>Listeria monocytogenes </it>is widely spread in the nature. <it>L. monocytogenes </it>was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO) is a <it>L. monocytogenes </it>major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between <it>L. monocytogenes </it>and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate <it>Tetrahymena pyriformis</it>.</p> <p>Results</p> <p>Wild type <it>L. monocytogenes </it>reduced <it>T. pyriformis </it>trophozoite counts and stimulated encystment. The effects were observed starting from 48 h of co-incubation. On the day 14, trophozoites were eliminated from the co-culture while about 5 × 10⁴cells/ml remained in the axenic <it>T. pyriformis </it>culture. The deficient in the LLO-encoding <it>hly </it>gene <it>L. monocytogenes </it>strain failed to cause mortality among protozoa and to trigger protozoan encystment. Replenishment of the <it>hly </it>gene in the mutant strain restored toxicity towards protozoa and induction of protozoan encystment. The saprophytic non-haemolytic species <it>L. innocua </it>transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. During the first week of co-incubation, LLO-producing <it>L. monocytogenes </it>demonstrated higher growth rates in association with <it>T. pyriformis </it>than the LLO-deficient isogenic strain. At latter stages of co-incubation bacterial counts were similar for both strains. <it>T. pyriformis </it>cysts infected with wild type <it>L. monocytogenes </it>caused listerial infection in guinea pigs upon ocular and oral inoculation. The infection was proved by bacterial plating from the internal organs.</p> <p>Conclusions</p> <p>The <it>L. monocytogenes </it>virulence factor LLO promotes bacterial survival and growth in the presence of bacteriovorous ciliate <it>T. pyriformis</it>. LLO is responsible for <it>L. monocytogenes </it>toxicity for protozoa and induction of protozoan encystment. <it>L. monocytogenes </it>entrapped in cysts remained viable and virulent. In whole, LLO activity seems to support bacterial survival in the natural habitat outside of a host.</p

Experimental Listeria–Tetrahymena–Amoeba food chain functioning depends on bacterial virulence traits

Abstract Background Some pathogenic bacteria have been developing as a part of terrestrial and aquatic microbial ecosystems. Bacteria are consumed by bacteriovorous protists which are readily consumed by larger organisms. Being natural predators, protozoa are also an instrument for selection of virulence traits in bacteria. Moreover, protozoa serve as a “Trojan horse” that deliver pathogens to the human body. Here, we suggested that carnivorous amoebas feeding on smaller bacteriovorous protists might serve as “Troy” themselves when pathogens are delivered to them with their preys. A dual role might be suggested for protozoa in the development of traits required for bacterial passage along the food chain. Results A model food chain was developed. Pathogenic bacteria L. monocytogenes or related saprophytic bacteria L. innocua constituted the base of the food chain, bacteriovorous ciliate Tetrahymena pyriformis was an intermediate consumer, and carnivorous amoeba Amoeba proteus was a consumer of the highest order. The population of A. proteus demonstrated variations in behaviour depending on whether saprophytic or virulent Listeria was used to feed the intermediate consumer, T. pyriformis. Feeding of A. proteus with T. pyriformis that grazed on saprophytic bacteria caused prevalence of pseudopodia-possessing hungry amoebas. Statistically significant prevalence of amoebas with spherical morphology typical for fed amoebas was observed when pathogenic L. monocytogenes were included in the food chain. Moreover, consumption of tetrahymenas fed with saprophytic L. innocua improved growth of A. proteus population while L. monocytogenes-filled tetrahymenas provided negative effect. Both pathogenic and saprophytic bacteria were delivered to A. proteus alive but only L. monocytogenes multiplied within amoebas. Observed differences in A. proteus population behaviour suggested that virulent L. monocytogenes might slow down restoration of A. proteus ability to hunt again and thus restrict the size of A. proteus population. Comparison of isogenic bacterial pairs that did or did not produce the haemolysin listeriolysin O (LLO) suggested a role for LLO in passing L. monocytogenes along the food chain. Conclusions Our results support the idea of protozoa as a means of pathogen delivery to consumers of a higher order and demonstrated a dual role of protozoa as both a “Trojan horse” and “Troy.