Hartmannella vermiformis inhibition of Legionella pneumophila cultivability.

Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page's amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log(10) colony forming unit (CFU) mL(-1) after 3 days of exposure compared to <0.5 log(10) CFU mL(-1) reduction of strain Lp02 (P < 0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P < 0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P < 0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.

Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella.

Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3-10. Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared. Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A. castellanii, A. polyphaga and H. vermiformis. Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns. Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM. Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures. The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila.

Legionella pneumophila is ingested by both human macrophages and amoebae, and it multiplies within similar endocytic compartments in both eukaryotic species. Inhibitors of eukaryotic protein synthesis, such as cycloheximide and emetine, had no effect on the uptake of L. pneumophila by macrophages but completely abolished ingestion by the amoeba Hartmannella vermiformis. Therefore, host cell protein synthesis is required for the bacterium to infect the amoeba but not human macrophages. To identify proteins expressed by H. vermiformis upon contact with L. pneumophila, we radiolabeled amoebal proteins after contact with bacteria in bacteriostatic concentrations of tetracycline to inhibit bacterial protein synthesis. We analyzed protein expression by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found that 33 amoebal proteins were induced; 12 of these were not detected in resting amoebae. Eleven other amoebal proteins were repressed; four of them became undetectable. In contrast, no phenotypic changes were observed in H. vermiformis upon contact with Escherichia coli or heat-killed L. pneumophila. An isogenic, avirulent variant of L. pneumophila, incapable of infecting either macrophages or amoebae, induced a different pattern of protein expression upon contact with H. vermiformis. Our data showed that amoebae manifested a specific phenotypic response upon contact with virulent L. pneumophila. This phenotypic modulation may be necessary for uptake of the bacteria into an endocytic compartment that permits bacterial survival and multiplication.