Characterization of virulence properties in the C. parapsilosis sensu lato species

Abstract

The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12 C. metapsilosis and 18 C. orthopsilosis isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the C. metapsilosis strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to C. orthopsilosis and C. metapsilosis isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and C. metapsilosis strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of Galleriamellonella larvae. The mortality rate of G. mellonella larvae infected with C. metapsilosis isolates was significantly lower than those infected with C. parapsilosis sensu stricto or C. orthopsilosis strains. Taken together, our findings demonstrate that C. metapsilosis is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions.

Figure 1. Diagrammatic presentation of the occurrence of lipase-, protease- and pseudohyphae-producer strains in the C. parapsilosis sensu lato complex.

n: number of isolates, upper number in fraction: number of pseudohypha-producer strains, lower number in fraction: number of pseudohypha negative strains.

Figure 2. Interactions of C. parapsilosis sensu lato isolates (see Table S1.) with J774.2 murine macrophages.

(A) Killing efficiency of C. parapsilosis sensu lato species determined by CFU determinations (Cp, C. parapsilosis sensu stricto; Co, C . orthopsilosis ; Cm, C . metapsilosis ), (B) killing efficiency of lipase producer vs. non-producer and pseudohypha positive vs. negative strains in the C. parapsilosis sensu lato group [lip+, lipase positive (regardless of pseudohypha production); lip-, lipase negative; psh+, pseudohyphae positive (regardless of lipase production); psh-, pseudohyphae negative], (C) killing efficiency of lipase or pseudohypha positive vs. negative isolates of C. parapsilosis sensu stricto, (D) killing efficiency of lipase or pseudohypha producer vs. non-producer strains of C . orthopsilosis , (E) host-cell damaging capacity of C. parapsilosis sensu lato species based on the release of LDH (lactate dehydrogenase), (F) host-cell damaging capacity of lipase or pseudohypha producer vs. non-producer strains in the C. parapsilosis sensu lato group, (G) host-cell damaging capacity of lipase or pseudohypha positive vs. negative isolates of C. parapsilosis sensu stricto, (H) host-cell damaging capacity of lipase or pseudohypha producer vs. non-producer strains of C . orthopsilosis , C.p., C. parapsilosis sensu stricto; C.o., C . orthopsilosis ; C.m., C . metapsilosis ; cont, control (macrophages without fungal cells). Data points on graphs represent individual strains. Experiments were performed in triplicates. Data were analyzed by the Kruskal-Wallis test (A, E) or the Mann-Whitney test (B, C, D, F, G, H). * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. Phagocytosis of one representative isolate each of C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis by J774.2 macrophages.

(A) Light microscopic picures of J774.2 macrophages phagocytosing C. parapsilosis sensu lato species, (B) phagocytosis of C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis by J774.2 macrophages assessed by quantitative imaging, R2: phagocytosing macrophage population discriminated by the presence of red fluorescence due to ingestion of Alexa Fluor 647-labeled yeast cells, (C) numbers of ingested yeast cell/macrophage in the R2 population in case of C. parapsilosis sensu stricto, C . orthopsilosis and C . metapsilosis determined by the spot-counting feature of IDEAS software.

Figure 4. Interactions of different C. parapsilosis sensu lato isolates (see Table S1.) with primary human monocyte-derived macrophages.

(A) Killing efficiency of C. parapsilosis sensu lato species based on CFU-determinations (Cp, C. parapsilosis sensu stricto; Co, C . orthopsilosis ; Cm, C . metapsilosis ), (B) killing efficiency of lipase producer vs. non-producer and pseudohypha positive vs. negative strains in the C. parapsilosis sensu lato group [lip+, lipase positive (regardless of pseudohypha production); lip-, lipase negative; psh+, pseudohyphae positive (regardless of lipase production); psh-, pseudohyphae negative], (C) killing efficiency of lipase or pseudohyphae positive vs. negative isolates of C. parapsilosis sensu stricto, (D) killing efficiency of lipase or pseudohyphae producer vs. non-producer strains of C . orthopsilosis , (E) host-cell damaging capacity of C. parapsilosis sensu lato species based on the release of LDH (lactate dehydrogenase), (F) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains in the C. parapsilosis sensu lato group, (G) host-cell damaging capacity of lipase or pseudohyphae positive vs. negative isolates of C. parapsilosis sensu stricto, (H) host-cell damaging capacity of lipase or pseudohyphae producer vs. non-producer strains of C . orthopsilosis . Cp, C. parapsilosis sensu stricto; Co, C . orthopsilosis ; Cm, C . metapsilosis . Data points on graphs represent individual strains. Experiments were performed in triplicates. Data were analyzed by the Kruskal-Wallis test (A, E), the Mann-Whitney test (B, D, F, G, H) or the Wilcoxon rank sum test (C). * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. Survival of Galleria mellonella larvae infected with different isolates of (A) C. parapsilosis sensu stricto, (B) C . orthopsilosis and (C) C . metapsilosis .

For C. parapsilosis sensu stricto isolates, each survival group contained 8 larvae, whereas 9 larvae per group were used to assess survival after C . orthopsilosis or C . metapsilosis infection. (D) Summarized survival curve of nine C. parapsilosis sensu lato; Cp, C. parapsilosis sensu stricto; Co, C . orthopsilosis ; and Cm, C . metapsilosis isolates. ns, not significant; * p<0.05, ** p<0.01, *** p<0.001 by the log-rank (Mantel-Cox) test.