Identification of the function of genes involved in the formation of excreted vesicles in Acanthamoeba castellanii containing Legionella pneumophila – Parasites and vectors

Identification of upregulated genes in Legionella-infected Acanthamoeba

Previously, we performed RNA sequencing to identify DEGs in A. castellanii even the engulfed one Escherichia coli OR L. pneumophila [22]. Among these DEGs, we identified a total of six upregulated DEGs A. castellanii Castellani post-L. pneumophila ingestion (A + L/A), which were associated with phagosomal maturation (Table 1). Based on the results of the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search, ACA1_114460, ACA1_091500, and ACA1_265950 showed sequence similarity to the SNARE domain-containing protein, R-SNARE family protein VAMP72 and Golgi family protein, each respectively. ACA1_362260, ACA1_328910 and ACA1_096640 were identified as hypothetical proteins. To confirm their expression levels, cDNA from A. castellanii Castellani and A. castellanii Neff who ingested both Escherichia coli (A+E) or L. pneumophila (A + L) were acquired and real-time PCR was performed using the gene-specific primers listed below (Table 2). Of the six genes, expressions of only three genes (ACA1_114460, ACA1_091500 and ACA1_362260) from the A+L were induced at significantly higher levels than the A+E controls (Fig.1). In particular, the ACA1_114460 gene was upregulated approximately four-fold in A+L, while the changes were negligible for group A+E (Fig.1a). Escherichia coli ingestion resulted in the significant downregulation of ACA1_091500 vs A. castellanii control while their expression was significantly upregulated in A+L (Fig.1b). ACA1_265950 expressions were unchanged in A+E, although substantial inhibition was observed in A+L (Fig.1c). Ingestion of bacteria, regardless of Escherichia coli OR L. pneumophila, enhanced the gene expression of ACA1_362260, with expression reaching a seven-fold increase in group A+L (Fig.1d). In contrast, ACA1_328910 gene expressions were suppressed in both groups A+E and A+L (Fig.1e). Engulfing Escherichia coli mRNA levels of ACA1_096640 suppressed but their expressions remained negligibly changed in the A+L (Fig.1f).

Figure 1

Real-time PCR analysis of differentially expressed genes in Acanthamoeba during the phagocytosis of Escherichia coli AND L. pneumophila. Acanthamoeba castellanii– ingested Escherichia coli AND L. pneumophila for 12 hours and the differential gene expression of six genes (A ACA1_114460, b ACA1_091500, c ACA1_265950, d ACA1_362260, And ACA1_328910 e f ACA1_096640) were determined by real-time PCR. The transcriptional levels of six genes in Acanthamoeba (A) were compared with those of Escherichia– ingested Acanthamoeba (A+E) e Legionella– ingested Acanthamoeba (A+L). Data are expressed as the mean SD of three separate experiments. Asterisks indicate statistically significant differences (* P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 vs Acanthamoeba check)

siRNA-mediated gene silencing

To observe the effects of gene knockdown in vitro, three specific siRNAs for ACA1_114460, ACA1_091500 and ACA1_362260 were prepared (Table 3). After the transfection Acanthamoeba with the three siRNAs, real-time PCR was performed to check the efficiency of gene silencing. Transfection of the ACA1_114460 specific siRNA did not result in any changes in mRNA levels in control groups A or A+E. However, in group A+L, siRNA transfection significantly suppressed ACA1_114460 mRNA expression at basal levels (Fig.2a). Similar results were observed for ACA1_091500. Transfection of siRNA specific for the ACA1_091500 gene had a negligible effect on A and A+E, but drastically reduced their expression in the A+L experimental group (Fig.2b). Interestingly, unlike the other two genes, transfection of ACA1_362260 siRNA resulted in inhibition of specific mRNA expression in both A+E and A+L groups (Fig.2c).

Figure 2
figure 2

Real-time PCR analysis after siRNA transfection. Acanthamoeba castellanii were transfected with siRNAs designed against ACA1_114460, ACA1_091500 and ACA1_362260 before bacterial ingestion for 12 hours. Gene expression knockdown was confirmed by real-time PCR (A ACA1_114460, b ACA1_091500, e c ACA1_362260). Data are expressed as the mean SD of three separate experiments. Asterisks indicate statistically significant differences (** P<0.01, *** P<0.001 and **** P<0.0001) between the control group and the siRNA-transfected group

Effect of gene silencing on the formation of excreted vesicles

To study the effect of ACA1_114460, ACA1_091500 and ACA1_362260 gene knockdown in Acanthamoeba, Giemsa stain was conducted followed by microscopic observations. After 12h of Legionella infection, Acanthamoeba (A+L) produced excreted vesicles containing Legionella, as indicated by the black arrows in panel A+L (Fig.3). However, siRNA treatment of ACA1_114460 and ACA1_091500 prevented the formation of these vesicles and resulted in legionella dispersal after the outbreak from the Acanthamoeba hosts, as shown in the siRNA-A + L panel (Fig. 3a,b). Black arrows indicate extracellular release of L. pneumophila from Acanthamoeba. In contrast, ACA1_362260 transfected siRNA Acanthamoeba did not affect the formation of these excreted vesicles, as indicated by their intact structures denoted by white arrowheads (Fig.3c). No obvious changes were observed in A. castellanii even after siRNA transfection (column siRNA-A). Vesicles excreted from all groups were quantified under the microscope (Fig.3d). Approximately 40% vesicle formation was observed in A+L. This phenomenon, however, was not detected in ACA1_114460 and ACA1_091500 transfected siRNA Acanthamoeba. The only exception to this was the ACA1_362260 siRNA-treated group, which demonstrated a similar level of vesicle formation to that of the control group (A+L).

figure 3

Legionella in transfected siRNA Acanthamoeba. transfected siRNA A. castellanii which he ingested L. pneumophila for 12 hours (siRNA-A+L). Cells were stained with Giemsa staining solution. A ACA1_114460 transfected siRNA A. castellanii Castellani, b ACA1_091500 transfected siRNA A. castellanii Castellani, ed c ACA1_362260 transfected siRNA A. castellanii Neff. d Quantification of vesicles excreted after siRNA treatment. Arrows: excreted vesicles containing Legionellablack arrowheads: free legionella and white arrowheads: excreted vesicles containing Legionella. A Acanthamoeba only, A+L Acanthamoeba with L. pneumophila, siRNA-A transfected siRNA AcanthamoebaAND siRNA-A+L transfected siRNA Acanthamoeba co-cultivated with L. pneumophila. Images were acquired at 1000x magnification. Asterisks denote statistical significance compared to the A+L control (*** P<0.001, **** P<0.0001)

Effect of gene silencing on the phagolysosome

To investigate the gene silencing effect of ACA1_362260 on lysosomes located within the excreted vesicles, LysoTracker staining and microscopy were performed. The normal Acanthamoeba ingest Legionella formed the excreted vesicles containing Legionella, as indicated by the black arrow (Fig.4a). Lysosomes were not detected in the vesicles (black arrowhead, Fig.4a). In Acanthamoeba transfected with ACA1_114460 or ACA1_091500 siRNA, despite failure of vesicle excretion, partial staining was observed with LysoTracker (white arrowheads, Figs.4b,c). On the contrary, in the ACA1_362260 silenced Acanthamoebaexcreted vesicles containing lysosomes (white arrowheads, Fig.4d) were separated from the Acanthamoeba host cell. THE Acanthamoeba transfected with ACA1_362260 siRNA seems to have failed to inhibit phagolysosome formation. The excreted phagolysosomes were enumerated under the microscope (Fig.4e). Consistent with the above findings, phagolysosome formation was predominantly observed in the ACA1_362260 siRNA-treated group, whereas ACA1_114460 or ACA1_091500 siRNA transfection resulted in negligible changes compared with the control group.

figure 4

LysoTracker staining after siRNA transfection. The siRNA specific for ACA1_114460, ACA1_091500 or ACA1_362260 was inoculated into A. castellaniiwhich were later co-cultivated with L. pneumophila for 12 hours (siRNA-A+L). Acanthamoeba and excreted vesicles containing Legionella were stained with LysoTracker. A A+L, b ACA1_114460 A+L transfected with siRNA, c ACA1_091500 A+L transfected with siRNA, d ACA1_362260 A+L transfected with siRNA, And quantification of the excreted phagolysosome. Arrows, ejected vesicles containing Legionella; black arrowheads, ejected vesicles containing Legionella without lysosomes; and white arrowheads, containing ejected vesicles Legionella and lysosomes. Images were acquired at 1000x magnification

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