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Identifying the function of genes involved in excreted vesicle formation in Acanthamoeba castellanii containing Legionella pneumophila



Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free legionellae or Legionella-filled vesicles. The vesicles support Legionella to survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes of Acanthamoeba infected by Legionella (ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape of Legionella from the Acanthamoeba.


After ingestion of Escherichia coli and Legionella pneumophila, expression levels of target genes in Acanthamoeba were measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation of Legionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain.


ACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion of Legionella in Acanthamoeba. ACA1_114460- and ACA1_091500-silenced Acanthamoeba failed to form the Legionella-containing excreted vesicles. Legionella was released as free legionellae from the Acanthamoeba. When the ACA1_362260 of Acanthamoeba was silenced, Legionella-containing excreted vesicles were fused with the lysosome.


These results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba played important roles in the formation of Legionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.

Graphical Abstract


Given the ubiquitous nature of Acanthamoeba, humans are prone to contacting this protozoan organism, which consequently leads to various amoeba-borne ocular diseases [1,2,3]. While Acanthamoeba ingests various microorganisms in the surrounding environment to promote its growth in the trophozoite stage, these trophozoites are transformed into highly drug-resistant cysts under unfavorable conditions [4, 5]. The propensity of this organism to encyst under growth-limiting conditions is of importance, as this contributes to the persistence of pathogenic intracellular bacteria such as Legionella pneumophila [6, 7]. Legionella pneumophila is a gram-negative bacillus prevalent in the aquatic environment and its infection causes severe respiratory diseases including pneumonia and Legionnaires’ disease in humans [8]. Because L. pneumophila can thrive within eukaryotic host cells including Acanthamoeba, irrespective of trophozoites or cyst stages, it serves as a suitable host organism for L. pneumophila. Furthermore, identifying factors that regulate intracellular bacterial growth in Acanthamoeba will contribute to developing a novel treatment option for diseases inflicted by L. pneumophila.

Unlike bacteria-containing phagosomes, L. pneumophila forms a Legionella-containing vacuole (LCV) upon successful host cell infiltration to prevent its digestion via lysosomal fusion [9, 10]. The formation of this specialized vacuole supports intracellular replication in the host cells and enhances the virulence of Legionella [10, 11]. Inhibiting the phagosome–lysosome fusion that occurs during L. pneumophila infection requires a functional intracellular multiplication/defective in organelle trafficking (icm/dot) type-IV secretion system [12,13,14]. Legionella pneumophila is capable of replicating in host cells, which subsequently release free legionellae or Legionella-filled vesicles that aid in bacterial transmission [6]. The release of Legionella-containing vesicles to the surrounding environment may be necessary for surviving for a long time and efficient transmission to other host cells. As such, investigating how L. pneumophila forms these vesicles within Acanthamoeba spp. or their egress from hosts would be beneficial to understanding their replication and transmission.

The molecular pathogenesis of L. pneumophila infection in Acanthamoeba and several other amoebic organisms are similar to those of macrophages, and as such, these protozoans were used as good models to investigate Legionella–macrophage interactions [10, 14, 15]. However, L. pneumophila grown in human monocytes and Acanthamoeba had different gene-expression profiles, thus suggesting that survival strategies employed by L. pneumophila may vary across hosts. For example, when L. pneumophila was cultured in THP-1 cells, expression of the pyroptotic protein flaA was downregulated and the pyroptosis-inhibiting sdhA protein expression which contributes to LCV stabilization was upregulated. Contrary to this finding, when L. pneumophila was permitted to grow within Acanthamoeba castellanii, sdhA gene expression was downregulated [16]. Numerous studies have delineated several factors involved in L. pneumophila vesicle formation or their release from Acanthamoeba. After Legionella has replicated enough in specialized vesicles, it is released via an exocytic pathway in protozoa that is either absent or unused in mammalian cells [17]. It has been demonstrated that Legionella transmission activator (LetA) was necessary for intracellular multiplication in A. castellanii, and letA mutants exhibited reduced infectivity [18]. The carboxy terminus of Legionella isoprenylcysteine carboxyl methyltransferase (IcmT) is essential for pore formation involved in bacterial egress, whereas its amino terminus is essential for the export of specific effectors [19, 20]. Legionella effector proteins, LepA and LepB, played a role in the non-lytic release of L. pneumophila from A. castellanii [21]. In addition to the aforementioned effectors, the genes of the icm/dot type-IV secretion system of Legionella required for LCV formation and multiplication in the host cells are well known. Nonetheless, as demonstrated above, gene expression-related studies involving L. pneumophila and Acanthamoeba spp. were predominantly focused on the former of the two. Specifically, Acanthamoeba-related factors contributing to the growth and release of Legionella remain largely elusive.

To address these limitations, in our previous study, we conducted a comparative analysis of differentially expressed genes (DEGs) in A. castellanii after ingesting either Escherichia coli or L. pneumophila [22]. Among these DEGs, 502 genes were upregulated in Acanthamoeba infected by Legionella. In the aforementioned study, we identified 22 genes involved in vesicle trafficking, membrane fusion, and phagocytosis. Interestingly, several of the 22 genes described above were upregulated in Legionella-infected Acanthamoeba but not when E. coli was taken up by the Acanthamoeba. Therefore, we hypothesized that these genes could be involved in vesicle formation which promotes L. pneumophila survival. To address this, in this study, we selected these upregulated genes in Legionella-infected Acanthamoeba and investigated their function in vesicle formation as well as their release from Acanthamoeba. Also, to account for L. pneumophila and host relationship issues, we used both environmental and clinical isolates of Acanthamoeba.


Cell culture and infection of bacteria

Acanthamoeba castellanii Castellani (ATCC 30868, clinical isolate) and Acanthamoeba castellanii Neff (ATCC 30011, environmental isolate) were obtained from the American Type Culture Collection and cultured axenically in peptone-yeast-glucose (PYG) medium at 25 °C. Legionella pneumophila Philadelphia-1 (ATCC 33152) was cultured on a buffered charcoal yeast extract (BCYE) agar plate at 37 °C with 5% CO2. Escherichia coli DH5α (Enzynomics, Seoul, Republic of Korea) was cultured in tryptone-yeast-NaCl lysogeny broth (LB) media at 37 °C using a shaking incubator. Acanthamoeba castellanii was infected by L. pneumophila at multiplicity of infection (MOI) of 100 as previously described [16]. Briefly, L. pneumophila was diluted in phosphate-buffered saline (PBS) until the optical density (OD)600 absorbance reading reached 1, which corresponds to 109 colony-forming units (CFU)/ml [23]. Next, 1 × 107 of Acanthamoeba were incubated with 1 ml of Legionella suspension at 37 °C with 5% CO2 for 1 h in PYG medium. After incubation, Acanthamoeba was washed with Page’s Amoeba Saline (PAS) and incubated with new PYG media containing 100 μg/ml of gentamicin for 2 h to kill extracellular Legionella. Acanthamoeba infected with Legionella (A+L) was washed with PAS twice and incubated with new PYG media for 12 h in a 25 °C incubator. Acanthamoeba infection of E. coli (A+E) was conducted in the same way.

Gene expression analysis via real-time polymerase chain reaction (PCR)

The expression of target genes (Table 1) was determined by real-time PCR analysis. The total RNA was purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and the complementary DNA (cDNA) was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Real-time PCR was conducted using a Magnetic Induction Cycler PCR machine (PhileKorea, Seoul, Republic of Korea) as previously described [22]: pre-incubation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. All reaction mixtures were made using a Luna Universal quantitative PCR (qPCR) Master Mix (New England Biolabs, Ipswich, MA, USA) with different sense and antisense primers (Table 2).

Table 1 Differentially expressed genes in Acanthamoeba during phagocytosis of E. coli and L. pneumophila by RNA sequencing analysis
Table 2 Primer sequences for real-time PCR

Gene silencing

Small interfering RNAs (siRNAs) targeting ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba were synthesized by Bioneer, Inc. (Bioneer, Daejeon, Republic of Korea), based on their cDNA sequences (Table 3). The siRNA (final concentration of 100 nM) was transfected into live Acanthamoeba trophozoites at a cell density of 4 × 105 cells using the Effectene transfection reagent (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The transfection efficiency of siRNA was determined by fluorescing cells under a fluorescent microscope (Leica, Wetzlar, Germany).

Table 3 siRNA sequences used in this study

Observation of excreted vesicles

Excreted vesicles of A. castellanii containing L. pneumophila were observed with Giemsa and LysoTracker staining. Acanthamoeba was transfected with siRNA (siRNA-A) and infected by Legionella (siRNA-A+L) as mentioned above. For the Giemsa stain, cells were fixed with methanol for 5 min and stained with Giemsa solution (Sigma Aldrich, Burlington, MA, USA) for 10 min. For the LysoTracker stain, cells were stained with 50 μM LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA, USA) for 1 h. Stained cells were observed under a fluorescent microscope. Vesicles or phagolysosomes were counted from a total of three randomly selected fields of view under the microscope at 1000× magnification.

Statistical analysis

Data are presented as mean ± standard deviation (SD) from three independent experiments. Student’s t-tests were performed using GraphPad Prism version 8 (Dotmatics, San Diego, CA, USA). Statistical significance between the means of groups was denoted using an asterisk. P-values less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


Identification of upregulated genes in Legionella-infected Acanthamoeba

Previously, we performed RNA sequencing to identify DEGs in A. castellanii that phagocytosed either E. coli or L. pneumophila [22]. Among these DEGs, we identified a total of six DEGs that were upregulated in A. castellanii Castellani post-L. pneumophila ingestion (A+L/A), which were associated with phagosomal maturation (Table 1). Based on the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) search results, ACA1_114460, ACA1_091500, and ACA1_265950 showed sequence similarity with SNARE domain-containing protein, R-SNARE VAMP72-family protein, 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 which ingested either E. 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, the expressions of only three genes (ACA1_114460, ACA1_091500, and ACA1_362260) from the A+L were induced to significantly higher levels than A+E controls (Fig. 1). Specifically, the ACA1_114460 gene was upregulated by approximately fourfold in A+L whereas changes were negligible for the A+E group (Fig. 1a). Escherichia coli ingestion resulted in the significant downregulation of ACA1_091500 compared to A. castellanii control while their expression was significantly upregulated in A+L (Fig. 1b). ACA1_265950 expressions were unchanged in A+E, though substantial inhibition was observed in the A+L (Fig. 1c). Ingestion of bacteria, irrespective of E. coli or L. pneumophila, enhanced the gene expression of ACA1_362260, with expression reaching a sevenfold increase in the A+L group (Fig. 1d). Conversely, ACA1_328910 gene expressions were suppressed in both A+E and A+L groups (Fig. 1e). Phagocytosing E. coli suppressed ACA1_096640 mRNA levels but their expressions remained negligibly changed in the A+L (Fig. 1f).

Fig. 1
figure 1

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

siRNA-mediated gene silencing

To observe the effects of gene knockdown in vitro, three siRNAs specific for ACA1_114460, ACA1_091500, and ACA1_362260 were prepared (Table 3). After transfecting Acanthamoeba with the three siRNAs, real-time PCR was performed to verify the gene silencing efficiency. Transfecting ACA1_114460-specific siRNA did not incur any changes to mRNA levels in the A or A+E control groups. However, in the A+L group, siRNA transfection significantly suppressed the ACA1_114460 mRNA expression to basal levels (Fig. 2a). Similar findings were observed for ACA1_091500. Transfection of siRNA specific to 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, ACA1_362260 siRNA transfection resulted in the inhibition of specific mRNA expression in both A+E and A+L groups (Fig. 2c).

Fig. 2
figure 2

Real-time PCR analysis after transfection of siRNA. Acanthamoeba castellanii were transfected with siRNA designed against ACA1_114460, ACA1_091500, and ACA1_362260 prior to bacterial ingestion for 12 h. Knockdown of gene expression was confirmed by real-time PCR (a ACA1_114460, b ACA1_091500, and c ACA1_362260). Data are expressed as mean ± SD of three separate experiments. Asterisks denote 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 excreted vesicle formation

To investigate the effect of ACA1_114460, ACA1_091500, and ACA1_362260 gene knockdown in Acanthamoeba, Giemsa staining was conducted followed by microscopic observations. After 12 h of Legionella infection, Acanthamoeba (A+L) produced excreted vesicles containing Legionella, as indicated by the black arrows in the A+L panel (Fig. 3). However, siRNA treatment of ACA1_114460 and ACA1_091500 prevented the formation of these vesicles and resulted in the dispersion of legionellae after bursting from the Acanthamoeba host, as shown in the siRNA-A+L panel (Fig. 3a, b). The black arrowheads indicate the extracellular release of L. pneumophila from Acanthamoeba. On the contrary, ACA1_362260 siRNA-transfected Acanthamoeba did not affect the formation of these excreted vesicles, as indicated by their intact structures denoted by white arrowheads (Fig. 3c). No noticeable changes were observed in A. castellanii even after siRNA transfection (siRNA-A column). Excreted vesicles from all groups were quantified under the microscope (Fig. 3d). In A+L, approximately 40% vesicle formation was observed. This phenomenon, however, was not detected in ACA1_114460 and ACA1_091500 siRNA-transfected 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).

Fig. 3
figure 3

Legionella in siRNA-transfected Acanthamoeba. siRNA-transfected A. castellanii which ingested L. pneumophila for 12 h (siRNA-A+L). Cells were stained with Giemsa staining solution. a ACA1_114460 siRNA-transfected A. castellanii Castellani, b ACA1_091500 siRNA-transfected A. castellanii Castellani, and c ACA1_362260 siRNA-transfected A. castellanii Neff. d Quantification of excreted vesicles following siRNA treatment. Arrows: excreted vesicles containing Legionella, black arrowheads: free legionellae, and white arrowheads: excreted vesicles containing Legionella. A Acanthamoeba only, A+L Acanthamoeba with L. pneumophila, siRNA-A siRNA-transfected Acanthamoeba, and siRNA-A+L siRNA-transfected Acanthamoeba co-cultured with L. pneumophila. Images were acquired at ×1000 magnification. Asterisks denote statistical significance compared to 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 the lysosomes localized within the excreted vesicles, LysoTracker staining, and microscopy were performed. The normal Acanthamoeba ingesting Legionella formed the excreted vesicles containing Legionella, as denoted by the black arrow (Fig. 4a). Lysosomes were not detected in the vesicles (black arrowhead, Fig. 4a). In Acanthamoeba transfected with either ACA1_114460 or ACA1_091500 siRNA, despite the vesicle excretion failure, partial staining with LysoTracker was observed (white arrowheads, Figs. 4b, c). Contrastingly, in the ACA1_362260 silenced Acanthamoeba, lysosome-containing excreted vesicles (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. Excreted phagolysosomes were enumerated under the microscope (Fig. 4e). Consistent with the above findings, phagolysosomal formation was predominantly observed in ACA1_362260 siRNA-treated group, whereas ACA1_114460 or ACA1_091500 siRNA transfection resulted in negligible changes compared to the control group.

Fig. 4
figure 4

LysoTracker staining after transfection of siRNA. ACA1_114460, ACA1_091500, or ACA1_362260-specific siRNA was inoculated into A. castellanii, which were subsequently co-cultured with L. pneumophila for 12 h (siRNA-A+L). Acanthamoeba and excreted vesicles containing Legionella were stained with LysoTracker. a A+L, b ACA1_114460 siRNA-transfected A+L, c ACA1_091500 siRNA-transfected A+L, d ACA1_362260 siRNA-transfected A+L, e excreted phagolysosome quantification. Arrows, excreted vesicles containing Legionella; black arrowheads, excreted vesicles containing Legionella without lysosomes; and white arrowheads, excreted vesicles containing Legionella and lysosomes. Images were acquired at ×1000 magnification


Legionella pneumophila is an intracellular pathogen that could replicate within eukaryotic host cells, and it can be transmitted to humans via aerosols containing infectious particles [6]. In this study, we investigated the role of several genes upregulated in Acanthamoeba following L. pneumophila ingestion. Our findings revealed that L. pneumophila replicated within A. castellanii and were released into the extracellular vicinity in a membrane-bound state. We also observed that siRNA targeting the ACA1_114460, ACA1_091500, and ACA1_362260 inhibited the formation of these excreted vesicles or lysosomal co-localization with the vesicles.

The DEGs ACA1_114460 and ACA1_091500 of A. castellanii were identified as a SNARE domain-containing protein and an R-SNARE VAMP72 family protein, respectively. Eukaryotic cells contain many internal organelles surrounded by membrane boundaries, and a large family of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) have been suggested to be involved in the mechanisms of vesicle trafficking, budding, and fusion [24,25,26,27]. SNAREs were functionally classified according to the membrane component associated with the vesicles (v-SNAREs) or the target compartment (t-SNAREs), and also could be structurally distinguished as Q-SNAREs and R-SNAREs based on the residue present in the center of the motif [28]. R-SNAREs can be subdivided into short vesicle-associated membrane proteins (VAMPs) and long VAMPs (longins), depending on whether they contain a short and variable domain or a conserved longin domain [28]. Given the nature of these SNAREs and their role in vesicle trafficking, we initially anticipated that knocking down these genes would affect L. pneumophila vesicle formation in A. castellanii. In line with this notion, our results confirmed that ACA1_114460 and ACA1_091500 of A. castellanii are involved in the formation of membrane-bound organelles (Fig. 3). ACA1_362260 of A. castellanii was identified as a hypothetical protein with an unknown function. Because transfection of ACA1_362260 siRNA did not inhibit vesicle formation, as was the case for ACA1_114460 and ACA1_091500, we reasoned that this hypothetical protein could be involved in a different intracellular vesicle-related process such as lysosome fusion. To ascertain this, Acanthamoeba ingesting Legionella was stained with LysoTracker post-siRNA treatment. Surprisingly, lysosomal conjugation of the vesicle was observed, and silencing the ACA1_362260 gene resulted in the release of lysosome-containing vesicles (Fig. 4d). Based on this observation, the ACA1_362260 gene is likely to be responsible for preventing lysosomal co-localization with the vesicles.

It is well known that L. pneumophila uses its icm/dot type-IV secretion system to translocate approximately 300 effector proteins into the host cell, which are required to establish the Legionella-containing vesicles necessary for its survival and replication [29,30,31]. Although earlier studies have revealed that these effectors of Legionella could control the vesicle trafficking of the host cell, the roles of effector-interacting proteins of the host cells remain unknown. Endosymbiont L. pneumophila within A. castellanii regulated the expression of numerous host genes including ACA1_114460, ACA1_091500, and ACA1_362260 [22]. Several interesting findings were reported from earlier studies, documenting the differences in L. pneumophila exocytic pathways between various organisms. For example, L. pneumophila induces apoptosis in macrophages and releases free-form L. pneumophila into the surroundings [32]. In Acanthamoeba spp., on the other hand, L. pneumophila can be released either as a free-living form or enclosed in spherical food vacuoles [4]. In support of this, our data shows that siRNA treatment inhibits Legionella-containing vacuole release. Based on this notion, we speculate that the VAMP and SNARE-like proteins that contributed to vacuole formation reported in our study are specific to Acanthamoeba. Though our findings did reveal that these genes are involved in L. pneumophila-containing vesicle formation and their release from A. castellanii, the exact function and mechanism of action for these genes remain unknown. Furthermore, in the case of ACA1_362260, the viability of legionellae contained within the lysosome-containing excreted vesicle needs to be confirmed. As such, more work is needed to identify effector molecules that control the function of these three proteins in addition to elucidating how these proteins affect L. pneumophila survival. These further studies investigating the molecular interactions will provide great information on the research of intracellular proliferation and transmission of Legionella.


In summary, we identified three highly expressed genes (ACA1_114460, ACA1_091500, and ACA1_362260) in A. castellanii ingesting L. pneumophila. ACA1_114460 and ACA1_091500 were involved in the formation of excreted vesicles containing Legionella. ACA1_362260 was involved in inhibiting the lysosome co-localization with the Legionella-containing vesicle. These results provided important information for studying the survival strategy of Legionella in Acanthamoeba and the transmission of Legionella to another host.

Availability of data and materials

The dataset supporting the conclusion of this article is included within the article.



Quantitative polymerase chain reaction


Small interfering RNA


Differentially expressed genes


Colony-forming unit


Complementary DNA


Standard deviation


Acanthamoeba castellanii


Acanthamoeba castellanii which ingested Escherichia coli


Acanthamoeba castellanii which ingested Legionella pneumophila


siRNA-transfected Acanthamoeba castellanii which ingested Legionella pneumophila


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This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MIST) (No. 2020R1F1A1068719).

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MJK performed the experiments, analyzed the data, and wrote the original manuscript. EKM designed the experiment, analyzed the data, and wrote and edited the manuscript. HJJ performed the experiments. FSQ analyzed the data and edited the revised manuscript. HHK designed the experiment, analyzed the data, acquired funding, and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Hyun-Hee Kong.

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Kim, MJ., Moon, EK., Jo, HJ. et al. Identifying the function of genes involved in excreted vesicle formation in Acanthamoeba castellanii containing Legionella pneumophila. Parasites Vectors 16, 215 (2023).

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