Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli

Moon, Eun‐Kyung; Kim, Min-Jeong; Lee, Hae-Ahm; Quan, Fu‐Shi; Kong, Hyun-Hee

doi:10.1016/j.exppara.2021.108188

Cited by 5 publications

(8 citation statements)

References 29 publications

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“…The total RNA was purified using an RNeasy Mini kit (Qiagen, Hilden, Germany), and the 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, Korea) as previously described [ 13 ] which included preincubation at 95°C for 1 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 30 sec. All reaction mixtures were made using a Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) with different sense and antisense primers ( Supplementary Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

“…In our previous study, differentially expressed genes (DEGs) acquired from A. castellanii during the phagocytosis of E. coli or endosymbiosis with L. pneumophila were compared [ 9 ]. Based on the findings of this previous study, it was hypothesized that the genes upregulated in E. coli -ingested Acanthamoeba were involved in the phagocytosis of Acanthamoeba and those upregulated in L. pneumophila -ingested Acanthamoeba were involved in promoting its survival and endosymbiosis within Acanthamoeba .…”

Section: Introductionmentioning

confidence: 99%

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Phagocytosis-associated genes in Acanthamoeba castellanii feeding on Escherichia coli

Kim,

Moon,

et al. 2023

Parasites Hosts Dis

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Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Phagocytosis-associated genes in Acanthamoeba castellanii feeding on Escherichia coli

Kim,

Moon,

et al. 2023

Parasites Hosts Dis

Self Cite

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“…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 ).…”

Section: Methodsmentioning

confidence: 99%

“…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 .…”

Section: Introductionmentioning

confidence: 99%

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

Kim

Moon

et al. 2023

Parasites Vectors

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Background 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. Methods 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. Results 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. Conclusions 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

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“…To date, many parasites transcriptomes have been sequenced using NGS technology, such as Acanthamoeba spp. [ 14 , 15 ]; Entamoeba histolytica [ 16 , 17 , 18 , 19 ]; Plasmodium spp. [ 20 ]; Clonorchis sinensis [ 21 , 22 , 23 ]; Schistosoma spp.…”

Section: Introductionmentioning

confidence: 99%

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing

Sohn

Kim

et al. 2023

Pathogens

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Naegleria fowleri is a pathogenic free-living amoeba, commonly found around the world in warm, fresh water and soil. N. fowleri trophozoites can infect humans by entering the brain through the nose and causing usually fatal primary amebic meningoencephalitis (PAM). Trophozoites can encyst to survive under unfavorable conditions such as cold temperature, starvation, and desiccation. Recent technological advances in genomics and bioinformatics have provided unique opportunities for the identification and pre-validation of pathogen-related and environmental resistance through improved understanding of the biology of pathogenic N. fowleri trophozoites and cysts at a molecular level. However, genomic and transcriptomic data on differential expression genes (DEGs) between trophozoites and cysts of N. fowleri are very limited. Here, we report transcriptome Illumina RNA sequencing (RNA-seq) for N. fowleri trophozoites and cysts and de novo transcriptome assembly. RNA-seq libraries were generated from RNA extracted from N. fowleri sampled from cysts, and a reference transcriptome was generated through the assembly of trophozoite data. In the database, the assembly procedure resulted in 42,220 contigs with a mean length of 11,254 nucleotides and a C+G content of 37.21%. RNA sequencing showed that 146 genes in cysts of N. fowleri indicated 2-fold upregulation in comparison with trophozoites of N. fowleri, and 163 genes were downregulated; these genes were found to participate in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The KEGG pathway included metabolic (131 sequences) and genetic information processing (66 sequences), cellular processing (43 sequences), environmental information processing (22 sequences), and organismal system (20 sequences) pathways. On the other hand, an analysis of 11,254 sequences via the Gene Ontology database showed that their annotations contained 1069 biological processes including the cellular process (228 sequences) and metabolic process (214 sequences); 923 cellular components including cells (240 sequences) and cell parts (225 sequences); and 415 molecular functions including catalytic activities (195 sequences) and binding processes (186 sequences). Differential expression levels increased in cysts of N. fowleri compared to trophozoites of N. fowleri, which were mainly categorized as serine/threonine protease, kinase, and lipid metabolism-related proteins. These results may provide new insights into pathogen-related genes or environment-resistant genes in the pathogenesis of N. fowleri.

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Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli

Cited by 5 publications

References 29 publications

Phagocytosis-associated genes in Acanthamoeba castellanii feeding on Escherichia coli

Phagocytosis-associated genes in Acanthamoeba castellanii feeding on Escherichia coli

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

De Novo Transcriptome Profiling of Naegleria fowleri Trophozoites and Cysts via RNA Sequencing

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