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Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties.

Biller L, Davis PH, Tillack M, Matthiesen J, Lotter H, Stanley SL, Tannich E, Bruchhaus I - BMC Genomics (2010)

Bottom Line: To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR.These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A.Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str, 74, 20359 Hamburg, Germany.

ABSTRACT

Background: The availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers.

Results: To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (> or = two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.

Conclusions: In this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.

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Schematic description of the putative AIG-Proteins of E. histolytica. GTP-binding motifs: G1 = GxxxxGKS, G2 = SET; G3 = VIDTPGL; Conserved domains: C1 = GL/I/VQG/AIV/II/LV/TL/MN, C2 = HVCIVWTKC, C3 = RSExEIERLI; TM: predicted transmembrane domain; Cc: Predicted coiled-coil motif.
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Figure 1: Schematic description of the putative AIG-Proteins of E. histolytica. GTP-binding motifs: G1 = GxxxxGKS, G2 = SET; G3 = VIDTPGL; Conserved domains: C1 = GL/I/VQG/AIV/II/LV/TL/MN, C2 = HVCIVWTKC, C3 = RSExEIERLI; TM: predicted transmembrane domain; Cc: Predicted coiled-coil motif.

Mentions: The oligonucleotide array that was used in this study covers about 75% of the annotated amoebic genes. It includes oligonucleotides for four putative aig1-GTPase genes, all expressed in higher levels in the pathogenic cell line B (Table 1, additional file 1). Basic Local Alignment Search Tool (BLAST) analyses indicated that the E. histolytica aig1 gene family consists of 47 members. The composed list of aig1genes includes 17 members where the RefSeq record was removed from NCBI as a result of standard genome annotation processes (Table 2). Nevertheless, some of the removed genes have been cloned in our laboratory [GenBank: XM_649824, XM_643009, XM_645223, XM_648115] and the respective sequences were also found in whole genome shotgun reads (Entamoeba histolytica HM-1:IMSS, taxid:294381). The E. histolyticaAIG1 family members show structural similarity to the GTPases of immunity-associated protein (GIMAPS)/immune-associated nucleotide-binding protein (IAN) family of AIG1-like GTPases, which are conserved between vertebrates and angiosperm plants [9]. The members of this family comprise 30-80 kDa proteins, characterized by an AIG1 domain (a GTP-binding motif) and coiled-coil motifs. The GTP-binding motif is composed of the G1 to G5 sequences and two conserved motifs (CB, consensus box and IAN motif) [9]. The E. histolytica AIG1 family members have a calculated molecular mass between 20 and 45 kDa. Most of the amoebic AIG1 molecules have the first three of the five GTP-binding sites. The CB motif and the IAN motif are not present within the amoebic proteins; instead they contain three specific domains, which are conserved throughout the amoebic protein family. As described for the GIMAP molecules, some amoebic AIG1 proteins have one or two putative coiled-coil domains. In addition, there is a subgroup that contains one to three C-terminal transmembrane domains. Some of the GIMAP members also contain hydrophobic regions at the C-terminus, which are thought to be involved in membrane anchoring [9] (Table 2; Figure 1).


Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties.

Biller L, Davis PH, Tillack M, Matthiesen J, Lotter H, Stanley SL, Tannich E, Bruchhaus I - BMC Genomics (2010)

Schematic description of the putative AIG-Proteins of E. histolytica. GTP-binding motifs: G1 = GxxxxGKS, G2 = SET; G3 = VIDTPGL; Conserved domains: C1 = GL/I/VQG/AIV/II/LV/TL/MN, C2 = HVCIVWTKC, C3 = RSExEIERLI; TM: predicted transmembrane domain; Cc: Predicted coiled-coil motif.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823695&req=5

Figure 1: Schematic description of the putative AIG-Proteins of E. histolytica. GTP-binding motifs: G1 = GxxxxGKS, G2 = SET; G3 = VIDTPGL; Conserved domains: C1 = GL/I/VQG/AIV/II/LV/TL/MN, C2 = HVCIVWTKC, C3 = RSExEIERLI; TM: predicted transmembrane domain; Cc: Predicted coiled-coil motif.
Mentions: The oligonucleotide array that was used in this study covers about 75% of the annotated amoebic genes. It includes oligonucleotides for four putative aig1-GTPase genes, all expressed in higher levels in the pathogenic cell line B (Table 1, additional file 1). Basic Local Alignment Search Tool (BLAST) analyses indicated that the E. histolytica aig1 gene family consists of 47 members. The composed list of aig1genes includes 17 members where the RefSeq record was removed from NCBI as a result of standard genome annotation processes (Table 2). Nevertheless, some of the removed genes have been cloned in our laboratory [GenBank: XM_649824, XM_643009, XM_645223, XM_648115] and the respective sequences were also found in whole genome shotgun reads (Entamoeba histolytica HM-1:IMSS, taxid:294381). The E. histolyticaAIG1 family members show structural similarity to the GTPases of immunity-associated protein (GIMAPS)/immune-associated nucleotide-binding protein (IAN) family of AIG1-like GTPases, which are conserved between vertebrates and angiosperm plants [9]. The members of this family comprise 30-80 kDa proteins, characterized by an AIG1 domain (a GTP-binding motif) and coiled-coil motifs. The GTP-binding motif is composed of the G1 to G5 sequences and two conserved motifs (CB, consensus box and IAN motif) [9]. The E. histolytica AIG1 family members have a calculated molecular mass between 20 and 45 kDa. Most of the amoebic AIG1 molecules have the first three of the five GTP-binding sites. The CB motif and the IAN motif are not present within the amoebic proteins; instead they contain three specific domains, which are conserved throughout the amoebic protein family. As described for the GIMAP molecules, some amoebic AIG1 proteins have one or two putative coiled-coil domains. In addition, there is a subgroup that contains one to three C-terminal transmembrane domains. Some of the GIMAP members also contain hydrophobic regions at the C-terminus, which are thought to be involved in membrane anchoring [9] (Table 2; Figure 1).

Bottom Line: To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR.These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A.Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str, 74, 20359 Hamburg, Germany.

ABSTRACT

Background: The availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers.

Results: To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (> or = two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.

Conclusions: In this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.

Show MeSH
Related in: MedlinePlus