Real time qRT-PCR profiles of candidate genes in N'Dama and Boran
In light of the wide range of biological processes over-represented in the ontology analysis in response to trypanosome infection, a diverse set of genes was subsequently selected from the output of the microarray data analysis for real time qRT-PCR validation. A list of 32 genes (see Table for major functions) involved in many different physiological processes including: regulation of transcription, regulation of protein biosynthesis, immune response, intracellular protein transport, protein phosphorylation, dephosphorylation, biosynthetic pathways and response to stress were selected for microarray data validation and further investigation of the differentially activated molecular mechanisms. The graphs shown in Figs. , and , which represent the mean fold changes, show the changing mRNA profiles for each breed over the entire time course. Overall, similar trends in mRNA expression emerged for subsets of profiles including, the most frequently observed trends of a general increase, or a general decrease in expression over time in one or both breeds. Another pattern of expression detected was an early increase (at 14 or 21 dpi) in expression followed by either a decrease or no further change in one or both breeds.
Biological process gene ontologies for genes validated by real time qRT-PCR.
Figure 4 Real time qRT-PCR results for nine selected genes from trypanotolerant N'Dama and trypanosusceptible Boran cattle across the trypanosome infection time course. Data analysis was carried out using the standard 2-ΔΔCt method . All C (more ...)
Figure 5 Real time qRT-PCR results for 14 selected genes from trypanotolerant N'Dama and trypanosusceptible Boran cattle across the trypanosome infection time course. Data analysis was carried out using the standard 2-ΔΔCt method . All Ct (more ...)
Figure 6 Real time qRT-PCR results for nine selected genes from trypanotolerant N'Dama and trypanosusceptible Boran cattle across the trypanosome infection time course. Data analysis was carried out using the standard 2-ΔΔCt method . All C (more ...)
The first nine genes from the panel of 32 are shown in Fig. and generally increase in expression for one or both groups of cattle and include the expression profiles for GFM1, CD19, RSF1, STX7, SCAMP1, CYBB, GMPS, MAP4K3 and LTB. The mRNA for the G elongation factor mitochondrial 1 protein (GFM1), one of three factors required by the elongation stage of the mitochondrial translation system, significantly increased in expression in PBMC from the N'Dama cattle over time and was consistently higher in N'Dama compared to Boran at all time points. The expression of the GFM1 gene failed to increase significantly in PBMC from the Boran cattle over the time course; PBMC from N'Dama, on the other hand, displayed highly significant increases at 25 and 29 dpi in particular compared to pre-infection levels (1.7-fold, P = 0.0095 and 1.8-fold, P = 0.0085 respectively). The difference in expression detected at 21 dpi, 2.4-fold higher in N'Dama compared to Boran (P = 0.0002), was one of the most significant breed differences detected in this study.
Significant, parallel increases in the mRNA expression level for the CD19 molecule, a membrane co-receptor found on all B cells were observed in N'Dama and Boran post-infection. The highly significant two- to three-fold increases were particularly evident after 14 dpi, when parasites were first apparent in the blood. In particular, the CD19 gene was highly significantly increased in expression in PBMC from N'Dama (P = 0.0000) and Boran (P = 0.0001) at 29 dpi relative to pre-infection. Endogenous mRNA levels of the chromatin remodelling and spacing factor 1 gene (RSF1) were significantly different between PBMC from N'Dama and Boran before infection (2.6-fold higher in Boran, P = 0.0109). However, following experimental infection, the profiles of RSF1 expression in both breeds showed very similar overall levels between breeds as N'Dama significantly increased in expression to a maximal level of 3.1-fold, P = 0.0126, at 25 dpi relative to pre-infection while expression in Boran remained relatively stable throughout.
The STX7 gene – encoding a protein involved in post-Golgi vesicle-mediated trafficking of proteins from the plasma membrane to endosomes and lysosomes – is another example of a gene with significantly increased expression in PBMC from N'Dama over time while remaining relatively stable in PBMC from Boran cattle. At 25 dpi, in particular, when maximum levels of STX7 mRNA were observed in N'Dama (2.2-fold, P = 0.0013) compared to pre-infection, N'Dama had 1.4-fold higher levels of STX7 mRNA relative to Boran (P = 0.0484). In addition to STX7, the secretory carrier membrane protein 1 gene (SCAMP1) is also categorized under the gene ontology biological process termed 'post-Golgi vesicle-mediated protein transport.' Before infection, the Boran group displayed a significant 1.9-fold higher level of SCAMP1 mRNA compared to N'Dama (P = 0.0109); however, expression levels remained reasonably constant after infection with no significant changes detected. Conversely, N'Dama, although starting with almost two-fold lower levels of the SCAMP1 transcript had significantly increased expression of SCAMP1 by 1.5 to 2.0-fold over the time course. At 29 dpi, in particular, a highly significant two-fold increase in expression of the SCAMP1 gene was detected in PBMC from the N'Dama group (P = 0.0031) relative to pre-infection levels.
The cytochrome b-245, beta polypeptide gene (CYBB) encodes a gp-91 phox (phagocyte oxidase) protein, which is a critical component of the microbicidal oxidase system of phagocytes. The CYBB gene was observed to increase in mRNA expression in PBMC from both breeds relative to pre-infection over the time course. However, the increase in expression in the N'Dama group was more uniform and sustained over time (although not significantly higher than the Boran at any time point measured) resulting in highly significant increases in CYBB expression from 21 dpi onwards (2.3-fold, P = 0.0007 at 21 dpi; 2.7-fold, P = 0.0000 at 25 dpi, 2.9-fold, P = 0.0002 at 29 dpi and 2.8-fold, P = 0.0000 at 34 dpi relative to pre-infection).
The guanine monophosphate synthetase gene (GMPS), which encodes a protein involved in the de novo synthesis of guanine nucleotides (essential for DNA and RNA synthesis and elevated in rapidly growing cells) was highly significantly increased in expression in PBMC from N'Dama after 14 dpi post-infection until the end of the time course relative to pre-infection levels. During this time, the expression levels in Boran were never significantly different to pre-infection levels. N'Dama showed an increase in expression that was particularly evident at 21 and 25 dpi, (1.7-fold, P = 0.0005 and 1.7-fold, P = 0.0000) respectively, which resulted in significant breed differences at those times (1.5-fold, P = 0.0022 at 21 dpi and 1.4-fold, P = 0.0052 at 25 dpi, higher in N'Dama). Therefore, although the absolute mean mRNA levels were similar before infection and at 34 dpi for both breeds, during the first wave of parasitaemia, N'Dama expressed significantly higher levels of the GMPS transcript.
Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3), also known as germinal centre kinase like kinase (GLK), is a member of the Ser/Thr protein kinase family and is thought to function in response to stress, specifically activating the Jun N-terminal kinase (JNK) signalling pathway. Despite an almost significant 1.8-fold higher level of MAP4K3 mRNA in the Boran group relative to N'Dama before infection (P = 0.0617); The N'Dama group had significantly higher expression levels of MAP4K3 at 25 dpi (1.5-fold, P = 0.0000) 29 dpi (1.6-fold, P = 0.0166) and 34 dpi (1.3-fold, P = 0.0058) relative to the Boran. This was the result of highly significant increases in MAP4K3 expression in N'Dama that were evident at the time points after 14 dpi (3.0-fold, P = 0.0021 at 21 dpi; 4.2-fold, P = 0.0000 at 25 dpi; 5.4-fold, P = 0.0047 at 29 dpi and 3.7-fold, P = 0.0005 at 34 dpi). These changes in N'Dama were coupled with moderate and less significant increases in the Boran group (1.5-fold, P = 0.0293 at 21 dpi and 1.6-fold, P = 0.0416 at 25 dpi) relative to pre-infection levels.
The lymphotoxin beta gene (LTB) involved in the inflammatory response and the normal development of lymphoid tissues, showed increasing levels of expression in the N'Dama and Boran groups that were significant after 14 dpi. Increases were comparable for both breeds with maximal levels in N'Dama at 29 dpi (2.0-fold, P = 0.0089) and 25 dpi in Boran (1.8-fold, P = 0.0126) compared to pre-infection. A marginally higher response was observed for the N'Dama: a 1.2-fold higher level of LTB expression relative to Boran at 29 dpi (P = 0.0190).
Fourteen genes (CD3E, DUSP1, IFIT2, LTBR, PIR, FOS, CD14, CEBPB, TIMP3, ICAM3, MAPK14, SEPP1, NFIL3, and SLC40A1) had profiles of expression that generally decreased over time in PBMC from one or both breeds after infection (Fig. ). A subset of these genes (CD3E, DUSP1, IFIT2, LTBR, PIR and FOS) behaved similarly, displaying coordinated patterns of expression in PBMC from both the N'Dama and Boran groups with no significant breed differences in gene expression after trypanosome infection. Decreased expression was particularly marked for these genes from 21 dpi.
The CD3e molecule, epsilon (CD3-TCR complex) gene (CD3E) encodes a cell differentiation antigen, which is part of the TCR-CD3 complex of T-lymphocytes and is involved in the positive regulation of T cell proliferation through generation of intracellular signal when antigen is bound to the TCR. Highly significant decreases in CD3E mRNA were detected in PBMC at 21 dpi (3.1-fold, P = 0.0079 and 3.2-fold, P = 0.0001 in N'Dama and Boran respectively) relative to pre-infection levels, which was sustained throughout the time course. Similarly, the dual specificity phosphatase 1 gene (DUSP1), which is a key regulator of the immune response through its role in the dephosphorylation and inactivation of MAP kinases, had significantly decreased expression from 25 dpi relative to pre-infection in PBMC from the N'Dama and Boran groups. The interferon-induced protein with tetratricopeptide repeats 2 gene (IFIT2) displayed an mRNA expression profile that showed a sharp decrease in PBMC at 21 dpi compared to pre-infection that was comparable for both breeds.
The lymphotoxin beta receptor (TNFR superfamily, member 3) gene (LTBR) encodes a receptor for the heterotrimeric lymphotoxin membrane form (a complex containing the LTA and LTB proteins), which is involved in the development of lymphoid tissue, the immune response and apoptosis. The expression of LTBR in PBMC from both animal groups generally decreased after 21 dpi; interestingly, however, a modest increase (1.4-fold, P = 0.0510) was detected at 14 dpi relative to pre-infection in the N'Dama group. Fluctuations in PBMC mRNA expression were observed for the pirin (iron-binding nuclear protein) gene (PIR), which encodes a transcriptional cofactor that interacts with the protein product of the nuclear factor I/C (CCAAT-binding transcription factor) gene (NFIC). However, there was a clear tight co-ordinate response in PBMC expression between N'Dama and Boran over the whole time course. The v-fos FBJ murine osteosarcoma viral oncogene homolog gene (FOS) displayed an expression profile that showed highly significant and substantial decreases – again, in a coordinated fashion – between the N'Dama and Boran groups, which were particularly apparent from 21 dpi onwards relative to pre-infection levels.
Five of the subset of genes shown in Fig. that generally decreased in expression across the time course (CD14, CEBPB, TIMP3, ICAM3 and MAPK14) were similarly regulated in the N'Dama and Boran groups and were comparable to the genes described above; however, the magnitude of response varied between the breeds and resulted in significant differences between the N'Dama and Boran groups at one or more time points. The CD14 molecule gene (CD14), an LPS- and apoptotic cell-binding molecule that is preferentially expressed on monocytes and macrophages had 2.5-fold (P = 0.0031) and 2.7-fold (P = 0.0439) lower levels of expression at 21 and 25 dpi respectively in PBMC from the N'Dama group relative to the Boran animals.
The PBMC mRNA expression profiles of the CCAAT/enhancer binding protein (C/EBP), beta gene (CEBPB) in the N'Dama and Boran groups tracked each other across the time course with the exception of 21 dpi, where the Boran had 2.1-fold (P = 0.0071) higher levels of CEBPB mRNA relative to the N'Dama. The PBMC gene expression profiles of the TIMP metallopeptidase inhibitor 3 gene (TIMP3) diverged from 21 dpi, after which the Boran appeared to stabilize and the N'Dama continued to decrease in expression of TIMP3 mRNA. This breed divergence in TIMP3 expression subsequently resulted in higher levels of TIMP3 mRNA expression in the Boran relative to the N'Dama at 25 dpi (1.9-fold, P = 0.0101), 29 dpi (2.6-fold, P = 0.0329) and 34 dpi (2.7-fold, P = 0.0299).
The mRNA expression profiles for the intracellular adhesion molecule 3 gene (ICAM3) were consistently lower in PBMC from the N'Dama compared to the Boran animals at 14 dpi (2.0-fold, P = 0.0306), 21 dpi (2.1-fold, P = 0.0096), 29 dpi (2.9-fold, P = 0.0124) and 34 dpi (2.7-fold, P = 0.0087). Over the entire time course, modest changes in expression occurred for the mitogen-activated protein kinase 14 gene (MAPK14). Despite this, at 29 dpi a significantly greater mRNA abundance of MAPK14 was observed in PBMC from Boran relative to N'Dama (2.0-fold, P = 0.0008).
Three genes that generally decreased in expression over the time course displayed PBMC expression profiles that varied significantly between the N'Dama and Boran groups (SEPP1, NFIL3 and SLC40A1). Although selenoprotein P, plasma 1 gene (SEPP1) mRNA expression decreased in N'Dama and Boran over time; at almost all time points examined, Boran had significantly higher levels of SEPP1 mRNA relative to N'Dama. Similarly, expression of a transcriptional activator, the nuclear factor, interleukin 3 regulated gene (NFIL3) generally decreased in both breeds over time; however, at 21 dpi the Boran displayed a highly significant 3.8-fold (P = 0.0066) increase of NFIL3 mRNA relative to the N'Dama. The solute carrier family 40 (iron-regulated transporter), member 1 gene (SLC40A1), which plays an essential role in iron ion homeostasis produced PBMC mRNA expression profiles across the time course that were among the most divergent in terms of differences in response between breeds. Expression levels of SCL40A1 mRNA did not significantly change in Boran over time, although there was tendency to increased expression; however, expression levels in the N'Dama were markedly depressed at 29 dpi (5.0-fold, P = 0.0417) and 34 dpi (5.3-fold, P = 0.0398) relative to pre-infection levels. These changes resulted in significantly higher levels of SLC40A1 mRNA in Boran relative to N'Dama at 21 dpi (3.0-fold, P = 0.0059), 25 dpi (3.6-fold, P = 0.0098), 29 dpi (8.8-fold, P = 0.0043) and 34 dpi (10.3-fold, P = 0.0311).
The mRNA expression profiles of two genes across the time course (RAB35 and NFE2L2) showed a pattern of early coordinate expression followed by later divergence in PBMC from the two groups of animals (Fig. ). The RAB35, member RAS oncogene family gene (RAB35) encodes a member of the Rab family, which are major regulators of intracellular protein transport. Minor fluctuations in expression are observed before 25 dpi; however, by 29 dpi N'Dama displayed higher mRNA levels of RAB35 mRNA relative to Boran (1.5-fold, P = 0.0377). Conversely, the expression levels of the nuclear factor (erythroid-derived 2)-like 2 gene (NFE2L2) were reduced after 25 dpi in N'Dama (although not significantly) while they fluctuated in Boran to a large extent over the time course. The result at 34 dpi was a 3.5-fold higher level of NFE2L2 mRNA in the Boran relative to the N'Dama (P = 0.0164).
The final series of PBMC expression profiles consists of seven genes also shown in Fig. (GZMB, LYZ, XDH, GBP4, CTSS, NCR3 and BAFF) with mRNA levels that followed a trend of increasing early with a subsequent decrease or no further increase in one or both breeds. The first five of these genes (GZMB, LYZ, XDH and GBP4) have comparable expression profiles for N'Dama and Boran and therefore have no significant breed differences after infection; the last three (CTSS, NCR3 and BAFF), on the other hand, have diverging profiles and significant differences between N'Dama and Boran at either 14 or 21 dpi. The granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) gene (GZMB) encodes a protease necessary for target cell lysis in cell-mediated immune responses. The GZMB gene exhibited a significant peak in mRNA expression at 14 dpi in both breeds (3.7-fold, P = 0.0031 in N'Dama and 5.0-fold, P = 0.0291 in Boran) relative to pre-infection values. Expression levels of the lysozyme (renal amyloidosis) gene (LYZ) did not change significantly in PBMC from the Boran over time, although they did tend to decrease over time. In contrast to this, the N'Dama showed an initial 2.8-fold increase (P = 0.0286) in LYZ mRNA abundance at 14 dpi relative to pre-infection that was followed by later decreases in expression. A single, modest but significant, increase in xanthine dehydrogenase gene (XDH) expression was detected at 14 dpi in N'Dama relative to pre-infection levels (1.6-fold, P = 0.0382). Guanylate binding protein 4 gene (GBP4) PBMC expression profiles for the N'Dama and Boran were synchronized after infection, with an early highly significant peak in expression at 14 dpi (3.3-fold, P = 0.0002 in N'Dama and 2.6-fold, P = 0.0029 in Boran) followed by a reduction in expression in both breeds.
The three last gene expression profiles (CTSS, NCR3 and BAFF) exhibit trends of early increases in expression in one or both breeds with significant differences between N'Dama and Boran at 14 or 21 dpi. The cathepsin S gene (CTSS) encodes a thiol protease that is responsible for the removal of the invariant chain from MHC class II molecules, thereby functioning in MHC class II-associated chain processing and peptide loading in the immune response. The mRNA expression profiles of CTSS expression in PBMC from N'Dama and Boran over the time course were strikingly different. There was no increase in CTSS mRNA abundance in Boran post-infection; Conversely, a highly significant increase in CTSS gene expression was observed for the N'Dama group at 14 dpi relative to pre-infection (2.4-fold, P = 0.0004). These fluctuations resulted in a 1.5-fold higher level of CTSS mRNA in N'Dama relative to Boran at 14 dpi (P = 0.0258). The natural cytoxicity triggering receptor 3 gene (NCR3) that may contribute to the increased efficiency of activated NK cells to lyse cells in the inflammatory response, showed a significant increase in gene expression in the N'Dama group at 14 dpi (1.6-fold, P = 0.0258) relative to pre-infection. Additionally, although NCR3 mRNA expression was reduced in N'Dama at 21 dpi, there was a significant 1.4-fold higher level of NCR3 mRNA (P = 0.0190) in N'Dama relative to Boran at this time. The tumour necrosis factor (ligand) superfamily, member 13 b gene (TNFSF13B) that encodes a potent B cell activating cytokine (BAFF), which plays an important role in the proliferation and differentiation of B cells, is abundantly expressed in peripheral blood leukocytes. In this study, TNFSF13B mRNA expression was highly significantly elevated in PBMC from N'Dama at 14 dpi (4.7-fold, P = 0.0008) and significantly increased at 21 dpi (2.0-fold, P = 0.0169) relative to pre-infection levels. Despite moderate fluctuations in TNFSF13B expression over time in the Boran, no significant differences were detected relative to pre-infection levels. Finally, at 14 dpi PBMC from the N'Dama had 1.9-fold higher mRNA levels of TNFSF13B relative to the Boran (P = 0.0129).