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National Research Institute of Animal Production, Krakowska 1, 32-083 Balice, PolandUniversity of Agriculture in Krakow, University Centre of Veterinary Medicine Krakow, Al. Mickiewicza 24/28, 30-059 Krakow, Poland
Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, Postępu 36A, Jastrzębiec, 05-552 Magdalenka, PolandDepartment of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw
Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.
). Staphylococcus aureus is one of the most frequently isolated pathogens in bovine mastitis and may cause a severe or chronic subclinical form of the disease. This coagulase-positive bacterium may lead to pathogenic induction of gene expression in infected cells of the host (
). Methylation is a chemical modification of DNA that can block gene activity. Methylation of regulatory sequences enriched in CpG sites is associated with compacted chromatin, an environment that impedes binding of transcriptional factors. In contrast, a lack of methylation of regulatory elements facilitates gene transcription (
). Once inherited, DNA methylation patterns undergo changes throughout life under environmental influences and aging-relevant processes. As a result, alterations in methylation of regulatory sequences can indicate interactions between the environment and gene expression patterns.
With respect to mastitis-induced chronic inflammation of the mammary gland, in the current research we evaluated the importance of methylation and expression profiles of genes important for autophagy (ATG5;
). However, we wanted to determine whether links exist between DGAT1 activity and pathogen-induced inflammation related to mammary gland infection.
In this study, we evaluated RNA and DNA samples of parenchyma tissue of bovine mammary gland infected with coagulase-positive and coagulase-negative staphylococci and uninfected tissues. Inspected samples were previously tested against bacterial infection on the basis of microbiological examination. Detailed information on animals, their maintaining conditions, tissue sampling, and milk microbiological examination is presented in
). The experimental groups in the present report included 31 tissue sections obtained from 18 donor cows with confirmed infection with coagulase-positive staphylococci (CPS group) and 14 tissue sections infected with CNS obtained from 11 cows (CNS group; Supplemental Table S1; https://doi.org/10.3168/jds.2020-18404). The control group included 11 tissue sections free of bacteria obtained from 6 healthy cows (H group; Supplemental Table S1). Tissue sections came from different quarters of the udder, depending on the presence or lack of particular type of bacteria (as designated in the first column of Supplemental Table S1).
Total RNA of the parenchymal tissue samples was isolated using PureLink RNA Mini Kit (Applied Biosystems/ThermoFisher Scientific, Waltham, MA) including an RNA purification stage using PureLink DNase Set (Applied Biosystems/ThermoFisher Scientific). The quantity and quality of RNA were examined using a Nanodrop spectrophotometer (Thermo Scientific, Warsaw, Poland) and 2% gel electrophoresis. Reverse transcription was performed on 500 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems/ThermoFisher Scientific) according to the manufacturer's protocol. Reverse transcription (RT)-PCR primers were designed with the option to span at least one intron or to cover exon junctions using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi; Supplemental Table S2; https://doi.org/10.3168/jds.2020-18404). Qualitative RT-PCR was performed for the pool of 11 equimolar cDNA samples including 4 CPS (750PP, 782LT, 842PP, 855PP), 4 CNS (662LP, 750LT, 829PP, 857LT), and free H samples (719P, 749LT, 861LP; Supplemental Table S1), with the use of Hotstart Taq DNA polymerase (Qiagen, Hilden, Germany) and 35 cycles of amplification. Obtained PCR amplicons were visualized after electrophoresis in 3% agarose gel stained with ethidium bromide. Quantitative RT-PCR reactions were run on QuantStudio 7 Flex System (Applied Biosystems/ThermoFisher Scientific) in 45 cycles, performed in triplicate for each cDNA sample. Reactions were in a total volume of 10 μL using RT-PCR primers and Sensitive RT HS-PCR Mix EvaGreen (A&A Biotechnology, Gdynia, Poland). Quantification of mRNA levels was done using the comparative ΔΔCT method according to
). Normality of the distribution was tested using the Shapiro-Wilk test, and differences (in relative quantification values) between the CPS, CNS, and H groups of samples were calculated using the Mann Whitney U-test (results included in Table 1). Differences in expression were shown in boxplots.
Table 1P-values of fold change of expression differences between coagulase-positive staphylococci (CPS), CNS, combined CPS and CNS, and healthy (H) sample group
Prepared DNA (Sherlock A&A Biotechnology, Gdynia, Poland) was bisulfite converted (EpiTect Bisulfite Kit, Qiagen) and PCR amplified using bisulfite primers designed for converted DNA sequences in CpG islands (CGI) located upstream of the ATG5, DGAT1, and TERT genes, and inside the IGF1R gene sequence (Supplemental Table S2; https://doi.org/10.3168/jds.2020-18404). The CpG islands were defined using EMBOSS CpG Plot software (https://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/). Bisulfite (BS)PCR amplification was done with the use of Hotstart Taq DNA polymerase (Qiagen). The 2-stage protocol for BSPCR amplification (Supplemental Table S3; https://doi.org/10.3168/jds.2020-18404), included 5 cycles at 64°C and 35 cycles at 62°C using ATG5 primers, and 5 cycles at 60°C and 35 cycles at 58°C using DGAT1, IGF1R, and TERT primers. The BSPCR products were sequenced using bisulfite primers. Sanger sequencing products were separated by capillary electrophoresis on an ABI3500 xl Genetic Analyzer (ThermoFisher Scientific). Partially methylated CpG sites of inspected DNA fragments (TERT locus) were quantified. The first approach included calculation of percent of methylation (PM) values using the Mquant method (
) based on Sanger sequencing reads of BSPCR. Normality of the distribution was tested using the Shapiro-Wilk test and the differences between PM values of the CPS, CNS, and H groups were calculated on the basis of the Mann-Whitney U test (results included in Table 1).
The second approach relied on bisulfite sequencing using a cloning procedure of amplified DNA after equimolar pooling of samples representing each investigated group. Thirty nanograms of bisulfite-converted DNA from each of 18 CPS, 12 CNS, and 10 H samples were pooled (Supplemental Table S1; https://doi.org/10.3168/jds.2020-18404) and subjected to BSPCR amplification as described above in a 30-µL reaction volume. Amplicons were purified using Minelute PCR purification kit (Qiagen) and cloned using TOPO TA Cloning kit for Sequencing under manufacturer recommendations (ThermoFisher Scientific). Picked bacterial colonies (45 CPS, 27 CNS, and 18 H BSPCR colonies) were amplified with T3 and T7 universal primers, purified with ExoSaP IT (ThermoFisher Scientific), and sequenced using the Sanger method. Quantification of CpG methylation and generation of methylation patterns based on clone BSPCR sequencing reads within each of investigated group were done using BISMA software (
Table 2DNA methylation percent (PM) in TERT locus (gene ID: 518884) using the coagulase-positive staphylococci (CPS), CNS, and healthy (H) DNA bisulfite pooling approach for cloned bisulfite PCR sequencing
The relative quantification results did not reveal any significant differences in expression of DGAT1, ATG5, or IGF1R between the defined groups of udder secretory tissue samples (Figure 1). The Sanger bisulfite sequencing of these genes showed a simple pattern of either hypo- (DGAT1 and ATG5) or hypermethylation (IGF1R) of CGI in all parenchyma samples (examples shown in Supplemental Figures S1 to S3; https://doi.org/10.3168/jds.2020-18404).
The presence of clear RT-PCR product of DGAT1 of pooled cDNA samples of secretory tissue (Figure 2) was concordant with the lack of methylation of the DGAT1 upstream DNA sequence (Figure S1). Unaltered hypomethylation in DGAT1 might be associated with constant transcription of this gene, which has an embryonic background. The DGAT1 gene is crucial for mammary gland development, as found in model organisms, and its transcriptional activity is further maintained in the ontogeny, similarly to somatic tissues (
The hypomethylated state of the ATG5 upstream element (Supplemental Figure S2) coincided with the lowest amount of ATG5 transcript (Figure 2) compared with the other genes, probably because the investigated tissue sections came from lactating cows (
reported higher expression of ATG5 in the dry period due to the autophagy required for regeneration of mammary glandular tissue.
Complete methylation of the investigated element of bovine IGF1R (Supplemental Figure S3) located in the intronic sequence did not correspond to the presence of clear PCR product of IGF1R of pooled cDNA samples of mammary gland (Figure 2). In this case, the inspected DNA sequence might not be functionally relevant for the regulation of IGF1R expression, being fully methylated like most other DNA regions in mammalian genomes (
The hypermethylation of CGI (>70% of methylation) of the upstream element of TERT gene in single and pooled DNA samples of parenchyma tissue (Figure 3 and Supplemental Figure S4; https://doi.org/10.3168/jds.2020-18404) might be linked to low transcript abundance of TERT. This could be observed in the form of the weaker amplification product of pooled cDNAs compared with results of qualitative RT-PCR for DGAT1, IGF1R, and ACTB (Figure 2). A small amount of TERT mRNA has been reported in the majority of noncancerous somatic tissues (
). However, the results of quantitative RT-PCR showed a significant increase in expression of TERT in CNS samples compared with CPS samples (P = 0.04) and H samples (P = 0.02; Figure 1). Direct BSPCR sequencing and clone BSPCR sequencing results of the TERT upstream element did not show substantial differences between PM values of the CNS, CPS, and H groups of parenchyma samples (Figure 4). It is interesting that a whole-genome differential methylation study using methylated DNA immunoprecipitation (MeDIP-chip) microarray of mononuclear lymphocytes (PBMC) of Staph. aureus-infected cows revealed significant differential methylation of the bovine TERT locus between Staph. aureus-infected and control groups of animals, albeit with no difference in TERT mRNA abundance (
). In our study hypermethylation of TERT upstream element detected in infected as well as noninfected samples does not correspond to significant TERT upregulation in CNS samples in comparison to CPS and H sample group.
The TERT gene encodes a telomerase responsible for the maintenance of chromosome ends (telomeres) in the majority of somatic cells. In the pathological state, telomere shortening is associated with cellular senescence, which manifests as chronic inflammation (caused by the impairment of immune cells) that is often observed in aged individuals (
). Substantial telomere shortening is also associated with the occurrence of viral and bacterial infections, indicating that certain pathogenic microorganisms are able to alter the cell cycle process of the host (as reviewed by
). In the present report, we observed that the presence of coagulase-negative bacteria can induce TERT expression in host tissue. Based on previous articles, we hypothesize that the increase of telomerase activity caused by CNS in infected parenchyma samples might be induced by activation of genes of the MAPK signaling pathway in the cells of this secretory tissue. It is well known that Staph. aureus can increase mRNA expression of genes belonging to the MAP kinase pathway in epithelial cells due to the release of bacterial exotoxins (
). Moreover, in previous transcriptomic studies related to CPS- and CNS-infected mammary glands, upregulation of particular loci of the MAPK signaling pathway was shown in udder secretory tissue infected with coagulase-negative (FOS and EGR1 genes) and coagulase-positive bacteria (MAP3K;
). Interestingly, our results revealed that only the CNS group of bacteria could promote TERT expression in infected mammary gland. We confirmed that bacterial infection may modulate telomerase activity during inflammation via changes in TERT expression (
We conclude that regulation of expression the ATG5, DGAT1, IGF1R, and TERT genes through DNA methylation during inflammation induced by bacterial infection of bovine mammary gland is negligible. The question remains as to whether significant alterations of gene expression of TERT are associated with the molecular mechanisms underlying the etiology of bovine mastitis or are a side-effect induced primarily by bacterial metabolites.
The study was supported by the grant of The National Science Centre, Twardowskiego 16, Krakow, Poland, No. 2015/17/B/NZ9/01561. The authors have not stated any conflicts of interest.