DNA-Fragments Are Transcytosed across CaCo-2 Cells by Adsorptive Endocytosis and Vesicular Mediated Transport

<div><p>Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >10³ fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to <em>in vivo</em> conditions it could be hypothesized that this transport mechanism has a function in the immune system.</p> </div

Effect of inhibition of endocytosis on transcytosis of DNA-fragment (5 nM).

<p>Transcytosis of DNA fragments and Lucifer yellow (LY) in the apical to basolateral direction were quantified as described in Materials and Methods and tested statistically with a linear mixed-effects model. Inhibition factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). Control for ice-treatment is 37 °C and control for bafilomycin A1 (BafA1)-treatment is with vehicle (DMSO). TEER = trans-epithelial electric resistance.</p

Trans-epithelial electric resistance (TEER) in the presence of compounds increasing Lucifer yellow (LY)-transport.

<p>TEER was measured at the end of the experiment. Deviation (dev) is calculated as the absolute value of the difference between the two experiments divided by 2. The number <i>n</i> is the number of independent experiments and number <i>m</i> is the total number of wells analysed (observations). CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p

Competition for transcytosis of DNA-fragment (5 nM) by nucleic acids and anionic compounds.

<p>Transcytosis of DNA fragments in the apical to basolateral direction with (treated) and without (control) competitor were quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG and GpC ODN = short oligonucleotides rich in CpG and GpC dinucleotide motifs, respectively.</p

Transcytosis of Lucifer yellow (LY) in the presence of compounds affecting transcytosis of DNA-fragment.

<p>Transcytosis of LY in the apical to basolateral direction with (treated) and without (control) competitor was quantified after 90 minutes of incubation as described in Materials and Methods and tested statistically with a linear mixed-effects model. Factor is calculated as control divided by treated. The number <i>n</i> is the number of independent experiments and the number <i>m</i> is the total number of wells analysed (observations). pUC19 lin = linearized pUC19 plasmid. CpG ODN = short oligonucleotide rich in CG dinucleotide motifs. Control for Cytochalasin D (CytD)-treatment is with vehicle (DMSO).</p

Differentiation of CaCo-2 cells.

<p>A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm²) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.</p

Supplement 1. S-Plus code used to generate Figs. 2-8 and Table 2.

<h2>File List</h2><blockquote> <p>S-Plus code:</p> <p> <a href="estimation.txt">estimation.q</a><br> <a href="testing.txt">testing.q</a><br> <a href="plotting.txt">plotting.q</a><br> </p> <p>Download all files at once: <a href="blowfly-code.zip">blowfly-code.zip</a></p> <p>All individual files are in ASCII text. The .zip file requires WinZip to extract. </p> </blockquote><h2>Description</h2>Following are the S-Plus files (in ascii format) that were used in conjunction with the time series described in the paper to generate Figs. 2-8 and Table 2. The file estimation.q contains the code to estimate all functions and parameters in the model in Eqs. 5-10. After execution the list fit.all contains estimates found on the basis of individual populations, whereas the list fit.cmb contains estimates found on the basis of combining the replicates. Note that the S-Plus code in estimation.q has to be executed before executing the other S-Plus files described below. <p> The file testing.q contains the code to generate the entries of Table 2, i.e., code for applying the Mann-Whitney <i>U</i> test and the Kolmogorov-Smirnov test to estimated parameters as described in the paper. </p><p> The file plotting.q contains the code to generate Figs. 2-8 based on the estimates found using estimation.q. </p

Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

<div><p>Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (<i>Salmo salar</i>). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q₂₆₆→L₂₆₆ substitution or insertion next to the putative cleavage site (R₂₆₇) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L₂₆₆ codons in screening samples and Q₂₆₆ codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.</p> </div