Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

Immunomodulatory Effect of Lactobacillus reuteri (Limosilactobacillus reuteri) and Its Exopolysaccharides Investigated on Epithelial Cell Line IPEC-J2 Challenged with Salmonella Typhimurium

The gastrointestinal tract is the largest and most complex component of the immune system. Each component influences the production and regulation of cytokines secreted by intestinal epithelial cells. The aim of this study was to see how the probiotic strain Limosilactobacillus reuteri L26 and its exopolysaccharide (EPS) affect porcine intestinal-epithelial cells IPEC-J2 infected with Salmonella Typhimurium. The results revealed that Salmonella infection up-regulated all studied pro-inflammatory cytokines such as TNF-&alpha;, IL-8, IL-6 and TLR4, TLR5 signaling pathways, while decreasing the expression of TGF-&beta;. An immunosuppressive activity was found in EPS-treated wells, since the transcriptional levels of the studied pro-inflammatory cytokines were not increased, and the pretreatment with EPS was even able to attenuate up-regulated pro-inflammatory genes induced by Salmonella infection. However, there was a significant increase in the expression of mRNA levels of IL-8 and TNF-&alpha; in L26-treated cells, although this up-regulation was suppressed in the case of pretreatment. The immunoregulatory function of L. reuteri was also confirmed by the increased level of mRNA expression for TGF-&beta;, a known immunosuppressive mediator. The most relevant finding of this ex vivo study was a case of immunity modulation, where the probiotic strain L. reuteri stimulated the innate immune-cell response which displayed both anti- and pro-inflammatory activities, and modulated the expression of TLRs in the IPEC-J2 cell line. Our findings also revealed that the pretreatment of cells with either EPS or live lactobacilli prior to infection has a suppressive effect on the inflammatory response induced by Salmonella Typhimurium

Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

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Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair

Exo 1 is involved in the pathways controlled by Pso2 and Mph1-Mgm101-MutSα.

<p>(<b>A and B</b>) HN2 sensitivity of combinations of <i>pso2</i> and <i>exo1</i> disruptants, and <i>exo1-D173A</i> mutants treated with HN2. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean. (<b>C</b>) HN2-induced chromatin recruitment of Exo1-FLAG in wt, <i>pso2</i>, <i>mph1</i> and <i>pso2 mph1</i> disruptants. Mcm2 is shown as a loading control for chromatin associated protein. Chromatin bound material is labelled chr, and that from whole cell extract labelled wce. (<b>D</b>) HN2-induced chromatin recruitment of Rad51 in wild type cells, <i>pso2</i> and <i>exo1</i> single and double disruptants. Mcm2 is shown as a loading control for chromatin associated protein. Chromatin bound material is labelled chr, and that from whole cell extract labelled wce.</p

Pso2 and Mph1-Mgm101-MutSα protect against ICL-induced GCRs.

<p>(A) Left panel - schematic of the genomically-integrated (at the <i>HXT13</i> locus on chromosome V) substrate used to measure GCRs. Simultaneous loss of both the <i>CAN1</i> and <i>URA3</i> genes can only occur <i>via</i> GCRs, producing cells able to form colonies able to grow on media containing canavanine and 5-FOA. See text for further details. Right panel - GCRs rates in wild type, <i>pso2</i>, <i>mgm101</i>, <i>mph1</i> and their respective double mutants, both spontaneously occurring and induced by a sub-lethal dose of HN2 (10 mM). Rates were determined as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002884#s4" target="_blank">Materials and Methods</a> section. (B) Accumulation of DSBs in HN2-treated wild type, <i>pso2</i>, <i>pso2 msh2</i>, <i>mgm101</i>, <i>pso2 mgm101</i> and <i>pso2 mgm101 msh2</i> cells. Exponentially growing cells were treated with 100 mM HN2 for 2 hours at 28°C, or mock-treated (U) with water, and subsequently allowed to repair in minimal medium for 2, 4 and 24 hours. The U24 sample was mock-treated allowed to repair for 24 hours. Samples were analysed on PFGE gels. (C) Rad53 phosphorylation following treatment with 100 mM HN2 and up to 2 hours recovery in wild type, <i>pso2</i>, <i>msh2</i>, <i>mph1</i>, <i>pso2 msh2</i> and <i>pso2 mph1</i> mutants.</p

A model for the two major ICL processing pathways in budding yeast, one controlled by Pso2 and the other by Mph1-Mgm101-MutSα in collaboration with Exo1.

<p>See associated text for details.</p

The FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are both involved in the ICL repair pathway controlled by Mph1-Mgm101-MutSα.

<p>(<b>A–D</b>) HN2 sensitivity analysis of cells disrupted for <i>PSO2</i> and <i>MSH2</i> in combination with the FANCJ-related factor <i>CHL1</i> and FANCP related factor <i>SLX4</i>. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean.</p

Pso2 and Mph1-Mgm101-MutSα control redundant ICL repair pathways.

<p>(A to G) Analysis of the ICL sensitivity of combinations of <i>PSO2</i> with <i>MPH1</i>, <i>MGM101</i>, <i>MSH6</i> and <i>MSH2</i> gene disruptions, and an <i>mph1K113Q</i> mutant. The ICL-inducing agent was HN2. All results are the mean of at least three independent experiments and the error bars show the standard error of the mean.</p