Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Differential Cytotoxicity of Curcumin-Loaded Micelles on Human Tumor and Stromal Cells.

Although curcumin in the form of nanoparticles has been demonstrated as a potential anti-tumor compound, the impact of curcumin and nanocurcumin in vitro on normal cells and in vivo in animal models is largely unknown. This study evaluated the toxicity of curcumin-loaded micelles in vitro and in vivo on several tumor cell lines, primary stromal cells, and zebrafish embryos. Breast tumor cell line (MCF7) and stromal cells (human umbilical cord vein endothelial cells, human fibroblasts, and human umbilical cord-derived mesenchymal stem cells) were used in this study. A zebrafish embryotoxicity (FET) assay was conducted following the Organisation for Economic Co-operation and Development (OECD) Test 236. Compared to free curcumin, curcumin PM showed higher cytotoxicity to MCF7 cells in both monolayer culture and multicellular tumor spheroids. The curcumin-loaded micelles efficiently penetrated the MCF7 spheroids and induced apoptosis. The nanocurcumin reduced the viability and disturbed the function of stromal cells by suppressing cell migration and tube formation. The micelles demonstrated toxicity to the development of zebrafish embryos. Curcumin-loaded micelles demonstrated toxicity to both tumor and normal primary stromal cells and zebrafish embryos, indicating that the use of nanocurcumin in cancer treatment should be carefully investigated and controlled.

Colibactin-Producing Escherichia coli Induce the Formation of Invasive Carcinomas in a Chronic Inflammation-Associated Mouse Model.

BACKGROUND: Escherichia coli producing the genotoxin colibactin (CoPEC or colibactin-producing E. coli) abnormally colonize the colonic mucosa of colorectal cancer (CRC) patients. We previously showed that deficiency of autophagy in intestinal epithelial cells (IECs) enhances CoPEC-induced colorectal carcinogenesis in ApcMin/+ mice. Here, we tested if CoPEC trigger tumorigenesis in a mouse model lacking genetic susceptibility or the use of carcinogen. METHODS: Mice with autophagy deficiency in IECs (Atg16l1∆IEC) or wild-type mice (Atg16l1flox/flox) were infected with the CoPEC 11G5 strain or the mutant 11G5∆clbQ incapable of producing colibactin and subjected to 12 cycles of DSS treatment to induce chronic colitis. Mouse colons were used for histological assessment, immunohistochemical and immunoblot analyses for DNA damage marker. Results: 11G5 or 11G5∆clbQ infection increased clinical and histological inflammation scores, and these were further enhanced by IEC-specific autophagy deficiency. 11G5 infection, but not 11G5∆clbQ infection, triggered the formation of invasive carcinomas, and this was further increased by autophagy deficiency. The increase in invasive carcinomas was correlated with enhanced DNA damage and independent of inflammation. Conclusions: CoPEC induce colorectal carcinogenesis in a CRC mouse model lacking genetic susceptibility and carcinogen. This work highlights the role of (i) CoPEC as a driver of CRC development, and (ii) autophagy in inhibiting the carcinogenic properties of CoPEC.

Autophagy of Intestinal Epithelial Cells Inhibits Colorectal Carcinogenesis Induced by Colibactin-Producing Escherichia coli in ApcMin/+ Mice.

BACKGROUND & AIMS: Colibactin-producing Escherichia coli (CoPEC) colonize the colonic mucosa of a higher proportion of patients with vs without colorectal cancer (CRC) and promote colorectal carcinogenesis in susceptible mouse models of CRC. Autophagy degrades cytoplasmic contents, including intracellular pathogens, via lysosomes and regulates intestinal homeostasis. We investigated whether inhibiting autophagy affects colorectal carcinogenesis in susceptible mice infected with CoPEC. METHODS: Human intestinal epithelial cells (IECs) (HCT-116) were infected with a strain of CoPEC (11G5 strain) isolated from a patient or a mutant strain that does not produce colibactin (11G5ΔclbQ). Levels of ATG5, ATG16L1, and SQSTM1 (also called p62) were knocked down in HCT-116 cells using small interfering RNAs. ApcMin/+ mice and ApcMin/+ mice with IEC-specific disruption of Atg16l1 (ApcMin/+/Atg16l1ΔIEC) were infected with 11G5 or 11G5ΔclbQ. Colonic tissues were collected from mice and analyzed for tumor size and number and by immunohistochemical staining, immunoblot, and quantitative reverse transcription polymerase chain reaction for markers of autophagy, DNA damage, cell proliferation, and inflammation. We analyzed levels of messenger RNAs (mRNAs) encoding proteins involved in autophagy in colonic mucosal tissues from patients with sporadic CRC colonized with vs without CoPEC by quantitative reverse-transcription polymerase chain reaction. RESULTS: Patient colonic mucosa with CoPEC colonization had higher levels of mRNAs encoding proteins involved in autophagy than colonic mucosa without these bacteria. Infection of cultured IECs with 11G5 induced autophagy and DNA damage repair, whereas infection with 11G5ΔclbQ did not. Knockdown of ATG5 in HCT-116 cells increased numbers of intracellular 11G5, secretion of interleukin (IL) 6 and IL8, and markers of DNA double-strand breaks but reduced markers of DNA repair, indicating that autophagy is required for bacteria-induced DNA damage repair. Knockdown of ATG5 in HCT-116 cells increased 11G5-induced senescence, promoting proliferation of uninfected cells. Under uninfected condition, ApcMin/+/Atg16l1ΔIEC mice developed fewer and smaller colon tumors than ApcMin/+ mice. However, after infection with 11G5, ApcMin/+/Atg16l1ΔIEC mice developed more and larger tumors, with a significant increase in mean histologic score, than infected ApcMin/+ mice. Increased levels of Il6, Tnf, and Cxcl1 mRNAs, decreased level of Il10 mRNA, and increased markers of DNA double-strand breaks and proliferation were observed in the colonic mucosa of 11G5-infected ApcMin/+/Atg16l1ΔIEC mice vs 11G5-infected ApcMin/+ mice. CONCLUSION: Infection of IECs and susceptible mice with CoPEC promotes autophagy, which is required to prevent colorectal tumorigenesis. Loss of ATG16L1 from IECs increases markers of inflammation, DNA damage, and cell proliferation and increases colorectal tumorigenesis in 11G5-infected ApcMin/+ mice. These findings indicate the importance of autophagy in response to CoPEC infection, and strategies to induce autophagy might be developed for patients with CRC and CoPEC colonization.

Phosphorylation of the transcriptional regulator MYB44 by mitogen activated protein kinase regulates Arabidopsis seed germination.

The phytohormones abscisic acid (ABA) and gibberellic acid (GA) have antagonistic roles in the control of seed germination and seedling development. We report here that the transcriptional regulator MYB44 has a role in the control of seed germination in Arabidopsis thaliana. High levels of the MYB44 transcript are found in dry seeds but the transcript levels decrease during germination. The decrease in transcript level during germination is inhibited by the GA biosynthesis inhibitor paclobutrazol (PAC). MYB44 is phosphorylated by both recombinant and native forms of MPK3 and MPK6 at Ser(53) and Ser(145). Transgenic overexpression of MYB44 results in increased sensitivity of seed germination to ABA or PAC treatment. The PAC-insensitive germination phenotype of the myb44 mutant is complemented by overexpression of wild type MYB44 but not by overexpression of a mutant protein that lacks the MPK-target serines indicating that phosphorylation of MYB44 by MPKs is required for its biological function.

Phosphorylation by AtMPK6 is required for the biological function of AtMYB41 in Arabidopsis.

Mitogen-activated protein kinases (MPKs) are involved in a number of signaling pathways that control plant development and stress tolerance via the phosphorylation of target molecules. However, so far only a limited number of target molecules have been identified. Here, we provide evidence that MYB41 represents a new target of MPK6. MYB41 interacts with MPK6 not only in vitro but also in planta. MYB41 was phosphorylated by recombinant MPK6 as well as by plant MPK6. Ser(251) in MYB41 was identified as the site phosphorylated by MPK6. The phosphorylation of MYB41 by MPK6 enhanced its DNA binding to the promoter of a LTP gene. Interestingly, transgenic plants over-expressing MYB41(WT) showed enhanced salt tolerance, whereas transgenic plants over-expressing MYB41(S251A) showed decreased salt tolerance during seed germination and initial root growth. These results indicate that the phosphorylation of MYB41 by MPK6 is required for the biological function of MYB41 in salt tolerance.

Identification of a C2H2-type zinc finger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase.

Mitogen-activated protein kinases (MAPKs or MPKs) are one of the most important and conserved signaling molecules in plants. MPKs can directly modulate gene expression by the phosphorylation of transcription factors. However, only a few target substrates of MPKs have been isolated. Here, we identified a C(2)H(2)-type zinc finger transcription factor from Arabidopsis, ZAT10, as a substrate of MPKs. Using in vitro and in vivo protein-protein interaction analyses, we demonstrated that ZAT10 directly interacted with MPK3 and MPK6. ZAT10 was phosphorylated by recombinant Arabidopsis MPK3 and MPK6 in a kinase assay. Furthermore, ZAT10 was also phosphorylated by native MPK3 and MPK6 prepared from Arabidopsis plants in an in-gel kinase assay. Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. These sites were verified by site-directed mutagenesis and in vitro kinase assays.

Pathogen inducible voltage-dependent anion channel (AtVDAC) isoforms are localized to mitochondria membrane in Arabidopsis.

Voltage-dependent anion channels (VDACs) are reported to be porin-type, beta-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.