Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn''s disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH _LF82 (expressing FimH with an AIEC-associated mutation) with fimH _K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.     

Deletion of the mouse meprin beta metalloprotease gene diminishes the ability of leukocytes to disseminate through extracellular matrix

Meprins are metalloendopeptidases expressed by leukocytes in the lamina propria of the human inflamed bowel, that degrade extracellular matrix proteins in vitro implicating them in leukocyte transmigration events. The aims of these studies were to 1) examine the expression of meprins in the mouse mesenteric lymph node, 2) determine whether macrophages express meprins, and 3) determine whether deletion of the meprin beta gene (Mep-1beta) mitigated the ability of leukocytes to disseminate through extracellular matrix in vitro. These studies show that meprin alpha and beta are expressed in leukocytes of the mouse mesenteric lymph node, and meprin alpha, but not beta, decreased during intestinal inflammation. Deletion of Mep-1beta gene decreased the ability of leukocytes to migrate through matrigel compared with wild-type leukocytes. Meprin beta, but not alpha, was detected in cortical and medullary macrophages of the lymph node. Thus overall, meprin beta is expressed by leukocytes in the draining lymph node of the intestine, regardless of the inflammatory status of the animal, and is likely to contribute to leukocyte transmigration events important to intestinal immune responses. Thus, the expression of meprins by leukocytes of the intestinal immune system may have important implications for diseases such as inflammatory bowel diseases, which are aggravated by leukocyte infiltration.     

Meprin mRNA in rat intestine during normal and glucocorticoid-induced maturation: divergent patterns of expression of alpha and beta subunits

Meprin is a zinc-metalloendopeptidase expressed in intestinal epithelial cells. In rat jejunum collected from postnatal day 4 (P4) through P25 meprins alpha mRNA exhibited uniform levels for the first three postnatal weeks and then declined, whereas meprin beta mRNA showed a biphasic pattern with high levels in the first postnatal week followed by low levels from P7 through P19 and then a marked rise at P22 and P25. Dexamethasone treatment beginning at P10 had no significant effect on levels of meprins a mRNA, whereas this treatment caused a precocious increase in expression of meprin beta mRNA. These divergent patterns of expression of meprins alpha and beta mRNA suggest distinct roles for the two subunits during the suckling and weaning phases of rodent intestinal development.     

Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.     

Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu(744)-Val(745). Recombinant forms of rat meprin B and homomeric mouse meprin A had K(m) values for villin and actin of ～1 μM (0.6-1.2 μM). The k(cat) values varied substantially (0.6-128 s(-1)), resulting in different efficiencies for cleavage, with meprin B having the highest k(cat)/K(m) values (128 M(-1)·s(-1) × 10(6)). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.     

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.     

Origin and Diversification of Meprin Proteases

Meprins are astacin metalloproteases with a characteristic, easily recognizable structure, given that they are the only proteases with both MAM and MATH domains plus a transmembrane region. So far assumed to be vertebrate-specific, it is shown here, using a combination of evolutionary and genomic analyses, that meprins originated before the urochordates/vertebrates split. In particular, three genes encoding structurally typical meprin proteins are arranged in tandem in the genome of the urochordate Ciona intestinalis. Phylogenetic analyses showed that the protease and MATH domains present in the meprin-like proteins encoded by the Ciona genes are very similar in sequence to the domains found in vertebrate meprins, which supports them having a common origin. While many vertebrates have the two canonical meprin-encoding genes orthologous to human MEP1A and MEP1B (which respectively encode for the proteins known as meprin α and meprin β), a single gene has been found so far in the genome of the chondrichthyan fish Callorhinchus milii, and additional meprin-encoding genes are present in some species. Particularly, a group of bony fish species have genes encoding highly divergent meprins, here named meprin-F. Genes encoding meprin-F proteins, derived from MEP1B genes, are abundant in some species, as the Amazon molly, Poecilia formosa, which has 7 of them. Finally, it is confirmed that the MATH domains of meprins are very similar to the ones in TRAF ubiquitin ligases, which suggests that meprins originated when protease and TRAF E3-encoding sequences were combined.     

Probing the active sites and mechanisms of rat metalloproteases meprin A and B

Meprin A and B are highly regulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain alpha and beta subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physico-chemical factors. Homo-oligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homo-oligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.     

Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers,tetramers, and high molecular mass multimers

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin alpha homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.     

OmpC and the sigma(E) regulatory pathway are involved in adhesion and invasion of the Crohn's disease-associated Escherichia coli strain LF82

Ileal lesions of 36.4% of patients with Crohn's disease (CD), an inflammatory bowel disease in humans, are colonized by pathogenic adherent-invasive Escherichia coli (AIEC), and high levels of antibodies directed against E. coli OmpC are present in 37-55% of CD patients. We therefore investigated the expression of OmpC and its role in the interaction of CD-associated adherent-invasive E. coli strain LF82 with intestinal epithelial cells. High osmolarity induced a significant increase in the ability of LF82 bacteria to interact with Intestine-407 cells, which correlates with increased OmpC expression. Deletion of ompC gene markedly decreased the adhesion and invasion levels of the corresponding mutant. A LF82-DeltaompR mutant impaired in OmpC and OmpF expression, showed decreased adhesion and invasion, and unlike a K-12-negative OmpR mutant did not express flagella and type 1 pili. Interestingly, the wild-type phenotype was restored when OmpC or OmpF expression was induced in the LF82-DeltaompR mutant. Overexpression of RpoE in the LF82-DeltaompR isogenic mutant restored a full wild-type phenotype without restoring OmpC expression. Increased expression of RpoE was observed in wild-type strain LF82 at high osmolarity. Hence, the role of OmpC in the AIEC LF82 adhesion and invasion is indirect and involves the sigma(E) regulatory pathway.     

Mannan-binding protein blocks the activation of metalloproteases meprin alpha and beta

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.     

Targeted disruption of the meprin beta gene in mice leads to underrepresentation of knockout mice and changes in renal gene expression profiles

Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded alpha and/or beta subunits. Homooligomers of meprin alpha are secreted; oligomers containing meprin beta are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin beta is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin beta null mice were generated by targeted disruption of the meprin beta gene on mouse chromosome 18q12. Analyses of meprin beta knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin beta knockout mice lack membrane-associated meprin alpha in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin beta allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin alpha in kidney and intestine.     

Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.     

Prointerleukin-18 is activated by meprin beta in vitro and in vivo in intestinal inflammation

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin beta subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51-Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 microm and in Madin-Darby canine kidney cells transfected with meprin beta when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-kappaB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin beta knock-out (betaKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin betaKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin alphaKO mice, which express meprin beta but not alpha, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin beta and implicates meprins in the modulation of inflammation.