Significance This study provides insights into the physiological role of Sel1L, an adaptor protein for the ubiquitin ligase Hrd1 in endoplasmic reticulum-associated degradation (ERAD). Using both animal and cell models, this study provides unequivocal evidence for an indispensable role of Sel1L in Hrd1 stabilization, mammalian ERAD, endoplasmic reticulum homeostasis, protein translation, and cellular and organismal survival. Moreover, generation of inducible knockout mouse and cell models deficient in both Sel1L and Hrd1 provides an unprecedented opportunity to elucidate the functional importance of this key branch of ERAD in vivo and to identify its physiological substrates.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both intramembrane hydrophilic residues of IRE1α and lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1LΔIEC) are viable and appear normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signaling in vivo by managing its protein turnover.

Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEKextracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Phenotypic and genetic traits of porcine intestinal spirochete strain P43/6/78' (= ATCC 51139T) (T = type strain), which is pathogenic and weakly beta-hemolytic, were determined in order to confirm the taxonomic position of this organism and its relationships to previously described species of intestinal spirochetes. In BHIS broth, P43/6/7ST cells had a doubling time of 1 to 2 h and grew to a maximum cell density of 2 X 10' cells per ml at 37 to 42°C. They hydrolyzed hippurate, utilized D-glucose, D-fructose, sucrose, D-trehalose, u-galactose, D-mannose, maltose, N-acetyl-D-glucosamine, D-glucosamine, pyruvate, L-fucose, D-cellobiose, and D-ribose as growth substrates, and produced acetate, butyrate, ethanol, H,, and CO, as metabolic products. They consumed substrate amounts of oxygen and had a G+C content (24.6 mol%) similar to that of Serpulina hyodysenteriue B7ST (25.9 mol%). Phenotypic traits that could be used to distinguish strain P43/6/78' from S. hyodysenteriue and Serpulina innocens included its ultrastructural appearance (each strain P43/6/78' cell had 8 or 10 periplasmic flagella, with 4 or 5 flagella inserted at each end, and the cells were thinner and shorter and had more pointed ends than S. hyodysenteriae and S. innocens cells), its faster growth rate in liquid media, its hydrolysis of hippurate, its lack of P-glucosidase activity, and its metabolism of D-ribose. DNA-DNA relative reassociation experiments in which the Sl nuclease method was used revealed that P43/6/7ST was related to, but was genetically distinct from, both S. hyodysenteriae B78' (level of sequence homology, 25 to 32%) and S. innocens B256T (level of sequence homology, 24 to 25%). These and previous results indicate that intestinal spirochete strain P43/6/7ST represents a distinct Serpulina species. Therefore, we propose that strain P43/6/78 should be designated as the type strain of a new species, Serpulina pilosicoli.Porcine intestinal spirochetosis or spirochetal diarrhea is a disease of swine that are between 4 and 20 weeks old but typically occurs within 7 to 14 days after weaning (7, 39). The clinical signs of this disease include mucus-containing, usually nonbloody diarrhea; poor feed conversion; and depressed growth rates (1, 7, 31,40). The characteristic histological feature that distinguishes porcine intestinal spirochetosis is a dense mat or false brush border of spirochete cells which are closely packed parallel to one another and are attached by one end to the colonic epithelium (4, 7, 14,40). Such cells are not typical of infections caused by Serpulina hyodysenteriae (the agent of swine dysentery), but have been found in humans colonized by intestinal spirochetes, where their clinical significance is unclear (8, 22).The etiologic agent of porcine intestinal spirochetosis was first described in 1980 by Taylor et al. (40). These investigators successfully isolated an intestinal spirochete, designated strain P43/6/78T (T = type strain), and reproduced clinical signs and lesions typical of the disease in pigs that were o...