Equine bronchial epithelial cells are susceptible to cell entry with a SARS-CoV-2 pseudovirus but reveal low replication efficiency.

OBJECTIVE: To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). SAMPLE: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. METHODS: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were transduced with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media for 96 hours. After 96 hours, eGFP expression in EBECs was assessed by fluorescence microscopy of cell cultures and quantitative PCR. RESULTS: ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean ΔCt values from quantitative PCR were significantly (P < .0001) higher in EBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. CLINICAL RELEVANCE: Equine respiratory tract cells were susceptible to cell entry with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infective to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.

Serum 3-phenyllactic acid level is reduced in benign multiple sclerosis and is associated with effector B cell ratios.

BACKGROUND: 3-phenyllactic acid (PLA) is produced by both intestinal bacteria and the human host. PLA exists in its D- and L- chiral forms. It modulates human immune functions, thereby acting as a mediator of bacterial-host interactions. We aim to determine the amount and potential influence of PLA on clinical and immunological features of MS. METHODS: We measured D- and L-PLA levels in bacterial supernatants and in sera of 60 MS patients and 25 healthy controls. We investigated potential associations between PLA levels, clinical features of MS, serum cytokine levels and ratios of peripheral blood lymphocyte subsets. RESULTS: Multiple gut commensal bacteria possessed the capacity to generate D- and L-PLA. MS patients with benign phenotype showed markedly lower PLA levels than healthy controls or other MS patients. Fingolimod resistant patients had higher PLA levels at baseline. Furthermore, MS patients with higher PLA levels tended to display increased memory B and plasma cell ratios, elevated IL-4 levels and increased ratios of IL-4 and IL-10 producing T cell subsets. CONCLUSION: Collectively, our work indicates that reduced serum levels of PLA could be associated with a favorable clinical course in MS and possibly be used as a biomarker.

Peppermint oil effects on the gut microbiome in children with functional abdominal pain.

Peppermint oil (PMO) is effective in the treatment of functional abdominal pain disorders, but its mechanism of action is unclear. Evidence suggests PMO has microbicidal activity. We investigated the effect of three different doses of PMO on gut microbiome composition. Thirty children (7-12 years of age) with functional abdominal pain provided a baseline stool sample prior to randomization to 180, 360, or 540 mg of enteric coated PMO (10 participants per dose). They took their respective dose of PMO (180 mg once, 180 mg twice, or 180 mg thrice daily) for 1 week, after which the stool collection was repeated. Baseline and post-PMO stools were analyzed for microbiome composition. There was no difference in alpha diversity of the gut microbiome between the baseline and post-PMO treatment. Principal coordinate analysis revealed no significant difference in overall bacterial composition between baseline and post-PMO samples, as well as between the PMO dose groups. However, the very low abundant Collinsella genus and three operational taxonomic units (one belonging to Collinsella) were significantly different in samples before and after PMO treatment. The Firmicutes/Bacteroidetes ratio was lower in children who received 540 mg of PMO compared to the 180 mg and 360 mg dose groups (p = 0.04). Network analysis revealed separation between pre- and post-PMO fecal samples with the genus Collinsella driving the post-PMO clusters. PMO administration appeared to impact only low abundance bacteria. The 540 mg PMO dose differentially impacted the Firmicutes/Bacteroidetes ratio. A higher dose and/or longer duration of treatment might yield different results.

ClC transporter activity modulates histidine catabolism in Lactobacillus reuteri by altering intracellular pH and membrane potential.

BACKGROUND: Histamine is a key mediator of the anti-inflammatory activity conferred by the probiotic organism Lactobacillus reuteri ATCC PTA 6475 in animal models of colitis and colorectal cancer. In L. reuteri, histamine synthesis and secretion requires L-histidine decarboxylase and a L-histidine/histamine exchanger. Chloride channel (ClC)-family proton/chloride antiporters have been proposed to act as electrochemical shunts in conjunction with amino acid decarboxylase systems, correcting ion imbalances generated by decarboxylation through fixed ratio exchange of two chloride ions for one proton. This family is unique among transporters by facilitating ion flux in either direction. Here we examine the histidine decarboxylase system in relation to ClC antiporters in the probiotic organism Lactobacillus reuteri. RESULTS: In silico analyses reveal that L. reuteri possesses two ClC transporters, EriC and EriC2, as well as a complete histidine decarboxylase gene cluster (HDC) for the synthesis and export of histamine. When the transport activity of either proton/chloride antiporter is disrupted by genetic manipulation, bacterial histamine output is reduced. Using fluorescent reporter assays, we further show that ClC transporters affect histamine output by altering intracellular pH and membrane potential. ClC transport also alters the expression and activity of two key HDC genes: the histidine decarboxylase (hdcA) and the histidine/histamine exchanger (hdcP). CONCLUSIONS: Histamine production is a potentially beneficial feature for intestinal microbes by promoting long-term colonization and suppression of inflammation and host immune responses. ClC transporters may serve as tunable modulators for histamine production by L. reuteri and other gut microbes.

Distinct Microbiome-Neuroimmune Signatures Correlate With Functional Abdominal Pain in Children With Autism Spectrum Disorder.

BACKGROUND & AIMS: Emerging data on the gut microbiome in autism spectrum disorder (ASD) suggest that altered host-microbe interactions may contribute to disease symptoms. Although gut microbial communities in children with ASD are reported to differ from individuals with neurotypical development, it is not known whether these bacteria induce pathogenic neuroimmune signals. METHODS: Because commensal clostridia interactions with the intestinal mucosa can regulate disease-associated cytokine and serotonergic pathways in animal models, we evaluated whether microbiome-neuroimmune profiles (from rectal biopsy specimens and blood) differed in ASD children with functional gastrointestinal disorders (ASD-FGID, n = 14) compared with neurotypical (NT) children with FGID (NT-FGID, n = 15) and without abdominal pain (NT, n = 6). Microbial 16S ribosomal DNA community signatures, cytokines, and serotonergic metabolites were quantified and correlated with gastrointestinal symptoms. RESULTS: A significant increase in several mucosa-associated Clostridiales was observed in ASD-FGID, whereas marked decreases in Dorea and Blautia, as well as Sutterella, were evident. Stratification by abdominal pain showed multiple organisms in ASD-FGID that correlated significantly with cytokines (interleukin [IL]6, IL1, IL17A, and interferon-γ). Group comparisons showed that IL6 and tryptophan release by mucosal biopsy specimens was highest in ASD children with abdominal pain, whereas serotonergic metabolites generally were increased in children with FGIDs. Furthermore, proinflammatory cytokines correlated significantly with several Clostridiales previously reported to associate with ASD, as did tryptophan and serotonin. CONCLUSIONS: Our findings identify distinctive mucosal microbial signatures in ASD children with FGID that correlate with cytokine and tryptophan homeostasis. Future studies are needed to establish whether these disease-associated Clostridiales species confer early pathogenic signals in children with ASD and FGID.

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion.

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins.

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP(6)- and inositol pyrophosphate (InsP(7))-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP(6) enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

The heads of the measles virus attachment protein move to transmit the fusion-triggering signal.

The measles virus entry system, consisting of attachment (hemagglutinin, H) and fusion proteins, operates by means of a variety of natural and targeted receptors; however, the mechanism that triggers fusion of the viral envelope with the plasma membrane is not understood. Here, we tested a model proposing that the two heads of an H dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface with additional intermolecular disulfide bonds prevented membrane fusion, an effect that was reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided that it engaged the H dimer at locations proximal to where the natural receptors bind and distal to the H-dimer interface. We discuss how receptors may force H-protein heads to switch partners and transmit the fusion-triggering signal.

NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU).

Picornaviruses have a 22-24 amino acid peptide, VPg, bound covalently at the 5' end of their RNA, that is essential for replication. VPgs are uridylylated at a conserved tyrosine to form VPgpU, the primer of RNA synthesis by the viral polymerase. This first complete structure for any uridylylated VPg, of poliovirus type 1 (PV1)-VPgpU, shows that conserved amino acids in VPg stabilize the bound UMP, with the uridine atoms involved in base pairing and chain elongation projected outward. Comparing this structure to PV1-VPg and partial structures of VPg/VPgpU from other picornaviruses suggests that enteroviral polymerases require a more stable VPg structure than does the distantly related aphthovirus, foot and mouth disease virus (FMDV). The glutamine residue at the C-terminus of PV1-VPgpU lies in back of the uridine base and may stabilize its position during chain elongation and/or contribute to base specificity. Under in vivo-like conditions with the authentic cre(2C) hairpin RNA and Mg(2+), 5-methylUTP cannot compete with UTP for VPg uridylyation in an in vitro uridylyation assay, but both nucleotides are equally incorporated by PV1-polymerase with Mn(2+) and a poly-A RNA template. This indicates the 5 position is recognized under in vivo conditions. The compact VPgpU structure docks within the active site cavity of the PV-polymerase, close to the position seen for the fragment of FMDV-VPgpU with its polymerase. This structure could aid in design of novel enterovirus inhibitors, and stabilization upon uridylylation may also be pertinent for post-translational uridylylation reactions that underlie other biological processes.

Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed.

The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.

Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1.

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3' blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg(2+). However, the Ser99 mutant has normal activity in 2 mM Mg(2+). Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg(2+), with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg(2+). This effect due to the mutation of Cys99, approximately 16 A from the active site, on the DNA binding and activity is surprising. Mg(2+) at the B site promotes stabilization of the C99S mutant complex. At higher Mg(2+) concentration the A site is also filled, causing the B-site Mg(2+) to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5' site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg(2+)]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.

Dynamic interaction of the measles virus hemagglutinin with its receptor signaling lymphocytic activation molecule (SLAM, CD150).

The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.

NMR structure of the viral peptide linked to the genome (VPg) of poliovirus.

VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 A RMSD (versus 6.6 A in buffer alone), with 10-30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.