Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

Inside-out Signalling From Aminopeptidase N (CD13) To Complement Receptor 3 (CR3, CD11b/CD18)

Upon ligand engagement, certain receptors can activate an integrin through a mechanism called inside-out signalling. This phenomenon prepares the cell for the next steps of the process it will perform. CR3 (Complement receptor 3), the most abundant β2 integrin in monocytes and macrophages, and CD13 (aminopeptidase N) are two immune receptors with overlapping activities: adhesion, phagocytosis of opsonized particles, and respiratory burst induction. They can be found together in functional signalling microdomains, or lipid rafts, on the surface of human leukocytes. Thus, given their common functions, shared physical location and the fact that some phagocytic and adhesion receptors activate a selection of integrins, we hypothesized that CD13 could activate CR3 through an inside-out signalling mechanism. To test this hypothesis, we first ascertained the activation of CR3 after CD13 crosslinking in human monocyte-derived macrophages. We used an integrated analysis of bioinformatics and experimental data to suggest two possible signalling cascades that could explain the phenomenon. Finally, we show that the non-receptor tyrosine kinase Syk is a key attenuator of this pathway. Our results demonstrated that, even in the absence of canonical signalling motifs, and despite having a noticeably short cytoplasmic tail (7-10 amino acids), CD13 was capable of triggering an inside-out signalling cascade, adding a new function to those already known for this moonlighting protein.

Antibody-Mediated Targeting of Antigens to Intestinal Aminopeptidase N Elicits Gut IgA Responses in Pigs

Many pathogens enter the host via the gut, causing disease in animals and humans. A robust intestinal immune response is necessary to protect the host from these gut pathogens. Despite being best suited for eliciting intestinal immunity, oral vaccination remains a challenge due to the gastrointestinal environment, a poor uptake of vaccine antigens by the intestinal epithelium and the tolerogenic environment pervading the gut. To improve uptake, efforts have focused on targeting antigens towards the gut mucosa. An interesting target is aminopeptidase N (APN), a conserved membrane protein present on small intestinal epithelial cells shown to mediate epithelial transcytosis. Here, we aimed to further optimize this oral vaccination strategy in a large animal model. Porcine APN-specific monoclonal antibodies were generated and the most promising candidate in terms of epithelial transcytosis was selected to generate antibody fusion constructs, comprising a murine IgG1 or porcine IgA backbone and a low immunogenic antigen: the F18-fimbriated E. coli tip adhesin FedF. Upon oral delivery of these recombinant antibodies in piglets, both mucosal and systemic immune responses were elicited. The presence of the FedF antigen however appeared to reduce these immune responses. Further analysis showed that F18 fimbriae were able to disrupt the antigen presenting capacity of intestinal antigen presenting cells, implying potential tolerogenic effects of FedF. Altogether, these findings show that targeted delivery of molecules to epithelial aminopeptidase N results in their transcytosis and delivery to the gut immune systems. The results provide a solid foundation for the development of oral subunit vaccines to protect against gut pathogens.

The roles of two major domains of the porcine deltacoronavirus spike subunit 1 in receptor binding and neutralization

Determination of the mechanisms of interspecies transmission is of great significance for the prevention of epidemic diseases caused by emerging coronaviruses (CoVs). Recently, porcine deltacoronavirus (PDCoV) was shown to exhibit broad host-cell range mediated by surface expression of aminopeptidase N (APN), and humans have been reported to be at risk of PDCoV infection. In the present study, we first demonstrated that overexpression of APN orthologues from various species including mice and felines in the APN-deficient swine small intestine epithelial cells permitted PDCoV infection, confirming that APN broadly facilitates PDCoV cellular entry and perhaps subsequent interspecies transmission. PDCoV was able to limitedly infect mice in vivo , distributing mainly in enteric and lymphoid tissues, suggesting that mice may serve as a susceptible reservoir of PDCoV. Furthermore, elements (two glycosylation sites and four aromatic amino acids) on the surface of domain B (S1 B ) of the PDCoV spike glycoprotein S1 subunit were identified to be critical for cellular surface binding of APN orthologues. However, both the domain A (S1 A ) and S1 B were able to elicit potent neutralizing antibodies against PDCoV infection. The antibodies against S1 A inhibited the hemagglutination activity of PDCoV using erythrocytes from various species, which might account for the neutralizing capacity of S1 A antibodies partially through a blockage of sialic acid binding. The study reveals the tremendous potential of PDCoV for interspecies transmission and the role of two major PDCoV S1 domains in receptor binding and neutralization, providing a theoretical basis for development of intervention strategies. Importance Coronaviruses exhibit a tendency for recombination and mutation, which enables them to quickly adapt to various novel hosts. Previously, orthologues of aminopeptidase N (APN) from mammalian and avian species were found to be associated with porcine deltacoronavirus (PDCoV) cellular entry in vitro . Here we provide in vivo evidence that mice are susceptible to PDCoV limited infection. We also show that two major domains (S1 A and S1 B ) of the PDCoV spike glycoprotein involved in APN receptor binding can elicit neutralizing antibodies, identifying two glycosylation sites and four aromatic amino acids on the surface of the S1 B domain critical for APN binding, and demonstrating neutralization activity of S1 A antibodies is partially attributed to blockage of sugar binding activity. Our findings further implicate PDCoV’s great potential for interspecies transmission, and the data of receptor binding and neutralization may provide a basis for development of future intervention strategies.

Abstract MP29: A Cloned Anti-aminopeptidase N Scfv Antibody Blocks Enzymatic Activity And Rescues A Low Sodium Phenotype In Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants

Inverse Salt Sensitivity (ISS), defined as the paradoxical increase in blood pressure of individuals on a low sodium diet compared to a high sodium diet, may be associated with the increase in mortality and morbidity found in individuals on a low sodium diet. Our group has previously found that urine derived human renal proximal tubule cells (RPTC) isolated from ISS participants express higher Aminopeptidase N (APN) protein than cells isolated from salt resistant (SR) participants. An anti-Aminopeptidase N (APN) hybridoma was used construct a single chain variable fragment (scFv) into a bacterial expression system. The purified anti-APN scFv bound to live human renal proximal tubule cells, detected using an Alexa594 directly labelled anti-myc monoclonal antibody by immunofluorescent confocal microscopy. The anti-APN scFv was then tested to determine if it blocks APN enzymatic activity. We have previously shown that under low sodium conditions the amount of reduced-glutathione as determined by live cell fluorescence staining with the redox sensitive dye, monochlorobimane (mBCl), is decreased only in ISS cells under low sodium conditions at the two-hour time point. We hypothesized that the decreased mBCl signal may be due to the increased APN expression and activity altering the Ang II/Ang III ratio and reducing the Ang III activation of the AT 2 R. Low sodium reduced the mBCl signal in ISS (-29.2±4.3%, ISS vs SR, N=3 per group, p<0.05) and the addition of the anti-APN scFv at 1 ug/ml completely blocked the mBCl signal back to levels found in SR in normal salt conditions. In order to verify that the full rescue of this ISS specific response is due to enhanced Ang III – AT 2 R signaling, we next tried to block the effect of the anti-APN scFv by the addition of the AT 2 R antagonist, PD123319 (PD, 1 uM). Addition of PD alone to the ISS LS RPTCs did not significantly alter the mBCl signal, but when PD and anti-APN scFv are added to the LS treated ISS cells, there is complete reversal of the effect of anti-APN scFv alone (-38.1±3.9%, ISS anti-APN scFv + PD vs ISS anti-APN scFv, N=3 per group, p<0.05). The anti-APN scFv has potential therapeutic value by reducing APN enzymatic activity in ISS individuals and inducing the protective AT 2 R arm of the renin angiotensin system in low sodium conditions.

Aminopeptidase N is an entry co-factor triggering porcine deltacoronavirus entry via an endocytotic pathway

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the role of porcine APN (pAPN) during PDCoV infection of non-susceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter non-susceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly co-localized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters non-susceptible cells, but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate anti-viral drugs.

O-148 Sperm Aminopeptidase N as a predictive biomarker of blastocyst development and embryo viability

Study question To evaluate human sperm APN as a prognostic factor for determining high-quality embryos. Summary answer The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality and viable embryos. What is known already Prognosis and diagnosis of male fertility is one of the major concerns in reproductive medicine. Approximately 30%-40% of men with otherwise normal fertility parameters are still unable to achieve pregnancy. The predictive clinical value of a semen analysis to identify fertile or infertile males is limited; therefore, new sperm diagnostic or prognostic methodologies are urgently required. Sperm Aminopeptidase N (APN) may be a relevant molecular marker due to its high concentration in sperm cells and its role in sperm physiology, such as motility, acrosome reaction, and embryo development. Study design, size, duration A prospective study that involves a total of 81 couples and 611 embryos who underwent oocyte-donation cycles at the Clínica IVI Bilbao (Spain) between September 2014 and July 2015. Participants/materials, setting, methods This study was set in an assisted reproduction unit and in an academic research laboratory. All semen samples were examined and classified following WHO guidelines. Spermatozoa were isolated from semen on discontinuous colloidal silica gradient (45%-90%) technique. Embryo quality and development were determined according to the Spanish Association of Reproduction Biology Studies (ASEBIR) criteria. Flow cytometry analyses of quantitative and semi-quantitative sperm human APN levels. Main results and the role of chance The obtained results proved that the most evolved and viable blastocysts were associated with low sperm APN levels. Expanding, expanded, hatching/hatched and viable blastocysts come from semen samples which showed lower APN levels than early blastocysts, blocked and non viable blastocyst. The cumulative probability of having more evolved blastyocysts increased 1.38-fold at day 5 and 1.98-fold at day 6 of embryo development as well as the likelihood of having viable embryo increased 1.48-fold when semen samples with low APN levels are used during the ICSI technique. Limitations, reasons for caution Data obtained from a single Fertility Clinic. A multi-centrum study will be required. Wider implications of the findings The human sperm APN has the potential to become new molecular prognostic biomarker for having high-quality embryos that could help to diagnose male infertility, especially when seminal parameters are close to the threshold values. Trial registration number Not applicable