NAPRT Expression Regulation Mechanisms: Novel Functions Predicted by a Bioinformatics Approach

The nicotinate phosphoribosyltransferase (NAPRT) gene has gained relevance in the research of cancer therapeutic strategies due to its main role as a NAD biosynthetic enzyme. NAD metabolism is an attractive target for the development of anti-cancer therapies, given the high energy requirements of proliferating cancer cells and NAD-dependent signaling. A few studies have shown that NAPRT expression varies in different cancer types, making it imperative to assess NAPRT expression and functionality status prior to the application of therapeutic strategies targeting NAD. In addition, the recent finding of NAPRT extracellular form (eNAPRT) suggested the involvement of NAPRT in inflammation and signaling. However, the mechanisms regulating NAPRT gene expression have never been thoroughly addressed. In this study, we searched for NAPRT gene expression regulatory mechanisms in transcription factors (TFs), RNA binding proteins (RBPs) and microRNA (miRNAs) databases. We identified several potential regulators of NAPRT transcription activation, downregulation and alternative splicing and performed GO and expression analyses. The results of the functional analysis of TFs, RBPs and miRNAs suggest new, unexpected functions for the NAPRT gene in cell differentiation, development and neuronal biology.

Persistence alters the interaction between Chlamydia trachomatis and its host cell.

In response to stress, the obligate intracellular pathogen Chlamydia trachomatis stops dividing and halts its biphasic developmental cycle. The infectious, extracellular form of this bacterium is highly susceptible to killing by the host immune response, and by pausing development Chlamydia can survive in an intracellular, ‘aberrant’ state for extended periods of time. The relevance of these aberrant forms has long been debated, and many questions remain concerning how they contribute to the persistence and pathogenesis of the organism. Using reporter cell lines, fluorescence microscopy, and a di-peptide labeling strategy, we measured the ability of C. trachomatis to synthesize, assemble, and degrade peptidoglycan under various aberrance-inducing conditions. We found that all aberrance-inducing conditions affect chlamydial peptidoglycan, and that some actually halt the biosynthesis pathway early enough to prevent the release of an immunostimulatory peptidoglycan component, muramyl tripeptide. In addition, utilizing immunofluorescence and electron microscopy, we determined that the induction of aberrance can detrimentally affect the development of the microbe’s pathogenic vacuole (the inclusion). Taken together, our data indicate that aberrant forms of Chlamydia generated by different environmental stressors can be sorted into two broad categories based on their ability to continue releasing peptidoglycan-derived, immunostimulatory muropeptides and their ability to secrete effector proteins that are normally expressed at the mid- and late- stages of the microbe’s developmental cycle. Our findings reveal a novel, immuno-evasive feature inherent to a subset of aberrant chlamydial forms and provide clarity and context to the numerous persistence mechanisms employed by these ancient, genetically-reduced microbes.

P072 The involvement of extracellular Nicotinamide Phosphoribosyltransferase (eNAMPT) and Nicotinate Phosphoribosyltransferase (eNAPRT) in inflammatory bowel disease

Background Nicotinamide phosphoribosyltrasferase (NAMPT) is a pleiotropic enzyme which catalyses the first and rate-limiting step in the biosynthesis of NAD. It is present in two different forms: an intracellular form, called iNAMPT, (Chiarugi et al., 2012), and an extracellular form, eNAMPT. eNAMPT is considered an important factor for granulocyte-colony stimulating factor-(G-CSF)-induced myeloid differentiation, with paracrine and autocrine effects on different cell types (i.e. immune and cancer cells), binding TLR4. NAMPT is structurally and functionally related to the enzyme nicotinate phosphoribosyltransferase (iNAPRT), which is rate-limiting in the NAD salvage pathway that starts from nicotinic acid. The NAD biosynthetic pathways controlled by NAMPT and NAPRT are closely interconnected and can compensate for each other. Also, NAPRT is identified as an extracellular ligand (eNAMPRT) for TLR4 and a mediator of inflammation (Managò et al., 2020). Importantly, iNAMPT and eNAMPT levels are increased in several pathologies, included inflammatory bowel disease (IBD). It has been reported that serum eNAMPT levels correlate with the stage of the pathology: in an active state of the disease the levels of NAMPT are very high, however its levels are partially reduced in a remission stage (Moschen et al., 2007). Methods First, we investigated the role of eNAMPT and eNAPRT in murine IBD models (especially in DNBS and DSS model). We took into account phenotypic effect as weight loss and colon shortening, but also the reduction of mRNA of inflammatory genes with RT-PCR, tissue damage with H&E and IHC analysis and systemic and local production through colon explant. Secondly, we determined serum eNAMPT and eNAPRT levels in a cohort of adult IBD patients. Results Both eNAMPT and eNAPRT have been found elevated in 180 IBD patients, as proinflammatory marker of the pathologies. These levels are also elevated in serum and colonic explant of DSS and DNBS preclinical models, associated to an active state of the disease, as a pro-inflammatory response developed locally and systemically. Moreover, we performed ELISA analysis on sera of 100 IBD patients, eligible for anti-TNF treatment, both pediatric and adults. Serum eNAMPT levels are increased before the treatment, responsive patients verified a reduction of these levels, while no-responsive ones verified higher levels. Conclusion eNAMPT and eNAPRT could be considered pro-inflammatory markers of IBD and possible druggable targets.

Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2

For anterior gradient 2 (AGR2), normal cells express the intracellular form iAGR2 localized to the endoplasmic reticulum while cancer cells express the extracellular form eAGR2 localized on the cell surface and secreted. Antibodies targeting eAGR2+ cancer cells for eradication will spare normal cells. Two AGR2 monoclonal antibodies, P1G4 and P3A5, were shown to recognize specifically eAGR2+ pancreatic tumors implanted in mice. In addition, P1G4 showed enhancement in drug inhibition of tumor growth. Human:mouse chimeric antibodies of IgG1, IgG2, IgG4 were generated for both antibodies. These human IgG were shown to lyse eAGR2+ prostate cancer cells in vitro with human serum. AGR2 has an important function in distal spread of cancer cells, and is highly expressed in prostate, pancreatic, bladder metastases. Therefore, immunotherapy based on AGR2 antibody-mediated ADCC and CDC is highly promising. Cancer specificity of eAGR2 predicts possibly minimal collateral damage to healthy tissues and organs. Moreover, AGR2 therapy, once fully developed and approved, can be used to treat other solid tumors since AGR2 is an adenocarcinoma antigen found in many common malignancies.

Polinton-like viruses are abundant in aquatic ecosystems

Background Polintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited. Results Here, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae. Conclusions Our study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups.

Delayed Release of Intracellular Microcystin Following Partial Oxidation of Cultured and Naturally Occurring Cyanobacteria

Oxidation processes can provide an effective barrier to eliminate cyanotoxins by damaging cyanobacteria cell membranes, releasing intracellular cyanotoxins, and subsequently oxidizing these toxins (now in extracellular form) based on published reaction kinetics. In this work, cyanobacteria cells from two natural blooms (from the United States and Canada) and a laboratory-cultured Microcystis aeruginosa strain were treated with chlorine, monochloramine, chlorine dioxide, ozone, and potassium permanganate. The release of microcystin was measured immediately after oxidation (t ≤ 20 min), and following oxidant residual quenching (stagnation times = 96 or 168 h). Oxidant exposures (CT) were determined resulting in complete release of intracellular microcystin following chlorine (21 mg-min/L), chloramine (72 mg-min/L), chlorine dioxide (58 mg-min/L), ozone (4.1 mg-min/L), and permanganate (391 mg-min/L). Required oxidant exposures using indigenous cells were greater than lab-cultured Microcystis. Following partial oxidation of cells (oxidant exposures ≤ CT values cited above), additional intracellular microcystin and dissolved organic carbon (DOC) were released while the samples remained stagnant in the absence of an oxidant (>96 h after quenching). The delayed release of microcystin from partially oxidized cells has implications for drinking water treatment as these cells may be retained on a filter surface or in solids and continue to slowly release cyanotoxins and other metabolites into the finished water.

2176. A New Rapid Test for Detection of The Cefazolin Inoculum Effect (CIE) in Methicillin-Susceptible Staphylococcus aureus (MSSA)

Background Most MSSA harbor one of the four different variants of β-lactamase (BlaZ) (A, B, C and D). The CIE is defined as an MIC >16 μg/mL when a high inoculum (107 CFU/mL) is used and depends on the presence of BlaZ. The presence of the CIE has been associated with therapeutic failure in invasive MSSA infections. In some countries of South America, the prevalence of CIE is high, ranging from 36% to 51% (Colombia and Argentina, respectively). Type A BlaZ is most often associated with the CIE due to its high affinity for cefazolin. Here, we developed a rapid test based on the premise that the extracellular form of BlaZ is responsible for the CIE. We aimed to identify invasive MSSA that exhibit the CIE and validate the test in two cohorts of isolates from patients in Colombia and Argentina Methods 152 MSSA clinical isolates were collected from Colombia (n = 71) and Argentina (n = 81). We determined MIC at standard and high inoculum. We developed a test using induction of BlaZ with ampicillin (150 μg/mL) for 20 minutes and, using the supernatant for incubation with nitrocefin for 30 min. A change in color from yellow to red was considered positive. MSSA TX0117 (BlaZ +, with the CIE), ATCC 29213 (BlaZ-negative) and ATCC 25923 (BlaZ + lacking the CIE) were used as controls. BlaZ typing of all Argentinian isolates was available by sequencing Results A high proportion (43%) of MSSA exhibited the CIE (34% and 52% of Colombian and Argentinian isolates, respectively) by MIC. The rapid test identified 76% of isolates exhibiting the CIE and correctly ruled out all isolates lacking the CIE (sensitivity 80%, specificity 100%). Furthermore, the rapid test detected all isolates with the CIE that harbored Type A BlaZ from Argentina. Conversely, the test failed to identify the CIE in Argentinian isolates that produce type B and C BlaZ. The sensitivity and specificity of the rapid test for the Colombian isolates whose BlaZ type was unknown were 89% and 100%, respectively. Conclusion A rapid test of less than 2 h can readily identify MSSA isolates exhibiting the CIE. For isolates carrying type A BlaZ, which is highly associated with the CIE, the test had a sensitivity and specificity of 100%. Rapid identification of MSSA with the CIE may have important therapeutic consequences in deep-seated infections Disclosures All authors: No reported disclosures.

Annexin A1 Contained in Extracellular Vesicles Promotes the Activation of Keratinocytes by Mesoglycan Effects: An Autocrine Loop Through FPRs

We have recently demonstrated that mesoglycan, a fibrinolytic compound, may be a promising pro-healing drug for skin wound repair. We showed that mesoglycan induces migration, invasion, early differentiation, and translocation to the membrane of keratinocytes, as well as the secretion of annexin A1 (ANXA1), further involved in keratinocytes activation. These events are triggered by the syndecan-4 (SDC4)/PKCα pathway. SDC4 also participates to the formation and secretion of microvesicles (EVs) which may contribute to wound healing. EVs were isolated from HaCaT cells, as human immortalized keratinocytes, and then characterised by Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. Their autocrine effects were investigated by Wound-Healing/invasion assays and confocal microscopy to analyse cell motility and differentiation, respectively. Here, we found that the mesoglycan increased the release of EVs which amplify its same effects. ANXA1 contained in the microvesicles is able to promote keratinocytes motility and differentiation by acting on Formyl Peptide Receptors (FPRs). Thus, the extracellular form of ANXA1 may be considered as a link to intensify the effects of mesoglycan. In this study, for the first time, we have identified an interesting autocrine loop ANXA1/EVs/FPRs in human keratinocytes, induced by mesoglycan.

Unconventional secretion of FABP4 by endosomes and secretory lysosomes

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum–Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.