Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

Helicobacter pylori Pathogen-Associated Molecular Patterns: Friends or Foes?

Microbial infections are sensed by the host immune system by recognizing signature molecules called Pathogen-Associated Molecular Patterns-PAMPs. The binding of these biomolecules to innate immune receptors, called Pattern Recognition Receptors (PRRs), alerts the host cell, activating microbicidal and pro-inflammatory responses. The outcome of the inflammatory cascade depends on the subtle balance between the bacterial burn and the host immune response. The role of PRRs is to promote the clearance of the pathogen and to limit the infection by bumping inflammatory response. However, many bacteria, including Helicobacter pylori, evolved to escape PRRs' recognition through different camouflages in their molecular pattern. This review examines all the different types of H. pylori PAMPs, their roles during the infection, and the mechanisms they evolved to escape the host recognition.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there is no information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis.

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

TFF1 Induces Aggregation and Reduces Motility of Helicobacter pylori.

Gastric cancer is considered one of the most common malignancies in humans and Helicobacter pylori infection is the major environmental risk factor of gastric cancer development. Given the high spread of this bacterium whose infection is mostly asymptomatic, H. pylori colonization persists for a long time, becoming chronic and predisposing to malignant transformation. The first defensive barrier from bacterial infection is constituted by the gastric mucosa that secretes several protective factors, among which is the trefoil factor 1 (TFF1), that, as mucin 5AC, binds the bacterium. Even if the protective role of TFF1 is well-documented, the molecular mechanisms that confer a beneficial function to the interaction among TFF1 and H. pylori remain still unclear. Here we analyze the effects of this interaction on H. pylori at morphological and molecular levels by means of microscopic observation, chemiotaxis and motility assays and real-time PCR analysis. Our results show that TFF1 favors aggregation of H. pylori and significantly slows down the motility of the bacterium across the mucus. Such aggregates significantly reduce both flgE and flaB gene transcription compared with bacteria not incubated with TFF1. Finally, our results suggest that the interaction between TFF1 and the bacterium may explain the frequent persistence of H. pylori in the human host without inducing disease.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

NMR Structure of the FIV gp36 C-Terminal Heptad Repeat and Membrane-Proximal External Region.

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR-MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR-MPER is characterized by a helix-turn-helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR-MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR-MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

Determining the Effect of Pterostilbene on Insulin Secretion Using Chemoproteomics.

Pterostilbene, the 3,5-dimethoxy derivative of resveratrol, is a well-known polyphenolic compound, mainly found in blueberries, grapevines, and Pterocarpus marsupium heartwood, which has recently attracted a great deal of attention due to its wide bio-pharmacological profile. Moreover, pterostilbene is more lipophilic than resveratrol, with a consequently better bioavailability and a more interesting therapeutic potential. In this work, a chemoproteomic approach, based on affinity chromatography, was applied on pterostilbene in the attempt to identify the biological targets responsible for its bioactivity. On this basis, syntaxins, a group of proteins involved in the formation of SNARE complexes mediating vesicles exocytosis, were selected among the most interesting pterostilbene interactors. In vitro and in cell assays gave evidence of the pterostilbene ability to reduce insulin secretion on glucose-stimulated pancreatic beta cells, opening the way to potential applications of pterostilbene as a supplement in the care of insulin-dependent metabolic disorders.

TFF1 Promotes EMT-Like Changes through an Auto-Induction Mechanism.

Trefoil factor 1 (TFF1) is a small secreted protein expressed in the gastrointestinal tract where, together with the other two members of its family, it plays an essential role in mucosal protection and repair against injury. The molecular mechanisms involved in the protective function of all three TFF proteins are not fully elucidated. In this paper, we investigated the role of TFF1 in epithelial to mesenchymal transition (EMT) events. The effects of TFF1 on cellular models in normoxia and/or hypoxia were evaluated by western blot, immunofluorescence, qRT-PCR and trans-well invasion assays. Luciferase reporter assays were used to assess the existence of an auto-regulatory mechanism of TFF1. The methylation status of TFF1 promoter was measured by high-resolution melting (HRM) analysis. We demonstrate a TFF1 auto-induction mechanism with the identification of a specific responsive element located between -583 and -212 bp of its promoter. Our results suggest that TFF1 can regulate its own expression in normoxic, as well as in hypoxic, conditions acting synergistically with the hypoxia-inducible factor 1 (HIF-1α) pathway. Functionally, this auto-induction mechanism seems to promote cell invasion and EMT-like modifications in vitro. Additionally, exogenously added human recombinant TFF1 protein was sufficient to observe similar effects. Together, these findings suggest that the hypoxic conditions, which can be induced by gastric injury, promote TFF1 up-regulation, strengthened by an auto-induction mechanism, and that the trefoil peptide takes part in the epithelial-mesenchymal transition events eventually triggered to repair the damage.

Annexin A1 May Induce Pancreatic Cancer Progression as a Key Player of Extracellular Vesicles Effects as Evidenced in the In Vitro MIA PaCa-2 Model System.

Pancreatic Cancer (PC) is one of the most aggressive malignancies worldwide. As annexin A1 (ANXA1) is implicated in the establishment of tumour metastasis, the role of the protein in PC progression as a component of extracellular vesicles (EVs) has been investigated. EVs were isolated from wild type (WT) and ANXA1 knock-out (KO) PC cells and then characterised by multiple approaches including Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. The effects of ANXA1 on tumour aggressiveness were investigated by Wound-Healing and invasion assays and microscopic analysis of the Epithelial to Mesenchymal Transition (EMT). The role of ANXA1 on angiogenesis was also examined in endothelial cells, using similar approaches. We found that WT cells released more EVs enriched in exosomes than those from cells lacking ANXA1. Notably, ANXA1 KO cells recovered their metastatic potential only when treated by WT EVs as they underwent EMT and a significant increase of motility. Similarly, human umbilical vein endothelial cells (HUVEC) migrated and invaded more rapidly when treated by WT EVs whereas ANXA1 KO EVs weakly induced angiogenesis. This study suggests that EVs-related ANXA1 is able to promote cell migration, invasion, and angiogenesis, confirming the relevance of this protein in PC progression.

Proteasome as a New Target for Bio-Inspired Benzo[k,l]xanthene Lignans.

Mass spectrometry-based chemical proteomics is a powerful tool for the target discovery of small molecules. Here, the application of this approach is presented to define the target profile of bio-inspired synthetic benzo[k,l]xanthene lignans endowed with interesting biological properties. Proteasome has been identified as a new main interactor for this class of compounds. A combination of molecular docking with in vitro and in cell fluorescence assays gave insights on the molecular mechanism of the interaction, highlighting the tendency of these lignans to inhibit the proteasome.

Determination of Gymnemic Acid I as a Protein Biosynthesis Inhibitor Using Chemical Proteomics.

The plant Gymnema sylvestre has been used widely in traditional medicine as a remedy for several diseases, and its leaf extract is known to contain a group of bioactive triterpene saponins belonging to the gymnemic acid class. Gymnemic acid I (1) is one of the main components among this group of secondary metabolites and is endowed with an interesting bioactivity profile. Since there is a lack of information about its specific biological targets, the full interactome of 1 was investigated through a quantitative chemical proteomic approach, based on stable-isotope dimethyl labeling. The ribosome complex was found to be the main partner of compound 1, and a full validation of the proteomics results was achieved by orthogonal approaches. Further biochemical and biological investigations revealed an inhibitory effect of 1 on the ribosome machinery.

Identification and mechanism of action analysis of the new PARP-1 inhibitor 2″-hydroxygenkwanol A.

BACKGROUND: Poly(ADP-ribose) polymerase 1 (PARP-1) activity has been implicated in the pathogenesis of numerous diseases as cancer, inflammation, diabetes and neurodegenerative disorders, therefore the research for new PARP-1 inhibitors is still an active area. METHODS: To identify new potential PARP-1 inhibitors, we performed a screening of a small-molecule library consisting of polyphenols isolated from plants used in the traditional medicine, by Surface Plasmon Resonance (SPR). Biochemical and cellular assays were performed to confirm SPR results and select the promising candidate(s). Finally, limited proteolysis and ligand docking analyses allowed defining the protein region involved in the interaction with the putative inhibitor(s). RESULTS: The dimeric spiro-flavonoid 2″-hydroxygenkwanol A, member of a relatively recently discovered class of flavonoids containing a spirane C-atom, has been identified as possible PARP-1 inhibitor. This compound showed a high affinity for the polymerase (KD: 0.32±0.05µM); moreover PARP-1 activity in the presence of 2″-hydroxygenkwanol A was significantly affected both when using the recombinant protein and when measuring the cellular effects. Finally, our study suggests this compound to efficiently interact with the protein catalytic domain, into the nicotine binding pocket. CONCLUSION: 2″-hydroxygenkwanol A efficiently binds and inhibits PARP-1 at submicromolar concentrations, thus representing a promising lead for the design of a new class of PARP-1 modulators, useful as therapeutic agents and/or biochemical tools. GENERAL SIGNIFICANCE: Our study has identified an additional class of plant molecules, the spiro-biflavonoids, with known beneficial pharmacological properties but with an unknown mechanism of action, as a possible novel class of PARP-1 activity inhibitors.

Transcriptome analysis of macrophages during Brucella abortus infection clarifies the survival mechanisms of the bacteria.

Brucellosis is a critical zoonotic disease impacting humans and animals globally, causing symptoms like fever and arthritis in humans and reproductive issues in animals. The disease stems from the Brucella genus, adept at evading the immune system and proliferating within host cells. This study explores how Brucella abortus manipulates host cellular mechanisms to sustain infection, focusing on the interaction with murine macrophages over 24 h. Initial host defenses involve innate immune responses, while Brucella's survival strategies include evading lysosomal degradation and modulating host cell functions through various pathways. The research identified significant transcriptional changes in macrophages post-infection, highlighting pathways such as cytokine storm, pyroptosis signaling, Toll-like receptor pathways, and LXRs/RXRs signaling. The findings shed light on Brucella's complex mechanisms to undermine host defenses and underscore the need for further investigation into therapeutic targets to combat brucellosis.

H3K56 acetylation affects Candida albicans morphology and secreted soluble factors interacting with the host.

In recent years, epigenetics has been revealed as a mechanism able to modulate the expression of virulence traits in diverse pathogens, including Candida albicans. Indeed, epigenetic regulation can sense environmental changes, leading to the rapid and reversible modulation of gene expression with consequent adaptation to novel environments. How epigenetic changes can impact expression and signalling output, including events associated with mechanisms of morphological transition and virulence, is still poorly studied. Here, using nicotinamide as a sirtuin inhibitor, we explored how the accumulation of the H3K56 acetylation, the most prominent histone acetylation in C. albicans, might affect its interaction with the host. Our experiments demonstrate that H3K56 acetylation profoundly affects the production and/or secretion of soluble factors compromising actin remodelling and cytokine production. ChIP- and RNA-seq analyses highlighted a direct impact of H3K56 acetylation on genes related to phenotypic switching, biofilm formation and cell aggregation. Direct and indirect regulation also involves genes related to cell wall protein biosynthesis, ß-glucan and mannan exposure, and hydrolytic secreted enzymes, supporting the hypothesis that the fluctuations of H3K56 acetylation in C. albicans might impair the macrophage response to the yeast and thus promote the host-immune escaping.

High levels of soluble CD73 unveil resistance to BRAF inhibitors in melanoma cells.

Melanoma cells express high levels of CD73 that produce extracellular immunosuppressive adenosine. Changes in the CD73 expression occur in response to tumor environmental factors, contributing to tumor phenotype plasticity and therapeutic resistance. Previously, we have observed that CD73 expression can be up-regulated on the surface of melanoma cells in response to nutritional stress. Here, we explore the mechanism by which melanoma cells release soluble CD73 under low nutrient availability and whether this might be affected by agents targeting the proto-oncogene B-Raf (BRAF). We found that starved melanoma cells can release high levels of CD73, able to convert AMP into adenosine, and this activity is abrogated by selective CD73 inhibitors, APCP or PSB-12489. The release of CD73 from melanoma cells is mediated by the matrix metalloproteinase MMP-9. Indeed, MMP-9 inhibitors significantly reduce the levels of CD73 released from the cells, while its surface levels increase. Of relevance, melanoma cells, harboring an activating BRAF mutation, upon treatment with dabrafenib or vemurafenib, show a strong reduction of CD73 cell expression and reduced levels of CD73 released into the extracellular space. Conversely, melanoma cells resistant to dabrafenib show high expression of membrane-bound CD73 and soluble CD73 released into the culture medium. In summary, our data indicate that CD73 is released from melanoma cells. The expression of CD73 is associated with response to BRAF inhibitors. Melanoma cells developing resistance to dabrafenib show increased expression of CD73, including soluble CD73 released from cells, suggesting that CD73 is involved in acquiring resistance to treatment.

Differential in gel electrophoresis (DIGE) comparative proteomic analysis of macrophages cell cultures in response to perthamide C treatment.

Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform.

Tatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3-methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and ß-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism.

Design of an Herbal Preparation Composed by a Combination of Ruscus aculeatus L. and Vitis vinifera L. Extracts, Magnolol and Diosmetin to Address Chronic Venous Diseases through an Anti-Inflammatory Effect and AP-1 Modulation.

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.