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Serine peptidases and metallopeptidases are the primary toxins found in Bothrops snakes venoms, which act on proteins in the tissues of victims or prey, and release of peptides formed through proteolytic activity. Various studies have indicated that these peptides, released by the proteolytic activity of heterologous enzymes, generate molecules with unidentified functions, referred to as cryptids. To address this, we purified serine peptidases from Bothrops jararaca venom using molecular exclusion chromatography and then incubated them with the endogenous substrate myoglobin. As a control, we also incubated the substrate with trypsin. The resulting proteolytic fragments were analyzed, separated, and collected via HPLC. These fractions were then tested on cell cultures, the active fractions were sequenced (ALELFR and TGHPETLEK) and synthesized. After confirming their activity, the peptides underwent sequencing and synthesis for additional cell tests, including the increase of cell viability, cycle phases, proliferation, signaling, growth kinetics, angiogenesis, and migration. The results revealed that the synthesized peptides exhibited cellular repair properties, suggesting a potential role in tissue repair in the range of 0.05-5 µ M. Additionally, the effects of fragments resulting from myoglobin degradation isolated (ALELFR and TGHPETLEK) revealed a regenerative action on tissue.

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BACKGROUND: Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain (G. lingzhi) and an Amazonian isolate (Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study. METHODS: Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). RESULTS: Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDS-- PAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor. CONCLUSION: Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities.

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The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.

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Abstract: Background: Metastatic melanoma stands out as the most lethal form of skin cancer because of its high propensity to spread and its remarkable resistance to treatment methods. Methods: In this review article, we address the incidence of melanoma worldwide and its staging phases. We thoroughly investigate the different melanomas and their associated risk factors. In addition, we underscore the principal therapeutic goals and pharmacological methods that are currently used in the treatment of melanoma. Results: The implementation of targeted therapies has contributed to improving the approach to patients. However, because of the emergence of resistance early in treatment, overall survival and progression-free periods continue to be limited. Conclusions: We provide new insights into plant serine protease inhibitor therapeutics, supporting high-throughput drug screening soon, and seeking a complementary approach to explain crucial mechanisms associated with melanoma.

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Serine peptidases and metallopeptidases are the primary toxins found in Bothrops snakes venoms, which act on proteins in the tissues of victims or prey, and release of peptides formed through proteolytic activity. Various studies have indicated that these peptides, released by the proteolytic activity of heterologous enzymes, generate molecules with unidentified functions, referred to as cryptids. To address this, we purified serine peptidases from Bothrops jararaca venom using molecular exclusion chromatography and then incubated them with the endogenous substrate myoglobin. As a control, we also incubated the substrate with trypsin. The resulting proteolytic fragments were analyzed, separated, and collected via HPLC. These fractions were then tested on cell cultures, the active fractions were sequenced (ALELFR and TGHPETLEK) and synthesized. After confirming their activity, the peptides underwent sequencing and synthesis for additional cell tests, including the increase of cell viability, cycle phases, proliferation, signaling, growth kinetics, angiogenesis, and migration. The results revealed that the synthesized peptides exhibited cellular repair properties, suggesting a potential role in tissue repair in the range of 0.05–5 μ M. Additionally, the effects of fragments resulting from myoglobin degradation isolated (ALELFR and TGHPETLEK) revealed a regenerative action on tissue.

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To corroborate the ontogenetic shift in the venom composition of the Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) previously reported through the census approach, we evaluated the shift in the protein profile, lethality, and proteolytic and phospholipase activities of four venom samples obtained in 2015, 2018, 2019, and 2021 from one C. m. nigrescens individual (CMN06) collected in Durango, Mexico. We demonstrated that the venom of C. m. nigrescens changed from a myotoxin-rich venom to a phospholipase A2 and snake venom metalloproteinase-rich venom. Additionally, the proteolytic and phospholipase activities increased with age, but the lethality decreased approximately three times.

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Fusarium is a genus of ubiquitous fungi that comprises mycotoxigenic animal and plant pathogens. These fungi have the ability to exploit a wide range of substrates and hosts, indicating their great potential for enzyme production; however, this aspect is understudied. Therefore, the present study aimed for revaluating the identity of twenty-three Fusarium strains maintained in the University Recife Mycology (URM) culture collection, Brazil, and to evaluate their potential for proteases production and the milk-clotting activity of these proteases. According to phylogenetic analysis of translation elongation factor 1-alpha (TEF1) gene partial sequences, these strains belonged to 12 species representing four species complexes: Fusarium concolor, F. fujikuroi, F. incarnatum-equiseti, and F. oxysporum. Four of these species are putatively novel to science. Notably, novel associations of Fusarium spp. with certain hosts/substrates were documented. The proteolytic activity ranged from 1.67 U ml-1 to 22.03 U ml-1 among the evaluated fungal isolates, with specific proteolytic activity reaching 205.86 U mg-1. The values for coagulant activity and specific activity were up to 157.14 U ml-1 and 1,424.11 U mg-1, respectively. These results indicate the potential of URM Fusarium strains as a source for the production of enzymes of industrial interest. Additionally, they reinforce the importance of applying DNA-based methods for reviewing the identification of fungal strains preserved in biodiversity repositories.

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Trichomonas vaginalis is responsible for 156 million new cases per year worldwide. When present asymptomatically, the parasite can lead to serious complications, such as development of cervical and prostate cancer. As infection increases the acquisition and transmission of HIV, the control of trichomoniasis represents an important niche for the discovery and development of new antiparasitic molecules. This urogenital parasite synthesizes several molecules that allow the establishment and pathogenesis of infection. Among them, peptidases occupy key roles as virulence factors, and the inhibition of these enzymes has become an important mechanism for modulating pathogenesis. Based on these premises, our group recently reported the potent anti-T. vaginalis action of the metal-based complex [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione). In the present study, we evaluated the influence of Cu-phendione on the modulation of proteolytic activities produced by T. vaginalis by biochemical and molecular approaches. Cu-phendione showed strong inhibitory potential against T. vaginalis peptidases, especially cysteine- and metallo-type peptidases. The latter revealed a more prominent effect at both the post-transcriptional and post-translational levels. Molecular Docking analysis confirmed the interaction of Cu-phendione, with high binding energy (-9.7 and -10.7 kcal·mol-1, respectively) at the active site of both TvMP50 and TvGP63 metallopeptidases. In addition, Cu-phendione significantly reduced trophozoite-mediated cytolysis in human vaginal (HMVII) and monkey kidney (VERO) epithelial cell lineages. These results highlight the antiparasitic potential of Cu-phendione by interaction with important T. vaginalis virulence factors.

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Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

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Extreme halophilic archaea (haloarchaea) have adapted their physiology and biomolecules to thrive in saline environments (>2 M NaCl). Many haloarchaea produce extracellular hydrolases (including proteases) with potential biotechnological applications, which require unusual high salt concentrations to attain their function and maintain their stability. These conditions restrict many of the standard methods used to study these enzymes such as activity determination and/or protein purification. Here, we describe basic protocols to detect and measure extracellular proteolytic activity in haloarchaea including casein hydrolysis on agar plates, quantitative proteolytic activity determination by the azocasein assay and gelatin zymography in presence of the compatible solute glycine-betaine.

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Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

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Fungal fibrinolytic enzymes, secreted by some Agaricomycetes, are recognized as important thrombolytic agents due to their ability to rapidly dissolve thromboembolic clots. The present work evaluated fibrinolytic and proteolytic secretion abilities of 35 Agaricomycetes isolates from the Paranaense rainforest (Misiones, Argentina). We detected proteolytic activity in 40% of the strains while nine strains showed fibrinolytic activity. Schizophyllum commune LBM 026, Schizophyllum commune LBM 223, and Hornodermoporus martius LBM 224 exhibited the highest levels of fibrinolytic activity. Fibrin zymography from S. commune LBM 026 and LBM 223 showed an enzyme of 27.5 kDa, while H. martius LBM 224 presented an enzyme of 29 kDa. The evaluation of the enzymatic stability of culture supernatant of these strains revealed that the fibrinolytic activity was highly stable over a wide temperature and pH range. Long-term stability of fibrinolytic activity at physiological conditions evidenced that the strains had a half-life of at least 72 h. Fibrinolytic enzymes produced by S. commune LBM 026 and LBM 223 were inhibited in the presence of EDTA indicating that they are metalloproteases. This work reveals the potential of S. commune LBM 026, S. commune LBM 223, and H. martius LBM 224 as an unconventional source of thrombolytic agents.

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High proteolytic activity and several biological functions (antimicrobial, antioxidant, antihypertensive, among others) have been attributed to lactic-acid bacteria (LAB) isolated from fish and peptides obtained from proteolysis. Therefore, the objective of this research was isolating, characterizing, and identifying LAB with proteolytic activity by spontaneous fermentation from common carp (Cyprinus carpio) reared in ponds and wild ones obtained from Lago de Chapala, Jalisco, Mexico. Spontaneous fermentation from complete carp specimens was observed, considering two sampling points (skin and intestines) at 15 °C at 5 and 10 days. Isolated LAB-from both reared and wild specimens-were identified and morphologically characterized; identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Proteolytic activity was assessed by the presence of the proteolytic halo. A total of five genera and eight different LAB proteolytic species were isolated from all the carp samples. At 10 days, greater proteolytic LAB diversity was obtained from the intestine (Tukey's, p < 0.05); the proteolytic halo with the greatest diameter was recorded in wild carp skin with Lactiplantibacillus plantarum S5P2 (2.8 cm) at 5 days of fermentation, followed by Leuconostoc mesenteroides S5I1 (2.73 cm) and Leuconostoc pseudomesenteroides S5P2 (2.66 cm) (p < 0.05). In conclusion, proteolytic capability of LAB isolated from carp (Cyprimus carpio)-both wild and reared-is influenced by the ecosystem where they develop. These proteolytic LAB may be used in biotechnological industries to obtain bioactive peptides by fermenting substrates rich in proteins.

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Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

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Pepper seeds are slow to germinate and emergence is often non-uniform and incomplete, reducing gains from this cash crop. This study investigated the effects of pineapple stem- derived protease (stem bromelain) based priming on pepper seed germination in relation to reserve mobilization (specifically, proteins and amino acids), germination, emergence and plant growth. These parameters were compared across two controls, (1) unsoaked seeds and (2) seeds soaked in deionized water, and seeds soaked in pineapple stem bromelain crude extract (treatment). Seeds were soaked in bromelain crude extract possessing a proteolytic activity of 6.25 tU or deionized water (first control) for 3 h at 35 °C. Light microscopy revealed an abundance of protein bodies in the endosperm of the seeds prior to imbibition. When observed for a period of 96 h, these bodies were progressively degraded, with the rate of this degradation being fastest in bromelain-treated seeds. Quantitative analysis of protein levels confirmed this observation: 17.2 mg proteins/g FW at 120 h after priming in bromelain-treated seeds compared with 22.1 mg/g FW in controls (average). The bromelain treatment also increased levels of free amino acids from 3.9 mg/g FW in the controls to 4.6 mg/g FW after 120 h of imbibition. Germination and emergence percentages were initially higher in bromelain-treated seeds: 92.0% germination in bromelain-treated seeds vs. ~ 52.2% in the controls at 18 d; 100% emergence in protease-treated seeds vs. ~ 72.2% in the controls at 18 d. However, these parameters were comparable across the treatment and the controls at 28 d. Importantly, plant fresh and dry weights were significantly higher when seeds were primed with bromelain. The results suggest the use of bromelain extracts for priming pepper seeds based on their proteolytic activity, since germination is dependent on the availability of crude protein and essential amino acids. The benefits of bromelain seed priming appear to translate into improved seedling growth as well.

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Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05T proteases which have potential for the valorization of organic wool as well as for other industrial applications.

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Proteolytic starter cultures with intrinsic immunomodulatory activities are desirably features for the development of functional foods, which would significantly reduce the cost of their production (one-strain starter) having an additional beneficial effect on the host. In this work, Lactobacillus delbrueckii strains were selected according to their ability to efficiently hydrolyse ß-casein and to modulate the immune system. Among 36 strains evaluated, the highest proteolytic activities were found for L. delbrueckii subsp. lactis CRL581 and L. delbrueckii subsp. bulgaricus CRL656. The immunomodulatory effect of both strains and their ß-casein hydrolysates (CRL581 and CRL656 hydrolysates, respectively) were studied in a murine model. Balb/c mice were fed lactobacilli or their hydrolysates for three days. One day after the last lactobacilli or hydrolysate treatments, mice were challenged with the Toll-like receptor 3 (TLR3) agonist poly(I:C) by intraperitoneal injection. Before and after poly(I:C) challenge the phagocytic and microbicidal activity of peritoneal macrophages, intestinal immunoglobulin A (IgA), cytokine profile, and histological analysis of the intestine were analysed. L. delbrueckii subsp. lactis CRL581 significantly increased the activation of peritoneal macrophages as well as the levels of intestinal IgA, interleukin (IL)-10 and interferon (IFN)-γ when compared to untreated controls. In addition, the CRL581 strain was able to significantly reduce the intestinal inflammatory damage triggered by TLR3 activation. L. delbrueckii CRL581 increased the levels of IL-10, IFN-γ and IFN-ß, and reduced tumour necrosis factor alpha and IL-6 concentrations in the intestine of poly(I:C)-challenged mice. No immunomodulatory effects were observed for the CRL656 strain or for the CRL581 or CRL656 hydrolysates. The results of this work show that the technologically relevant and high proteolytic strain L. delbrueckii CRL581 is able to beneficially modulate the intestinal innate antiviral immune response. Although further studies with the CRL581 strain are required to corroborate and deepen its immunological effects, this bacterium is an interesting alternative for the development of new functional foods with antiviral capabilities.

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The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.

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AIM: To determine the protective capacity against Salmonella infection in mice of the cell-free fraction (postbiotic) of fermented milk, produced at laboratory and industrial level. METHODS AND RESULTS: The proteolytic activity (PA) of 5 commercial cultures and 11 autochthonous Lactobacillus strains was evaluated. The DSM-100H culture displayed the highest PA and it was selected for further studies. The capacity of the postbiotics produced by pH-controlled fermentation to stimulate the production of secretory IgA in faeces and to protect mice against Salmonella infection was evaluated. A significant increase in secretory IgA in faeces of mice fed 14 days the postbiotic obtained at the laboratory (F36) was detected compared to control animals. A significantly higher survival was observed in mice fed the F36 and the FiSD (industrial product) compared to controls. CONCLUSION: The postbiotics obtained showed immunomodulatory and protective capacity against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The pH-controlled milk fermentation by the proteolytic DSM-100H culture could be a suitable strategy to obtain a food ingredient to be added to a given food matrix, not adequate to host viable cells of probiotics, to confer it enhanced functionality and thus expand the functional food market.

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Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

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