A proteomic perspective on the resistance response of Klebsiella pneumoniae to antimicrobial peptide PaDBS1R1.

BACKGROUND: The synthetic antimicrobial peptide, PaDBS1R1, has been reported as a powerful anti-Klebsiella pneumoniae antimicrobial. However, there is only scarce knowledge about whether K. pneumoniae could develop resistance against PaDBS1R1 and which resistance mechanisms could be involved. OBJECTIVES: Identify via label-free shotgun proteomics the K. pneumoniae resistance mechanisms developed against PaDBS1R1. METHODS: An adaptive laboratory evolution experiment was performed to obtain a PaDBS1R1-resistant K. pneumoniae lineage. Antimicrobial susceptibility was determined through microdilution assay. Modifications in protein abundances between the resistant and sensitive lineages were measured via label-free quantitative shotgun proteomics. Enriched Gene Ontology terms and KEGG pathways were identified through over-representation analysis. Data are available via ProteomeXchange with identifier PXD033020. RESULTS: K. pneumoniae ATCC 13883 parental strain challenged with increased subinhibitory PaDBS1R1 concentrations allowed the PaDBS1R1-resistant K. pneumoniae lineage to emerge. Proteome comparisons between PaDBS1R1-resistant K. pneumoniae and PaDBS1R1-sensitive K. pneumoniae under PaDBS1R1-induced stress conditions enabled the identification and quantification of 1702 proteins, out of which 201 were differentially abundant proteins (DAPs). The profiled DAPs comprised 103 up-regulated proteins (adjusted P value < 0.05, fold change ≥ 2) and 98 down-regulated proteins (adjusted P value < 0.05, fold change ≤ 0.5). The enrichment analysis suggests that PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery could be relevant resistance mechanisms against PaDBS1R1. CONCLUSIONS: Based on experimental evolution and a label-free quantitative shotgun proteomic approach, we showed that K. pneumoniae developed resistance against PaDBS1R1, whereas PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery appear to be relevant resistance mechanisms against PaDBS1R1.

Repurposing a peptide toxin from wasp venom into antiinfectives with dual antimicrobial and immunomodulatory properties.

Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials.

The use of host defense peptides in root canal therapy in rats.

OBJECTIVES: In order to evaluate host defense peptides (HDPs) HHC-10 and synoeca-MP activity in in vitro osteoclastogenesis process and in vivo induced apical periodontitis, testing the effect of molecules in the inflammatory response and in apical periodontitis size/volume after root canal treatment. MATERIALS AND METHODS: In vitro osteoclastogenesis was assessed on bone marrow cell cultures extracted from mice, while in vivo endodontic treatment involved rats treated with Ca(OH)2 or HDPs. In vitro osteoclasts were subjected to TRAP staining, and in vivo samples were evaluated by radiographic and tomographic exams, as well as histologic analysis. RESULTS: None of the substances downregulated the in vitro osteoclastogenesis. Nevertheless, all treatments affected the average of apical periodontitis size in rats, although only teeth treated with HDPs demonstrated lower levels of the inflammatory process. These results demonstrated the in vivo potential of HDPs. Radiographic analysis suggested that HHC-10 and synoeca-MP-treated animals presented a similar lesion size than Ca(OH)2-treated animals after 7-day of endodontic treatment. However, tomography analysis demonstrated smaller lesion volume in synoeca-MP-treated animals than HHC-10 and Ca(OH)2-treated animals, after 7 days. CONCLUSIONS: These molecules demonstrated an auxiliary effect in endodontic treatment that might be related to its immunomodulatory ability, broad-spectrum antimicrobial activity, and possible induction of tissue repair at low concentrations. These results can encourage further investigations on the specific mechanisms of action in animal models to clarify the commercial applicability of these biomolecules for endodontic treatment. CLINICAL SIGNIFICANCE: HDPs have the potential to be adjuvant substances in endodontic therapy due to its potential to reduce inflammation in apical periodontitis.

A short peptide with selective anti-biofilm activity against Pseudomonas aeruginosa and Klebsiella pneumoniae carbapenemase-producing bacteria.

Biofilm-related infections represent an enormous clinical challenge nowadays. In this context, diverse studies are underway to develop effective antimicrobial agents targeting bacterial biofilms. Here, we describe the antibacterial and anti-biofilm activities of a short, cationic peptide named R5F5, obtained from sliding-window analysis based on a peptide (PcDBS1R5) derived from Plasmodium chabaudi. Ten fragments were generated (R5F1 to F10) and submitted to initial antibacterial assays against Pseudomonas aeruginosa. As a result, R5F5 showed the highest antimicrobial activity. We therefore carried out further antibacterial and anti-biofilm assays against P. aeruginosa and Klebsiella pneumoniae carbapenemase-producing bacterial strains. R5F5 revealed selective anti-biofilm activity, as the peptide inhibited >60% biofilm formation in all cases from 8 to 64 µg·mL-1. Moreover, R5F5 was not hemolytic against mice erythrocytes at 640 µg mL-1. Cytotoxic effects on human lung fibroblast cells were not detected at 160 µg·mL-1. Structural studies revealed that R5F5 presents random coil conformations in water and 50% 2,2,2-trifluoroethanol (TFE)/water (v/v), whereas amphipathic, extended conformations were observed in contact with sodium dodecyl sulfate (SDS) micelles. Thus, here we report a novel peptide with selective anti-biofilm activity against susceptible and resistant bacterial strains, with no toxicity toward mammalian cells and that adopts a stable structure in anionic environment.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 µM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

A self-assembled clavanin A-coated amniotic membrane scaffold for the prevention of biofilm formation by ocular surface fungal pathogens.

Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface reconstruction. Sometimes it may create a major problem with associated infections after biofilm formation over the membrane. To overcome this problem, AM was coated with the antimicrobial peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical, morphological and antifungal characteristics, indicating potential applicability for ocular surface infection control.

Characterization of a Bioactive Acyclotide from Palicourea rigida.

The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of ∼2.5 µM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.

Clavanin A improves outcome of complications from different bacterial infections.

The rapid increase in the incidence of multidrug-resistant infections today has led to enormous interest in antimicrobial peptides (AMPs) as suitable compounds for developing unusual antibiotics. In this study, clavanin A, an antimicrobial peptide previously isolated from the marine tunicate Styela clava, was selected as a purposeful molecule that could be used in controlling infection and further synthesized. Clavanin A was in vitro evaluated against Staphylococcus aureus and Escherichia coli as well as toward L929 mouse fibroblasts and skin primary cells (SPCs). Moreover, this peptide was challenged here in an in vivo wound and sepsis model, and the immune response was also analyzed. Despite displaying clear in vitro antimicrobial activity toward Gram-positive and -negative bacteria, clavanin A showed no cytotoxic activities against mammalian cells, and in acute toxicity tests, no adverse reaction was observed at any of the concentrations. Moreover, clavanin A significantly reduced the S. aureus CFU in an experimental wound model. This peptide also reduced the mortality of mice infected with E. coli and S. aureus by 80% compared with that of control animals (treated with phosphate-buffered saline [PBS]): these data suggest that clavanin A prevents the start of sepsis and thereby reduces mortality. These data suggest that clavanin A is an AMP that could improve the development of novel peptide-based strategies for the treatment of wound and sepsis infections.

Dentistry proteomics: from laboratory development to clinical practice.

Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease.

Effects of acute exercise over heart proteome from monogenic obese (ob/ob) mice.

Exercise is recognized to prevent and attenuate several metabolic and cardiovascular disorders. Obesity is commonly related to cardiovascular diseases, frequently resulting in heart failure and death. To elucidate the effects of acute exercise in heart tissue from obese animals, 12-week-old C57BL6/J obese (ob/ob) and non-obese (ob/OB) mice were submitted to a single bout of swimming and had their hearts analyzed by proteomic techniques. Mice were divided into three groups: control (ob/ob, n = 3; ob/OB, n = 3); a moderate intensity consisting of 20 min of swimming around 90% of Maximal Lactate Steady State (ob/ob, n = 3; ob/OB, n = 3), and a high intensity exercise performed as an incremental overload test (ob/ob, n = 3; ob/OB, n = 3). Obesity modulations were analyzed by comparing ob/ob and ob/OB control groups. Differential 2-DE analysis revealed that single session of exercise was able to up-regulate: myoglobin (ob/ob), aspartate aminotransferase (ob/OB) and zinc finger protein (ob/OB) and down-regulate: nucleoside diphosphate kinase B (ob/OB), mitochondrial aconitase (ob/ob and ob/OB) and fatty acid binding protein (ob/ob). Zinc finger protein and α-actin were up-regulated by the effect of obesity on heart proteome. These data demonstrate the immediate response of metabolic and stress-related proteins after exercise so as contractile protein by obesity modulation on heart proteome.

Preclinical data on morpholine (3,5-di-tertbutyl-4-hydroxyphenyl) methanone induced anxiolysis.

Trimetozine is used to be indicated for the treatment of mental illnesses, particularly anxiety. The present study provides data on the pharmacological profile of trimetozine derivative morpholine (3,5-di-tert-butyl-4-hydroxyphenyl) methanone (LQFM289) which was designed from molecular hybridization of trimetozine lead compound and 2,6-di-tert-butyl-hydroxytoluene to develop new anxiolytic drugs. Here, we conduct molecular dynamics simulations, docking studies, receptor binding assays, and in silico ADMET profiling of LQFM289 before its behavioral and biochemical assessment in mice within the dose range of 5-20 mg/kg. The docking of LQFM289 showed strong interactions with the benzodiazepine binding sites and matched well with receptor binding data. With the ADMET profile of this trimetozine derivative that predicts a high intestinal absorption and permeability to blood-brain barrier without being inhibited by the permeability glycoprotein, the oral administration of LQFM289 10 mg/kg consistently induced anxiolytic-like behavior of the mice exposed to the open field and light-dark box apparatus without eliciting motor incoordination in the wire, rotarod, and chimney tests. A decrease in the wire and rotarod´s fall latency coupled with an increase in the chimney test´s climbing time and a decrease in the number of crossings in the open field apparatus at the dose of 20 mg/kg of this trimetozine derivative suggest sedative or motor coordination impairment at this highest dose. The attenuation of the anxiolytic-like effects of LQFM289 (10 mg/kg) by flumazenil pretreatment implicates the participation of benzodiazepine binding sites. The lowering of corticosterone and tumor necrosis factor alpha (cytokine) in LQFM289-treated mice at a single oral (acute) dose of 10 mg/kg suggests that the anxiolytic-like effect of this compound also involves the recruitment of non-benzodiazepine binding sites/GABAergic molecular machinery.

Evaluation of Gastroprotective Activity of Linoleic Acid on Gastric Ulcer in a Mice Model.

BACKGROUND: Gastric ulcer has been a major cause of morbidity and mortality worldwide, and it has been linked to factors such as nutritional deficiency, smoking, stress, and continuous intake of non-steroidal antiinflammatory drugs (NSAIDs). The search for new anti-ulcer therapeutic agents has been the subject of several studies. Recently, the gastroprotective effect of Celtis iguanaea has been reported, with linoleic acid (LA) responsible for many of the therapeutic effects of this medicinal plant. AIMS: This study aims to investigate the gastroprotective activity and the possible mechanisms in which LA may be involved through different experimental assays in mice. METHODS: The gastroprotective activity of LA was evaluated in the ulcer induced by indomethacin, HCl/EtOH, hypothermic-restraint stress and pyloric ligation. For the investigation of gastroprotective mechanisms, the quantification of the volume (mL), pH and total acidity of gastric secretion were considered. RESULTS: The oral administrations of 25 mg/kg, 50 mg/kg or 100 mg/kg of body weight of LA were capable of protecting the gastric mucosa against HCl/ethanol (10 mL/kg p.o.), and oral/intraduodenal treatment administrations of 50 mg/kg LA showed protection from ulcers induced by indomethacin, hypothermic-restraint stress and pyloric ligation. CONCLUSION: The results of this study show the gastroprotective role of LA in gastric mucosal damage induced by all assayed distresses. The observed gastroprotection possibly occurs due to the mediated increase of mucosal defensive factors.

A Kunitz proteinase inhibitor from corms of Xanthosoma blandum with bactericidal activity.

Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus.

IL-4 absence triggers distinct pathways in apical periodontitis development.

Dental pulp is a specialized tissue able to respond to infectious processes. Nevertheless, infection progress and root canal colonization trigger an immune-inflammatory response in tooth-surrounding tissues, leading to apical periodontitis and bone tissue destruction, further contributing to tooth loss. In order to shed some light on the effects of IL-4 on periradicular pathology development modulation, microtomographic, histological and proteomic analyses were performed using 60 mice, 30 wild type and 30 IL-4-/-. For that, 5 animals were used for microtomographic and histological analysis, and another 5 for proteomic analysis for 0, 7 and 21 days with/without pulp exposure. The periapical lesions were established in WT and IL-4-/- mice without statistical differences in their volume, and the value of p < 0.05 was adopted as significant in microtomographic and histological analyses. Regarding histological analysis, IL-4-/- mice show aggravation of pulp inflammation compared to WT. By using proteomic analysis, we have identified 32 proteins with increased abundance and 218 proteins with decreased abundance in WT animals after 21 days of pulp exposure, compared to IL-4-/- animals. However, IL-4-/- mice demonstrated faster development of apical periodontitis. These animals developed a compensatory mechanism to overcome IL-4 absence, putatively based on the identification of upregulated proteins related to immune system signaling pathways. Significance: IL-4 might play a protective role in diseases involving bone destruction and its activity may contribute to host protection, mainly due to its antiosteoclastogenic action.

Involvement of the gabaergic, serotonergic and glucocorticoid mechanism in the anxiolytic-like effect of mastoparan-L.

Mastoparan-L (mast-L) is a cell-penetrating tetradecapeptide and stimulator of monoamine exocytosis. In the present study, we evaluated the anxiolytic-like effect of mast-L. Preliminary pharmacological tests were conducted to determine the most appropriate route of administration, extrapolate dose and detect potential toxic effects of this peptide. Oral and intracerebroventricular administration of mast-L (0.1, 0.3 or 0.9 mg.kg-1), diazepam (1 or 5 mg.kg-1), buspirone (10 mg.kg-1) or vehicle 10 mL.kg-1 was carried out prior to the exposure of mice to the anxiety models: open field, light-dark box and elevated plus-maze. To characterize the mechanism underlying the antianxiety-like effect of mast-L, pharmacological antagonism, blood plasma analysis, molecular docking, and receptor binding assays were performed. The absence of a neurotoxic sign, animal's death as well as lack of significant changes in the relative organ weight, hematological and biochemical parameters suggest that mast-L is relatively safe. The anxiolytic-like effect of mast-L was attenuated by flumazenil (antagonist of benzodiazepine binding site) and WAY100635 (selective antagonist of 5-HT1A receptors) pretreatments. Mast-L reduced plasma corticosterone and lowered the scoring function at GABAA -18.48 kcal/mol (Ki = 155 nM), 5-HT1A -22.39 kcal/mol (Ki = 130 nM), corticotropin-releasing factor receptor subtype 1 (CRF1) -11.95 kcal/mol (Ki = 299 nM) and glucocorticoid receptors (GR) -14.69 kcal/mol (Ki = 3552 nM). These data fit the binding affinity (Ki) and demonstrate the involvement of gabaergic, serotonergic and glucocorticoid mechanisms in the anxiolytic-like property of mast-L.

Perspectives on the Therapeutic Benefits of Arginine Supplementation in Cancer Treatment.

BACKGROUND: Arginine is considered a semi-essential amino acid in healthy adults and the elderly. This amino acid seems to improve the immune system, stimulate cell growth and differentiation, and increase endothelial permeability, among other effects. For those reasons, it has been theorized that arginine supplementation may be used as an adjuvant to conventional cancer therapy treatments. OBJECTIVE: This review aims to evaluate the existing knowledge of the scientific community on arginine supplementation in order to improve the efficacy of current cancer treatment. RESULTS: Despite the continued efforts of science to improve treatment strategies, cancer remains one of the greatest causes of death on the planet in adults and elderly people. Chemo and radiotherapy are still the most effective treatments but at the cost of significant side effects. CONCLUSION: Thus, new therapeutic perspectives have been studied in recent years, to be used in addition to traditional treatments or not, seeking to treat or even cure the various types of cancer with fewer side effects.

Cutting-Edge Search for Safer Opioid Pain Relief: Retrospective Review of Salvinorin A and Its Analogs.

Over the years, pain has contributed to low life quality, poor health, and economic loss. Opioids are very effective analgesic drugs for treating mild, moderate, or severe pain. Therapeutic application of opioids has been limited by short and long-term side effects. These side effects and opioid-overuse crisis has intensified interest in the search for new molecular targets and drugs. The present review focuses on salvinorin A and its analogs with the aim of exploring their structural and pharmacological profiles as clues for the development of safer analgesics. Ethnopharmacological reports and growing preclinical data have demonstrated the antinociceptive effect of salvinorin A and some of its analogs. The pharmacology of analogs modified at C-2 dominates the literature when compared to the ones from other positions. The distinctive binding affinity of these analogs seems to correlate with their chemical structure and in vivo antinociceptive effects. The high susceptibility of salvinorin A to chemical modification makes it an important pharmacological tool for cellular probing and developing analogs with promising analgesic effects. Additional research is still needed to draw reliable conclusions on the therapeutic potential of salvinorin A and its analogs.

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria.

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.

Host-defense peptides and their potential use as biomarkers in human diseases.

Since the early 19th century, host-defense peptides (HDPs) have been known to play a crucial role in innate host defense. Subsequent work has demonstrated their role in adaptive immunity as well as their involvement in cancer and also a number of inflammatory and/or autoimmune diseases. In addition to these multiple functional activities, several studies have shown that HDP accumulation might be correlated with various human diseases and, therefore, could be used as a biomarkers for such. Thus, research has aimed to validate the clinical use of HDPs for diagnosis, prognosis, and further treatment. In this review, we outline the most recent findings related to the use of HDPs as biomarkers, their clinical and epidemiological value, and the techniques used to determine the levels of HDPs.

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity.

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization⁻time-of-flight (MALDI⁻TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI.