Synthesis, characterization, antimicrobial activity, and toxicity evaluation of aminolevulinic acid-silver and silver-iron nanoparticles for potential applications in agriculture.

Aminolevulinic acid (ALA) is considered one of the most critical plants growth regulators and essential precursors for chlorophyll biosynthesis; besides, its photodynamic activity can be used to exterminate larvae and microorganisms in plants and soil. Silver nanoparticles (AgNPs) have unique physicochemical properties and potent antimicrobial, antiviral, and antifungal activities, and in agriculture, their application as nanopesticides has been proposed. In this study, silver and silver-iron nanoparticles capped/stabilized with aminolevulinic acid (ALAAgNPs and ALAAgFeNPs) were synthesized by the photoreduction method and characterized by UV-vis spectroscopy, transmission electron microscopy, and zeta potential analysis. The kinetics of 1O2 generation from ALAAgFeNPs were obtained. The ALANP toxicity was evaluated on stalks of E. densa by observing cell morphology changes and measuring chlorophyll content compared with water-treated plants. Antimicrobial activity was tested against E. coli, P. aeruginosa, and Candida albicans. The results suggested that ALANPs (prepared with [AgNO3] ≤ 0.2 mM and [ALA] ≤ 0.4 mM) could be suitable for applications in the agricultural sector. The presence of ∼0.3 mmol of iron in ALAAgNPs synthesis increased cell uptake and chlorophyll synthesis.

Antimicrobial Activity of Silver and Gold Nanoparticles Prepared by Photoreduction Process with Leaves and Fruit Extracts of Plinia cauliflora and Punica granatum.

The increased number of resistant microbes generates a search for new antibiotic methods. Metallic nanoparticles have emerged as a new platform against several microorganisms. The nanoparticles can damage the bacteria membrane and DNA by oxidative stress. The photoreduction process is a clean and low-cost method for obtaining silver and gold nanoparticles. This work describes two original insights: (1) the use of extracts of leaves and fruits from a Brazilian plant Plinia cauliflora, compared with a well know plant Punica granatum, and (2) the use of phytochemicals as stabilizing agents in the photoreduction process. The prepared nanoparticles were characterized by UV-vis, FTIR, transmission electron microscopy, and Zeta potential. The antimicrobial activity of nanoparticles was obtained with Gram-negative and Gram-positive bacteria, particularly the pathogens Staphylococcus aureus ATCC 25923; Bacillus subtilis ATCC 6633; clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis; Escherichia coli ATCC 25922; Escherichia coli O44:H18 EAEC042 (clinical isolate); Klebsiella pneumoniae ATCC 700603, Salmonella Thiphymurium ATCC 10231; Pseudomonas aeruginosa ATCC 27853; and Candida albicans ATCC 10231. Excellent synthesis results were obtained. The AgNPs exhibited antimicrobial activities against Gram-negative and Gram-positive bacteria and yeast (80-100%), better than AuNPs (0-87.92%), and may have the potential to be used as antimicrobial agents.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

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.

Eugenia uniflora L. Silver and Gold Nanoparticle Synthesis, Characterization, and Evaluation of the Photoreduction Process in Antimicrobial Activities.

Eugenia uniflora linnaeus, known as Brazilian cherry, is widely distributed in Brazil, Argentina, Uruguay, and Paraguay. E. uniflora L. extracts contain phenolic compounds, such as flavonoids, tannins, triterpenes, and sesquiterpenes. The antimicrobial action of essential oils has been attributed to their compositions of bioactive compounds, such as sesquiterpenes. In this paper, the fruit extract of E. uniflora was used to synthesize silver and gold nanoparticles. The nanoparticles were characterized by UV-Vis, transmission electron microscopy, elemental analysis, FTIR, and Zeta potential measurement. The silver and gold nanoparticles prepared with fruit extracts presented sizes of ~32 nm and 11 nm (diameter), respectively, and Zeta potentials of -22 mV and -14 mV. The antimicrobial tests were performed with Gram-negative and Gram-positive bacteria and Candida albicans. The growth inhibition of EuAgNPs prepared with and without photoreduction showed the important functional groups in the antimicrobial activity.

Antimicrobial activity of silver and gold nanoparticles prepared by photoreduction process with leaves and fruit extracts of Plinia cauliflora and Punica granatum

The increased number of resistant microbes generates a search for new antibiotic methods. Metallic nanoparticles have emerged as a new platform against several microorganisms. The nanoparticles can damage the bacteria membrane and DNA by oxidative stress. The photoreduction process is a clean and low-cost method for obtaining silver and gold nanoparticles. This work describes two original insights: (1) the use of extracts of leaves and fruits from a Brazilian plant Plinia cauliflora, compared with a well know plant Punica granatum, and (2) the use of phytochemicals as stabilizing agents in the photoreduction process. The prepared nanoparticles were characterized by UV-vis, FTIR, transmission electron microscopy, and Zeta potential. The antimicrobial activity of nanoparticles was obtained with Gram-negative and Gram-positive bacteria, particularly the pathogens Staphylococcus aureus ATCC 25923; Bacillus subtilis ATCC 6633; clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis; Escherichia coli ATCC 25922; Escherichia coli O44:H18 EAEC042 (clinical isolate); Klebsiella pneumoniae ATCC 700603, Salmonella Thiphymurium ATCC 10231; seudomonas aeruginosa ATCC 27853; and Candida albicans ATCC 10231. Excellent synthesis results were obtained. The AgNPs exhibited antimicrobial activities against Gram-negative and Gram-positive bacteria and yeast (80–100%), better than AuNPs (0–87.92%), and may have the potential to be used as antimicrobial agents.

Synthesis, characterization, antimicrobial activity, and toxicity evaluation of aminolevulinic acid–silver and silver–iron nanoparticles for potential applications in agriculture

Aminolevulinic acid (ALA) is considered one of the most critical plants growth regulators and essential precursors for chlorophyll biosynthesis; besides, its photodynamic activity can be used to exterminate larvae and microorganisms in plants and soil. Silver nanoparticles (AgNPs) have unique physicochemical properties and potent antimicrobial, antiviral, and antifungal activities, and in agriculture, their application as nanopesticides has been proposed. In this study, silver and silver–iron nanoparticles capped/stabilized with aminolevulinic acid (ALAAgNPs and ALAAgFeNPs) were synthesized by the photoreduction method and characterized by UV-vis spectroscopy, transmission electron microscopy, and zeta potential analysis. The kinetics of 1O2 generation from ALAAgFeNPs were obtained. The ALANP toxicity was evaluated on stalks of E. densa by observing cell morphology changes and measuring chlorophyll content compared with water-treated plants. Antimicrobial activity was tested against E. coli, P. aeruginosa, and Candida albicans. The results suggested that ALANPs (prepared with [AgNO3] ≤ 0.2 mM and [ALA] ≤ 0.4 mM) could be suitable for applications in the agricultural sector. The presence of ∼0.3 mmol of iron in ALAAgNPs synthesis increased cell uptake and chlorophyll synthesis.

Leptolysin, a leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity

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.

Eugenia uniflora L. silver and gold nanoparticle synthesis, characterization, and evaluation of the photoreduction process in antimicrobial activities

Eugenia uniflora linnaeus, known as Brazilian cherry, is widely distributed in Brazil, Argentina, Uruguay, and Paraguay. E. uniflora L. extracts contain phenolic compounds, such as flavonoids, tannins, triterpenes, and sesquiterpenes. The antimicrobial action of essential oils has been attributed to their compositions of bioactive compounds, such as sesquiterpenes. In this paper, the fruit extract of E. uniflora was used to synthesize silver and gold nanoparticles. The nanoparticles were characterized by UV–Vis, transmission electron microscopy, elemental analysis, FTIR, and Zeta potential measurement. The silver and gold nanoparticles prepared with fruit extracts presented sizes of ~32 nm and 11 nm (diameter), respectively, and Zeta potentials of −22 mV and −14 mV. The antimicrobial tests were performed with Gram-negative and Gram-positive bacteria and Candida albicans. The growth inhibition of EuAgNPs prepared with and without photoreduction showed the important functional groups in the antimicrobial activity.

Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.

Inactivation of the antimicrobial peptide LL-37 by pathogenic Leptospira

Leptospires are aerobic, Gram-negative spirochetes with a high invasive capacity. Pathogenic leptospires secrete proteases that inactivate a variety of host's proteins including molecules of the extracellular matrix and of the human complement system. This strategy, used by several pathogens of medical importance, contributes to bacterial invasion and immune evasion. In the current work we present evidence that Leptospira proteases also target human cathelicidin (LL-37), an antimicrobial peptide that plays an important role in the innate immune response. By using six Leptospira strains, four pathogenic and two saprophytic, we demonstrated that proteases present in the supernatants of pathogenic strains were capable of degrading LL-37 in a time-dependent manner, whereas proteolytic degradation was not observed with the supernatants of the two saprophytic strains. Inactivation of LL-37 was prevented by using the 1,10-phenanthroline inhibitor, thus suggesting the involvement of metalloproteinases in this process. In addition, the antibacterial activity of LL-37 against two Leptospira strains was evaluated. Compared to the saprophytic strain, a greater resistance of the pathogenic strain to the action of the peptide was observed. Our data suggest that the capacity to inactivate the host defense peptide LL-37 may be part of the virulence arsenal of pathogenic Leptospira, and we hypothesize that its inactivation by the bacteria may influence the outcome of the disease.

A new class of antimicrobial molecules derived from kefir, effective against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA) strains

Many studies have linked the antimicrobial properties of kefir with the presence of bacteriocins and organic acids. In the present work, results obtained from bacteriostatic and bactericidal studies, and from RP-HPLC, Mass Spectrometry and proton NMR analysis, show that a sample of milk kefir grains is able to produce an antimicrobial fraction, denoted FK-1000, composed of sugars and amino acids, predominantly polymers of alanine, doublets of tyrosine and phenylalanine. Since this fraction is a lyophilized product whose molecular profile is different from bacteriocins and simple carboxylic acids, its antimicrobial effect cannot be attributed to these molecules, or to alcohols or hydrogen peroxide. The fraction is bactericidal against weak-acid-resistant MRSA and weak-acid resistant P. aeruginosa at pH 5, and is bacteriostatic against both pathogens at pH 7. In combination formulation, the FK-1000 fraction is able to increase fivefold the effect of streptomycin against P. aeruginosa and it is not toxic to human epithelial cells at antimicrobial concentrations. 16 S rRNA microbiota analysis of antimicrobial-producing and non-producing kefir grains demonstrated that they are distinct. In summary, the results indicate that milk kefir grains can produce different classes of molecules with potent antibiotic activity against resistant bacteria.

Tryptophan Silver Nanoparticles Synthesized by Photoreduction Method: Characterization and Determination of Bactericidal and Anti-Biofilm Activities on Resistant and Susceptible Bacteria.

The high rates of antibiotics use in hospitals have resulted in a condition where multidrug-resistant pathogens have become a severe threat to the human health worldwide. Therefore, there is an increasing necessity to identify new antimicrobial agents that can inhibit the multidrug-resistant bacteria and biofilm formation. In this study, antibacterial and anti-biofilm activities of tryptophan silver nanoparticles (TrpAgNP) were investigated. The TrpAgNPs were synthesized by photoreduction method, and the influence of irradiation time and concentration of reagents were analyzed. The nanoparticles were characterized by transmission electron microscopy, Zeta Potential and (UV)-absorption spectra. The antibacterial activity of TrpAgNPs was tested for antibiotic-resistant and susceptible pathogens, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Salmonella typhimurium, and Pseudomonas aeruginosa, evaluating the influence of photoreduction parameters in bactericidal effect. The results have shown that TrpAgNPs solutions with lower tryptophan/silver nitrate (AgNO3) ratio and higher AgNO3 concentration have higher bactericidal action against bacteria with inhibition of ~100% in almost all studied bacterial strains. The antimicrobial activity of TrpAgNPs within biofilms generated under static conditions of antibiotic-resistant and susceptible strains of S. aureus, S. epidermidis, E. coli, K. pneumoniae, C. freundii, and P. aeruginosa was also investigated. The results showed that TrpAgNPs have an inhibitory effect against biofilm formation, exceeding 50% in the case of Gram-negative bacteria (E. coli, K. pneumoniae, C. freundii, and P. aeruginosa-54.8% to 98.8%). For Gram-positive species, an inhibition of biofilm formation of 68.7% to 72.2 % was observed for S. aureus and 20.0% to 40.2% for S. epidermidis.

Tryptophan silver nanoparticles synthesized by photoreduction method: characterization and determination of bactericidal and anti-biofilm activities on resistant and susceptible bacteria

The high rates of antibiotics use in hospitals have resulted in a condition where multidrug-resistant pathogens have become a severe threat to the human health worldwide. Therefore, there is an increasing necessity to identify new antimicrobial agents that can inhibit the multidrug-resistant bacteria and biofilm formation. In this study, antibacterial and anti-biofilm activities of tryptophan silver nanoparticles (TrpAgNP) were investigated. The TrpAgNPs were synthesized by photoreduction method, and the influence of irradiation time and concentration of reagents were analyzed. The nanoparticles were characterized by transmission electron microscopy, Zeta Potential and (UV)-absorption spectra. The antibacterial activity of TrpAgNPs was tested for antibiotic-resistant and susceptible pathogens, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Salmonella typhimurium, and Pseudomonas aeruginosa, evaluating the influence of photoreduction parameters in bactericidal effect. The results have shown that TrpAgNPs solutions with lower tryptophan/silver nitrate (AgNO3) ratio and higher AgNO3 concentration have higher bactericidal action against bacteria with inhibition of ~100% in almost all studied bacterial strains. The antimicrobial activity of TrpAgNPs within biofilms generated under static conditions of antibiotic-resistant and susceptible strains of S. aureus, S. epidermidis, E. coli, K. pneumoniae, C. freundii, and P. aeruginosa was also investigated. The results showed that TrpAgNPs have an inhibitory effect against biofilm formation, exceeding 50% in the case of Gram-negative bacteria (E. coli, K. pneumoniae, C. freundii, and P. aeruginosa—54.8% to 98.8%). For Gram-positive species, an inhibition of biofilm formation of 68.7% to 72.2 % was observed for S. aureus and 20.0% to 40.2% for S. epidermidis.

Tryptophan silver nanoparticles synthesized by photoreduction method: characterization and determination of bactericidal and anti-biofilm activities on resistant and susceptible bacteria

The high rates of antibiotics use in hospitals have resulted in a condition where multidrug-resistant pathogens have become a severe threat to the human health worldwide. Therefore, there is an increasing necessity to identify new antimicrobial agents that can inhibit the multidrug-resistant bacteria and biofilm formation. In this study, antibacterial and anti-biofilm activities of tryptophan silver nanoparticles (TrpAgNP) were investigated. The TrpAgNPs were synthesized by photoreduction method, and the influence of irradiation time and concentration of reagents were analyzed. The nanoparticles were characterized by transmission electron microscopy, Zeta Potential and (UV)-absorption spectra. The antibacterial activity of TrpAgNPs was tested for antibiotic-resistant and susceptible pathogens, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Salmonella typhimurium, and Pseudomonas aeruginosa, evaluating the influence of photoreduction parameters in bactericidal effect. The results have shown that TrpAgNPs solutions with lower tryptophan/silver nitrate (AgNO3) ratio and higher AgNO3 concentration have higher bactericidal action against bacteria with inhibition of ~100% in almost all studied bacterial strains. The antimicrobial activity of TrpAgNPs within biofilms generated under static conditions of antibiotic-resistant and susceptible strains of S. aureus, S. epidermidis, E. coli, K. pneumoniae, C. freundii, and P. aeruginosa was also investigated. The results showed that TrpAgNPs have an inhibitory effect against biofilm formation, exceeding 50% in the case of Gram-negative bacteria (E. coli, K. pneumoniae, C. freundii, and P. aeruginosa—54.8% to 98.8%). For Gram-positive species, an inhibition of biofilm formation of 68.7% to 72.2 % was observed for S. aureus and 20.0% to 40.2% for S. epidermidis.KeywoRDS: tryptophan, silver nanoparticles, photoreduction, antimicrobial activity, anti-biofilmReCeIV eD: December 20, 2018. ACCePTeD: January 20, 2019.TyPe: Original ResearchFuNDINg: The authors are grateful to the FAPESP (2014/06960-9) and CNPq, Brazilian funding agencies, for their financial support.DeClARATIoN oF C oNFlICTINg INTeReSTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.CoRReSPoNDINg AuTHoR: Lilia Coronato Courrol, Laboratório de Lasers e Óptica Biomédica Aplicada and Departamento de Física, Universidade Federal de São Paulo, Diadema SP 09972-270, Brazil. Email: lccourrol@gmail.com831677TRY0010.1177/1178646919831677International Journal of Tryptophan ResearchCourrol et alresearch-article2019

Optical properties and antimicrobial effects of silver nanoparticles synthesized by femtosecond laser photoreduction

Silver nanoparticles exhibit a powerful antimicrobial action showing a pronounced potential to be widely used against drug resistance bacteria. The present work describes the optical properties and antimicrobial effect of silver nanoparticles produced by femtosecond laser photoreduction of AgNO3 in the presence of tryptophan water solution. The advantages of this method are the absence of hazardous chemical reducing agents in the solution, and the versatile dimensional control achieved. The synthesized silver nanoparticles were characterized by absorption and fluorescence spectroscopy and their antibacterial activity were determined by monitoring the cell viability of Escherichia coli. The effects of the silver nanoparticles concentration and laser parameters (exposure time and pulse energy), on the formation of the nanoparticles, and its influence on the bacteria growth inhibition were studied. The prepared silver nanoparticles exhibited suitable antimicrobial properties. The results demonstrated that the nanoparticles concentration plays an important role in their bactericidal efficacy. The increase in the laser energy caused an increase in E. coli growth inhibition. Irradiations with energies around 300?µJ for 60?min presented high antimicrobial activity due to the presence of kynurenine, sub product of tryptophan photolysis. The first-time formation mechanism of tryptophan silver nanoparticles in high optical intensities was also discussed.

Optical properties and antimicrobial effects of silver nanoparticles synthesized by femtosecond laser photoreduction

Silver nanoparticles exhibit a powerful antimicrobial action showing a pronounced potential to be widely used against drug resistance bacteria. The present work describes the optical properties and antimicrobial effect of silver nanoparticles produced by femtosecond laser photoreduction of AgNO3 in the presence of tryptophan water solution. The advantages of this method are the absence of hazardous chemical reducing agents in the solution, and the versatile dimensional control achieved. The synthesized silver nanoparticles were characterized by absorption and fluorescence spectroscopy and their antibacterial activity were determined by monitoring the cell viability of Escherichia coli. The effects of the silver nanoparticles concentration and laser parameters (exposure time and pulse energy), on the formation of the nanoparticles, and its influence on the bacteria growth inhibition were studied. The prepared silver nanoparticles exhibited suitable antimicrobial properties. The results demonstrated that the nanoparticles concentration plays an important role in their bactericidal efficacy. The increase in the laser energy caused an increase in E. coli growth inhibition. Irradiations with energies around 300?µJ for 60?min presented high antimicrobial activity due to the presence of kynurenine, sub product of tryptophan photolysis. The first-time formation mechanism of tryptophan silver nanoparticles in high optical intensities was also discussed.

Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement proteins.

Leptospirosis is an infectious disease of public health importance. To successfully colonize the host, pathogens have evolved multiple strategies to escape the complement system. Here we demonstrate that the culture supernatant of pathogenic but not saprophytic Leptospira inhibit the three complement pathways. We showed that the proteolytic activity in the supernatants of pathogenic strains targets the central complement molecule C3 and specific proteins from each pathway, such as factor B, C2, and C4b. The proteases cleaved α and ß chains of C3 and work in synergy with host regulators to inactivate C3b. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. A recombinant leptospiral metalloprotease from the thermolysin family cleaved C3 in serum and could be one of the proteases responsible for the supernatant activity. We conclude that pathogenic leptospiral proteases can deactivate immune effector molecules and represent potential targets to the development of new therapies in leptospirosis.