United we stand, divided we fall: in-depth proteomic evaluation of the synergistic effect of Mo-CBP3-PepI and Ciprofloxacin against Staphylococcus aureus biofilms.

Staphylococcus aureus forms biofilms, a structure that protects bacterial cells, conferring more resistance to difficult treatment. Synthetic peptides surge as an alternative to overcome the biofilm of multidrug-resistant pathogens. Mo-CBP3-PepI, when combined with Ciprofloxacin, reduced preformed S. aureus biofilm by 50% at low concentrations (0.2 and 6.2 µg. mL-1, respectively). The goal of this study was to evaluate the proteomic profile of biofilms after treatment with the Mo-CBP3-PepI combined with ciprofloxacin. Here, proteomic analysis confirmed with more depth previously described mechanisms and revealed changes in the accumulation of proteins related to DNA and protein metabolism, cell wall biosynthesis, redox metabolism, quorum sensing, and biofilm formation. Some proteins related to DNA and protein metabolism were reduced, while other proteins, like redox system proteins, disappeared in Ciprofloxacin+Mo-CBP3-PepI treatment. Our results indicated a synergistic effect of these two molecules with several mechanisms against S. aureus biofilm and opened new doors for combined treatments with other drugs.

Proteomic analysis of Cryptostegia grandiflora latex, purification, characterization, and biological activity of two osmotin isoforms.

Although latex fluids are found in >20,000 plant species, the biochemical composition and biological function of their proteins are still poorly explored. Thus, this work aimed to conduct a proteomic analysis of Cryptostegia grandiflora latex (CgLP) for subsequent purification and characterization of an antifungal protein. After 2D-SDS-PAGE and mass spectrometry, 27 proteins were identified in CgLP, including a polygalacturonase inhibitor, cysteine peptidases, pathogenesis-related proteins (PR-4), and osmotins. Then, two osmotin isoforms (CgOsm) were purified, and a unique N-terminal sequence was determined (1ATFDIRSNCPYTVWAAAVPGGGRRLDRGQTWTINVAPGTA40). The PCR products revealed a cDNA sequence of 609 nucleotides for CgOsm, which encoded a polypeptide with 203 amino acid residues. The structure of CgOsm has features of typical osmotin or thaumatin-like proteins (TLPs), such as 16 conserved Cys residues, REDDD and FF motifs, an acidic cleft, and three main domains. Atomic force microscopy (AFM) and bioinformatics suggested that CgOsm is associated with three chain units. This result was interesting since the literature describes osmotins and TLPs as monomers. AFM also showed that Fusarium falciforme spores treated with CgOsm were drastically damaged. Therefore, it is speculated that CgOsm forms pores in the membrane of these cells, causing the leakage of cytoplasmic content.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Giving a Hand: Synthetic Peptides Boost the Antifungal Activity of Itraconazole against Cryptococcus neoformans.

Cryptococcus neoformans is a multidrug-resistant pathogen responsible for infections in immunocompromised patients. Here, itraconazole (ITR), a commercial antifungal drug with low effectiveness against C. neoformans, was combined with different synthetic antimicrobial peptides (SAMPs), Mo-CBP3-PepII, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA. The Mo-CBP3-PepII was designed based on the sequence of MoCBP3, purified from Moringa oleifera seeds. RcAlb-PepII and RcAlb-PepIII were designed using Rc-2S-Alb, purified from Ricinus communis seed cakes. The putative sequence of a chitinase from Arabidopsis thaliana was used to design PepGAT and PepKAA. All SAMPs have a positive liquid charge and a hydrophobic potential ranging from 41-65%. The mechanisms of action responsible for the combined effect were evaluated for the best combinations using fluorescence microscopy (FM). The synthetic peptides enhanced the activity of ITR by 10-fold against C. neoformans. Our results demonstrated that the combinations could induce pore formation in the membrane and the overaccumulation of ROS on C. neoformans cells. Our findings indicate that our peptides successfully potentialize the activity of ITR against C. neoformans. Therefore, synthetic peptides are potential molecules to assist antifungal agents in treating Cryptococcal infections.

No Chance to Survive: Mo-CBP3-PepII Synthetic Peptide Acts on Cryptococcus neoformans by Multiple Mechanisms of Action.

Multidrug-resistant Cryptococcus neoformans is an encapsulated yeast causing a high mortality rate in immunocompromised patients. Recently, the synthetic peptide Mo-CBP3-PepII emerged as a potent anticryptococcal molecule with an MIC50 at low concentration. Here, the mechanisms of action of Mo-CBP3-PepII were deeply analyzed to provide new information about how it led C. neoformans cells to death. Light and fluorescence microscopies, analysis of enzymatic activities, and proteomic analysis were employed to understand the effect of Mo-CBP3-PepII on C. neoformans cells. Light and fluorescence microscopies revealed Mo-CBP3-PepII induced the accumulation of anion superoxide and hydrogen peroxide in C. neoformans cells, in addition to a reduction in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) in the cells treated with Mo-CBP3-PepII. In the presence of ascorbic acid (AsA), no reactive oxygen species (ROS) were detected, and Mo-CBP3-PepII lost the inhibitory activity against C. neoformans. However, Mo-CBP3-PepII inhibited the activity of lactate dehydrogenase (LDH) ergosterol biosynthesis and induced the decoupling of cytochrome c (Cyt c) from the mitochondrial membrane. Proteomic analysis revealed a reduction in the abundance of proteins related to energetic metabolism, DNA and RNA metabolism, pathogenicity, protein metabolism, cytoskeleton, and cell wall organization and division. Our findings indicated that Mo-CBP3-PepII might have multiple mechanisms of action against C. neoformans cells, mitigating the development of resistance and thus being a potent molecule to be employed in the production of new drugs against C. neoformans infections.

Behind the Curtain: In Silico and In Vitro Experiments Brought to Light New Insights into the Anticryptococcal Action of Synthetic Peptides.

Cryptococcus neoformans is the pathogen responsible for cryptococcal pneumonia and meningitis, mainly affecting patients with suppressed immune systems. We have previously revealed the mechanism of anticryptococcal action of synthetic antimicrobial peptides (SAMPs). In this study, computational and experimental analyses provide new insights into the mechanisms of action of SAMPs. Computational analysis revealed that peptides interacted with the PHO36 membrane receptor of C. neoformans. Additionally, ROS (reactive oxygen species) overproduction, the enzymes of ROS metabolism, interference in the ergosterol biosynthesis pathway, and decoupling of cytochrome c mitochondrial membrane were evaluated. Three of four peptides were able to interact with the PHO36 receptor, altering its function and leading to ROS overproduction. SAMPs-treated C. neoformans cells showed a decrease in scavenger enzyme activity, supporting ROS accumulation. In the presence of ascorbic acid, an antioxidant agent, SAMPs did not induce ROS accumulation in C. neoformans cells. Interestingly, two SAMPs maintained inhibitory activity and membrane pore formation in C. neoformans cells by a ROS-independent mechanism. Yet, the ergosterol biosynthesis and lactate dehydrogenase activity were affected by SAMPs. In addition, we noticed decoupling of Cyt c from the mitochondria, which led to apoptosis events in the cryptococcal cells. The results presented herein suggest multiple mechanisms imposed by SAMPs against C. neoformans interfering in the development of resistance, thus revealing the potential of SAMPs in treating infections caused by C. neoformans.

New Insights into the Mechanism of Antibacterial Action of Synthetic Peptide Mo-CBP3-PepI against Klebsiella pneumoniae.

Klebsiella pneumoniae is a multidrug-resistant opportunistic human pathogen related to various infections. As such, synthetic peptides have emerged as potential alternative molecules. Mo-CBP3-PepI has presented great activity against K. pneumoniae by presenting an MIC50 at a very low concentration (31.25 µg mL-1). Here, fluorescence microscopy and proteomic analysis revealed the alteration in cell membrane permeability, ROS overproduction, and protein profile of K. pneumoniae cells treated with Mo-CBP3-PepI. Mo-CBP3-PepI led to ROS overaccumulation and membrane pore formation in K. pneumoniae cells. Furthermore, the proteomic analysis highlighted changes in essential metabolic pathways. For example, after treatment of K. pneumoniae cells with Mo-CBP3-PepI, a reduction in the abundance of protein related to DNA and protein metabolism, cytoskeleton and cell wall organization, redox metabolism, regulation factors, ribosomal proteins, and resistance to antibiotics was seen. The reduction in proteins involved in vital processes for cell life, such as DNA repair, cell wall turnover, and protein turnover, results in the accumulation of ROS, driving the cell to death. Our findings indicated that Mo-CBP3-PepI might have mechanisms of action against K. pneumoniae cells, mitigating the development of resistance and thus being a potent molecule to be employed in producing new drugs against K. pneumoniae infections.

Essential Oil from Croton blanchetianus Leaves: Anticandidal Potential and Mechanisms of Action.

Antimicrobial drugs are becoming ineffective given the resistance acquired by microorganisms. As such, it is imperative to seek new antimicrobial molecules that could provide a basis for the development of new drugs. Therefore, this work aimed to evaluate the antimicrobial potential and the mechanisms of action of the essential oil extracted from leaves of Croton blanchetianus (named CbEO) on different fungi and bacteria of clinical importance in both planktonic and biofilm lifestyles. GC-MS/MS analysis revealed the presence of twenty-two different compounds in the CbEO, which were identified using the Kovats retention index. Among these, the most abundant were amorphene (20.03%), spathulenol (5%), bicyclogermacrene (1.49%), caryophyllene oxide (4.55%), and eucalyptol (5.62%). CbOE (50 µg mL-1) barely inhibited the growth of Bacillus subtilis (23%), Pseudomonas aeruginosa (27%), and Salmonella enterica (28%), and no inhibition was obtained against Enterobacter aerogenes and Klebsiella pneumoniae. Additionally, no activity against bacterial biofilm was detected. In contrast, CbEO was active against Candida species. C. albicans and C. parapsilosis were inhibited by 78 and 75%, respectively. The antibiofilm potential also was favorable against C. albicans and C. parapsilosis, inhibiting 44 and 74% of biofilm formation and reducing around 41 and 27% of the preformed biofilm, respectively. CbOE caused membrane damage and pore formation, overproduction of ROS, and apoptosis on C. albicans and C. parapsilosis cells, as well as not inducing hemolysis in human red cells. The results obtained in this work raise the possibility of using the essential oil of C. blanchetianus leaves as an alternative to fight infections caused by C. albicans and C. parapsilosis.

Plant Lectins: A Review on their Biotechnological Potential Toward Human Pathogens.

The indiscriminate use of antibiotics is associated with the appearance of bacterial resistance. In light of this, plant-based products treating infections are considered potential alternatives. Lectins are a group of proteins widely distributed in nature, capable of reversibly binding carbohydrates. Lectins can bind to the surface of pathogens and cause damage to their structure, thus preventing host infection. The antimicrobial activity of plant lectins results from their interaction with carbohydrates present in the bacterial cell wall and fungal membrane. The data about lectins as modulating agents of antibiotic activity, potentiates the effect of antibiotics without triggering microbial resistance. In addition, lectins play an essential role in the defense against fungi, reducing their infectivity and pathogenicity. Little is known about the antiviral activity of plant lectins. However, their effectiveness against retroviruses and parainfluenza is reported in the literature. Some authors still consider mannose/ glucose/N-Acetylglucosamine binding lectins as potent antiviral agents against coronavirus, suggesting that these lectins may have inhibitory activity against SARS-CoV-2. Thus, it was found that plant lectins are an alternative for producing new antimicrobial drugs, but further studies still need to decipher some mechanisms of action.

Synergistic Antibiofilm Activity between Synthetic Peptides and Ciprofloxacin against Staphylococcus aureus.

Staphylococcus aureus is a human pathogen known to be resistant to antibiotics since the mid-20th century and is constantly associated with hospital-acquired infections. S. aureus forms biofilms, which are complex surface-attached communities of bacteria held together by a self-produced polymer matrix consisting of proteins, extracellular DNA, and polysaccharides. Biofilms are resistance structures responsible for increasing bacterial resistance to drugs by 1000 times more than the planktonic lifestyle. Therefore, studies have been conducted to discover novel antibacterial molecules to prevent biofilm formation and/or degrade preformed biofilms. Synthetic antimicrobial peptides (SAMPs) have appeared as promising alternative agents to overcome increasing antibiotic resistance. Here, the antibiofilm activity of eight SAMPs, in combination with the antibiotic ciprofloxacin, was investigated in vitro. Biofilm formation by S. aureus was best inhibited (76%) by the combination of Mo-CBP3-PepIII (6.2 µg mL-1) and ciprofloxacin (0.39 µg mL-1). In contrast, the highest reduction (60%) of the preformed biofilm mass was achieved with RcAlb-PepII (1.56 µg mL-1) and ciprofloxacin (0.78 µg mL-1). Fluorescence microscopy analysis reinforced these results. These active peptides formed pores in the cellular membrane of S. aureus, which may be related to the enhanced ciprofloxacin's antibacterial activity. Our findings indicated that these peptides may act with ciprofloxacin and are powerful co-adjuvant agents for the treatment of S. aureus infections.

Antifungal Potential of Synthetic Peptides against Cryptococcus neoformans: Mechanism of Action Studies Reveal Synthetic Peptides Induce Membrane-Pore Formation, DNA Degradation, and Apoptosis.

Cryptococcus neoformans is a human-pathogenic yeast responsible for pneumonia and meningitis, mainly in patients immunocompromised. Infections caused by C. neoformans are a global health concern. Synthetic antimicrobial peptides (SAMPs) have emerged as alternative molecules to cope with fungal infections, including C. neoformans. Here, eight SAMPs were tested regarding their antifungal potential against C. neoformans and had their mechanisms of action elucidated by fluorescence and scanning electron microscopies. Five SAMPs showed an inhibitory effect (MIC50) on C. neoformans growth at low concentrations. Fluorescence microscope (FM) revealed that SAMPs induced 6-kDa pores in the C. neoformans membrane. Inhibitory assays in the presence of ergosterol revealed that some peptides lost their activity, suggesting interaction with it. Furthermore, FM analysis revealed that SAMPs induced caspase 3/7-mediated apoptosis and DNA degradation in C. neoformans cells. Scanning Electron Microscopy (SEM) analysis revealed that peptides induced many morphological alterations such as cell membrane, wall damage, and loss of internal content on C. neoformans cells. Our results strongly suggest synthetic peptides are potential alternative molecules to control C. neoformans growth and treat the cryptococcal infection.

Luffa operculata seed proteins: Identification by LC-ESI-MS/MS and biotechnological potential against Candida albicans and C. krusei.

L: operculata is a plant commonly found in the North and Northeast of Brazil. Although the regional population knows its medicinal potential, there are few scientific studies about its antimicrobial potential. Thus, this study aimed to characterize the proteins from L. operculata seeds extracted using different solutions and evaluate their antimicrobial potentials. The protein extracts obtained with NaCl and sodium acetate buffer presented the best inhibitory activities against Candida albicans and C. krusei. The study of the mechanism of action revealed proteins from L. operculata seeds induced pore formation on the membrane and ROS overaccumulation. Scanning Electron Microscopy images also showed severe morphological changes in Candida albicans and C. krusei. Proteins from L.operculata seeds did not show antibacterial activity. The enzymatic assays revealed the presence of proteolytic enzymes, serine and cysteine protease inhibitors, and chitinases in both protein extracts. Proteomic analysis by LC-ESI-MS/MS identified 57 proteins related to many biological processes, such as defense to (a)biotic stress, energetic metabolism, protein folding, and nucleotide metabolism. In conclusion, the L. operculata seed proteins have biotechnological potential against the human pathogenic yeasts Candida albicans and C. krusei.