Elizabete de Souza Cândido

Plant storage proteins with antimicrobial activity: novel insights into plant defense mechanisms

Storage proteins perform essential roles in plant survival, acting as molecular reserves important for plant growth and maintenance, as well as being involved in defense mechanisms by virtue of their properties as insecticidal and antimicrobial proteins. These proteins accumulate in storage vacuoles inside plant cells, and, in response to determined signals, they may be used by the different plant tissues in response to pathogen attack. To shed some light on these remarkable proteins with dual functions, storage proteins found in germinative tissues, such as seeds and kernels, and in vegetative tissues, such as tubercles and leaves, are extensively discussed here, along with the related mechanisms of protein expression. Among these proteins, we focus on 2S albumins, Kunitz proteinase inhibitors, plant lectins, glycine‐rich proteins, vicilins, patatins, tarins, and ocatins. Finally, the potential use of these molecules in development of drugs to combat human and plant pathogens, contributing to the development of new biotechnology‐based medications and products for agribusiness, is also presented.—De Souza Cândido, E., Pinto, M. F. S., Pelegrini, P. B., Lima, T. B., Silva, O. N., Pogue, R., Grossi‐de‐Sá, M. F., Franco, O. L. Plant storage proteins with antimicrobial activity: novel insights into plant defense mechanisms. FASEB J. 25, 3290–3305 (2011). www.fasebj.org

The use of versatile plant antimicrobial peptides in agribusiness and human health.

Plant immune responses involve a wide diversity of physiological reactions that are induced by the recognition of pathogens, such as hypersensitive responses, cell wall modifications, and the synthesis of antimicrobial molecules including antimicrobial peptides (AMPs). These proteinaceous molecules have been widely studied, presenting peculiar characteristics such as conserved domains and a conserved disulfide bond pattern. Currently, many AMP classes with diverse modes of action are known, having been isolated from a large number of organisms. Plant AMPs comprise an interesting source of studies nowadays, and among these there are reports of different classes, including defensins, albumins, cyclotides, snakins and several others. These peptides have been widely used in works that pursue human disease control, including nosocomial infections, as well as for agricultural purposes. In this context, this review will focus on the relevance of the structural-function relations of AMPs derived from plants and their proper use in applications for human health and agribusiness.

Bacterial resistance mechanism: what proteomics can elucidate

Antibiotics are important therapeutic agents commonly used for the control of bacterial infectious diseases; however, resistance to antibiotics has become a global public health problem. Therefore, effective therapy in the treatment of resistant bacteria is necessary and, to achieve this, a detailed understanding of mechanisms that underlie drug resistance must be sought. To fill the multiple gaps that remain in understanding bacterial resistance, proteomic tools have been used to study bacterial physiology in response to antibiotic stress. In general, the global analysis of changes in the protein composition of bacterial cells in response to treatment with antibiotic agents has made it possible to construct a database of proteins involved in the process of resistance to drugs with similar mechanisms of action. In the past few years, progress in using proteomic tools has provided the most realistic picture of the infective process, since these tools detect the end products of gene biosynthetic pathways, which may eventually determine a biological phenotype. In most bacterial species, alterations occur in energy and nitrogen metabolism regulation; glucan biosynthesis is up‐regulated; amino acid, protein, and nucleotide synthesis is affected; and various proteins show a stress response after exposing these microorganisms to antibiotics. These issues have been useful in identifying targets for the development of novel antibiotics and also in understanding, at the molecular level, how bacteria resist antibiotics.—Lima, T. B., Pinto, M. F. S., Ribeiro, S. M., de Lima, L. A., Viana, J. C., Júnior, N. G., Cândido, E. D., Dias, S. C., and Franco, O. L. Bacterial resistance mechanism: what proteomics can elucidate. FASEB J. 27, 1291–1303 (2013). www.fasebj.org

Synthetic antibiofilm peptides.

Bacteria predominantly exist as multicellular aggregates known as biofilms that are associated with at least two thirds of all infections and exhibit increased adaptive resistance to conventional antibiotic therapies. Therefore, biofilms are major contributors to the global health problem of antibiotic resistance, and novel approaches to counter them are urgently needed. Small molecules of the innate immune system called host defense peptides (HDPs) have emerged as promising templates for the design of potent, broad-spectrum antibiofilm agents. Here, we review recent developments in the new field of synthetic antibiofilm peptides, including mechanistic insights, synergistic interactions with available antibiotics, and their potential as novel antimicrobials against persistent infections caused by biofilms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Deciphering the magainin resistance process of Escherichia coli strains in light of the cytosolic proteome.

ABSTRACT Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter−1), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways.

Shedding some light over the floral metabolism by arum lily (Zantedeschia aethiopica) spathe de novo transcriptome assembly.

Zantedeschia aethiopica is an evergreen perennial plant cultivated worldwide and commonly used for ornamental and medicinal purposes including the treatment of bacterial infections. However, the current understanding of molecular and physiological mechanisms in this plant is limited, in comparison to other non-model plants. In order to improve understanding of the biology of this botanical species, RNA-Seq technology was used for transcriptome assembly and characterization. Following Z. aethiopica spathe tissue RNA extraction, high-throughput RNA sequencing was performed with the aim of obtaining both abundant and rare transcript data. Functional profiling based on KEGG Orthology (KO) analysis highlighted contigs that were involved predominantly in genetic information (37%) and metabolism (34%) processes. Predicted proteins involved in the plant circadian system, hormone signal transduction, secondary metabolism and basal immunity are described here. In silico screening of the transcriptome data set for antimicrobial peptide (AMP) –encoding sequences was also carried out and three lipid transfer proteins (LTP) were identified as potential AMPs involved in plant defense. Spathe predicted protein maps were drawn, and suggested that major plant efforts are expended in guaranteeing the maintenance of cell homeostasis, characterized by high investment in carbohydrate, amino acid and energy metabolism as well as in genetic information.

Venom gland transcriptome analyses of two freshwater stingrays (Myliobatiformes: Potamotrygonidae) from Brazil

Stingrays commonly cause human envenoming related accidents in populations of the sea, near rivers and lakes. Transcriptomic profiles have been used to elucidate components of animal venom, since they are capable of providing molecular information on the biology of the animal and could have biomedical applications. In this study, we elucidated the transcriptomic profile of the venom glands from two different freshwater stingray species that are endemic to the Paraná-Paraguay basin in Brazil, Potamotrygon amandae and Potamotrygon falkneri. Using RNA-Seq, we identified species-specific transcripts and overlapping proteins in the venom gland of both species. Among the transcripts related with envenoming, high abundance of hyaluronidases was observed in both species. In addition, we built three-dimensional homology models based on several venom transcripts identified. Our study represents a significant improvement in the information about the venoms employed by these two species and their molecular characteristics. Moreover, the information generated by our group helps in a better understanding of the biology of freshwater cartilaginous fishes and offers clues for the development of clinical treatments for stingray envenoming in Brazil and around the world. Finally, our results might have biomedical implications in developing treatments for complex diseases.

Understanding, preventing and eradicating Klebsiella pneumoniae biofilms

The ability of pathogenic bacteria to aggregate and form biofilm represents a great problem for public health, since they present extracellular components that encase these micro-organisms, making them more resistant to antibiotics and host immune attack. This may become worse when antibiotic-resistant bacterial strains form biofilms. However, antibiofilm screens with different compounds may reveal potential therapies to prevent/treat biofilm infections. Here, we focused on Klebsiella pneumoniae, an opportunistic bacterium that causes different types of infections, including in the bloodstream, meninges, lungs, urinary system and at surgical sites. We also highlight aspects involved in the formation and maintenance of K. pneumoniae biofilms, as well as resistance and the emergence of new trends to combat this health challenge.

Comparative NanoUPLC-MS E analysis between magainin I-susceptible and -resistant Escherichia coli strains

In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and –resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and β-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple “omics” studies in order to elucidate the global molecular mechanisms involved in this resistance.

An acidic model pro-peptide affects the secondary structure, membrane interactions and antimicrobial activity of a crotalicidin fragment

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.