Project 1. Mechanistic of the phosphate-uptake and regulation: the close relationship between an ATP- Binding Cassette transporter and a two-component system

The Pho regulon is characterized as a set of genes controlled by a common regulator, whose proteins are involved in the absorption, assimilation and regulation of phosphate in cells. In E. coli, the PhoR/PhoB two-component system is activated when extracellular inorganic phosphate levels are less than 4 μmol/L. PhoR is a membrane protein that undergoes autophosphorylation in the absence of an ion and transfers the phosphoryl group to PhoB, a transcriptional regulator. PhoB has high affinity for regions called ‘phobox’, highly conserved in the promoters of genes related to phosphate metabolism. One of the targets of PhoB is the pstSCABphoU operon that encodes the Pst system (specific phosphate transport system), a high-affinity ABC-type transporter (Schramke et al, 2017), and its negative regulator, the PhoU protein. The PstSCAB-PhoU system has been linked in different organisms to mechanisms of virulence/pathogenicity, regulation of biofilm formation, invasion, antibiotic resistance and colonization. X. citri conserves the essential genes of the regulon, such as those encoding the alkaline phosphatase PhoA, considered a marker of the regulon, outer membrane proteins, enzymes, the two-component system PhoR/PhoB, the PstSCAB system and the regulatory protein PhoU (Pegos et al. al, 2014). The genes of the pstSCABphoU operon of energy for transport. The last gene of the operon encodes the PhoU protein, the negative regulator of the Pst system (Haddad, 2009).

We intend to answer questions related to the essential components of the Pho regulon of X. citri, but which can be extrapolated to other microorganisms, since the system is highly conserved. They are: (i) how does the interaction between PhoR, PhoU and PhoB happen? (ii) what would the interaction between the Pst-PhoU-PhoR systems be like? What would be the significant structural/physiological changes for this interaction?; (iii) Why does X. citri have two phosphate-binding proteins? Are the mechanisms of interaction with the permeases PstA and PstC, absorption and phosphate transport similar between PhoX and PstS?; (iv) Are PhoX and PstS proteins related to motility, biofilm formation and pathogenicity of X. citri?

For this Project, we have collaborative support of Prof. Dr. Albert Guskov from the University of Groningen (the Netherlands). We also have the support of Prof. Dr. Cristiano Oliveira – Instituto de Física/USP and the infrastructure of CNPEM through LNLS, LNBio, and LNNano.

Project 2. Functional and structural analysis of transporters involved with the uptake of sulfate and related compounds 

Sulfur is the fourth most abundant element on the planet (Beinert, 2000) and is part of the composition of numerous biomolecules such as cysteine ​​and methionine, co-factors: S- adenosyl methionine, CoA, biotin, lipoic acid, thiamine, iron clusters, metabolites: penicillin and glutathione (Campanini et al., 2015), being an essential element for the development of diseases and pathogenesis (Senaratne et al., 2006). Genome analysis of different microorganisms has shown that the genes involved in sulfate uptake are conserved, including in X. citri. In its reduced form, sulfur is used in the biosynthesis of methionine and cysteine. Cysteine ​​is incorporated into proteins, coenzymes and others. In Escherichia coli, Pseudomonas putida and Bacillus subtilis (Guillouard et al., 2002), sulfur assimilation can occur through the sulfate, thiosulfate, sulfonated alkane and aliphatic sulfonated uptake pathways, comprising a total of 26 genes that constitute the cys regulon. This regulon includes genes encoding three ABC transporters with the respective substrate oxidation-reduction enzymes. Furthermore, the sulfate ABC transporter and the sulfite ion reduction pathway (cysCDNHIJKMG genes) have been widely studied in other bacteria (Eichhorn et al., 1999; Stec et al., 2006) and identified as potential targets for design. of drugs in microorganisms and protozoa (Bankov et al., 1996; Walker and Barrett, 1997; Nozaki et al., 2005), as they are proven to be involved in growth and pathogenesis (Lestrate et al., 2000; Bogdan et al. , 2001; Yang et al., 2002; Ejim et al., 2004; Senaratne et al., 2006). Our group wants to understand from the structural point of view, how bacteria senses sulfate and sulfur-compounds, what are the transporters associated with the anion uptake and what are the mechanisms of transport regulation.

Project 3. Structural and Functional Characterization of M. tuberculosis Transporters involved in infection and pathogenesis

Tuberculosis is a chronic human disease responsible for more than 1.5 million annual deaths and latent infection in more than a third of the world’s human population, mainly due to the presence of several drug resistance mechanisms, including mutations and expression of several transporters. of the ABC type (Furin et al, 2019). The phenomenon of multiple drug resistance (MDR) in tuberculosis is responsible for resistance to first-line drugs such as isoniazid (INH) and rifampicin (RIF) (Gupta and Espinal, 2003), and extensive drug resistance (XDR-TB), with additional resistance to fluoroquinolone (FQ) and injectable drugs such as kanamycin (KAN), amikacin (AMI) and capriomycin (Louw et al, 2011). The genome of M. tuberculosis (strain H37Rv) was completely sequenced (Tekaia et al, 1999) revealing several genes responsible for the mechanisms of infection, pathogenesis and resistance. In this organism, whose genome is relatively small, ABC transporters represent 2.5%, highlighting their importance. There are at least 44 ABC-type transporters identified, of which 21 are exporters, including drug efflux pumps. From a structural point of view, only 3 complete transporters have been characterized to date (Oliveira and Balan, 2020). In our group, we previously selected a set of transporters relevant to the genus Mycobacterium spp. and we set up a cloning strategy for the respective genes and large-scale expression analysis. At the moment, we are working with two ABC transporters putatively involved with drug resistance but also, recycling of lipids through the membrane.

Collaborators: Dr. Isabel de Moraes (Crick Institute, London, UK); Prof. Dr. Cristiano Pinto (I. Physics), Prof. Luis Carlos de S. Ferreira (ICB), Prof. Albert Guskov (U. Groningen, ND).

Project 4. Validation and development of methodologies for inhibition of membrane proteins and ABC transporters

The main concern with resistant bacterial strains is a reduced response to available antimicrobials and prolonged treatment resulting in frequent and varied side effects (Johnson et al, 2006; Shah et al, 2007; WHO, 2010/2011). Obtaining new efficient, safe and rapid ways of blocking essential microorganism pathways is urgently needed. The mechanics of transport in ABC conveyors has been elucidated for both exporters and importers. Despite differences found in different types and in different organisms, it is canonical that the conformational changes undergone in permeases, as a result of interaction with the periplasmic binding protein, in the case of importers, or direct interaction with substrates, in the case of exporters, they activate nucleotide-binding domains or ATPases, which then promote the release of energy necessary for transport (Thomas et al, 2020). In this process, it is essential that ATPases are associated with an alpha-helical structure of permeases. In this project we are applying different approaches to validate targets and to inhibit membrane proteins: (i) use of the fragment-based drug development methodology (Fragment-Based Drug Discovery); (ii) development of antibodies against transporters permease using a platform based on a peptide presentation anchor protein; and (iii) development of de novo mini-proteins and sybodies that target essential permeases of export systems.

Collaborators: Prof. Dr. Luís Carlos de Souza Ferreira (ICB, USP), Departamento de Microbiologia, USP (produção de mutantes X. citri), Dr. Cristiano Oliveira, IF, USP), Prof. Dr. Marko Hyvonen (Dept. Biochemistry, U.Cambridge, UK), Prof. Dr. Albert Guskov (U. Groningen, The Netherlands), Dr. Luciana Cezar de Cerqueira Leite (I. Butantan), Dr. Ana Carolina Ramos Moreno (I. Butantan)


Project 1. Structural and functional studies of cyanide-degrading nitrilases (CHT) and their dependence on pH

Cyanide is a toxic compound that causes poisoning through inhalation, ingestion, or skin absorption. Due to its great affinity with metals, it is widely used as leaching for gold recovery. Cyanide is a weak acid; its dissociation constant is 9.22 at 25°C. This characteristic limits its bioremediation since it must necessarily be done at a pH greater than 9 to avoid its volatilization, and most cyanidases decrease their activity above alkaline pH. Nitrilases are enzymes that manage to break the carbon-nitrogen triple bond of CN. They are characterized by having an α-β-β-α fold and three catalytic residues: cysteine, glutamate, and lysine. The nitrilases that catalyze cyanide degradation can be divided into two types: cyanide hydratases (CHTs) and cyanide dihydratases (CynDs). The CHT group catalyzes the substrate, obtaining a formamide as a product. The CHT sequences analyzed to date show many similarities with each other with a percentage greater than 80% identity, keeping the C-terminal highly variable. On the other hand, the CynD group converts cyanide into ammonia and formic acid. They are enzymes that have spiral structures that associate to form rods of different sizes. It has been shown that as the pH of the medium increases, the oligomerization of the enzyme decreases. The main objective of this project is to compare the enzymatic activity of CHT enzymes purified and select the one with the best performance to characterize it structurally. In addition, mutant enzyme libraries will be prepared to search for variants with activity at alkaline pH. Working with enzymes capable of maintaining cyanidase activity at alkaline pH would be highly beneficial since it would allow degradation to be carried out under conditions that limit its volatilization.

Project 2. Functional and structural characterization of lipolytic enzymes from a microbial consortium degrading diesel oil

The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$789 million), food applications (US$634 million), and agriculture (US$237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. We are interested in the characterization of putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil.

Project 3. Development of a rapid system for generation of neutralyzing antibodies and monitoring of immunological aspects of SARS-CoV-2

The four structural proteins in SARS-CoV-2 (Membrane, Nucleocapsid,  Envelope and Spike) have been the largest targets for the development of drugs, vaccines and diagnostic tests. To overcome the problem of expressing heterologous proteins in eukaryotic systems – which in addition to being more costly, requires time and appropriate infrastructure – and the problems of producing synthetic epitopes that do not always acquire the native conformation, we chose the RAD Display technique for the production of peptides. The ease of cloning the sequences of interest on a large scale and the production of proteins in a short period of time (maximum of 40 days) stand out. The proteins obtained will form the SARS-CoV-2 peptide library and will be used for interaction studies with drugs and other proteins, as a complement to phenotypic screening approaches, serological assays and antibody production.