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Papers by Sílvia Sousa

International Journal of Molecular Sciences

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ ... more The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)2) and the counter-ion (Ph4P+ or Et4N+). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC50 values ranged from 0.1–8 μM (A2780) and 0.8–29 μM (OVCAR8). The complexes with the Ph4P+ ([P]) counter-ion are in general more active than their Et4N+ ([N]) analogues, presenting IC50 values in...

Antibiotics, 2021

Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge b... more Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge burden to healthcare systems worldwide. The ongoing COVID-19 pandemic, with the raised hospitalization of patients and the increased use of antimicrobial agents, boosted the emergence of difficult-to-treat multidrug-resistant (MDR) bacteria in hospital settings. Therefore, current available antibiotic treatments often have limited or no efficacy against nosocomial bacterial infections, and novel therapeutic approaches need to be considered. In this review, we analyze current antibacterial alternatives under investigation, focusing on metal-based complexes, antimicrobial peptides, and antisense antimicrobial therapeutics. The association of new compounds with older, commercially available antibiotics and the repurposing of existing drugs are also revised in this work.

The reaction catalyzed by PMIs is the first step of the mannose pathway leading to the generation... more The reaction catalyzed by PMIs is the first step of the mannose pathway leading to the generation of guanosine diphosphate (GDP)-D-mannose (Fig. 2). In this pathway, M6P is subsequently converted into mannose-1-phosphate (M1P) by the phosphomannomutase (PMM, E.C.5.4.2.8) enzymatic activity, followed by the conversion of M1P into GDP-Dmannose by GDP-D-mannose pyrophosphorylase (GMP, E.C.2.7.7.22). GDP-D-mannose is an important precursor of many mannosylated structures such as glycoproteins, nucleotide sugars, glycolipids, cell wall components found in fungi, and bacterial polysaccharides (Dunwell et al., 2000). As shown in Fig. 2, GDP-D-mannose is also the precursor of the activated sugar nucleotides GDP-L-fucose, GDP-D-rhamnose, GDP-colitose, and GDPperosamine, that are required for the biosynthesis of several glycoconjugates, including lipopolysaccharide (LPS) O-antigens, exopolysaccharides (EPS) and glycoproteins (Richau et al., 2000b; Vinion-Dubiel and Goldberg, 2003, Fig. 2).

Metallomics, 2020

The anticancer, antimicrobial and antiplasmodial activities of six gold(iii) bis(dithiolene) comp... more The anticancer, antimicrobial and antiplasmodial activities of six gold(iii) bis(dithiolene) complexes were studied. Complexes 1–6 showed relevant anticancer properties against A2780/A2780cisR ovarian cancer cells (IC50 values of 0.08–2 μM), also being able to overcome cisplatin resistance in A2780cisR cells. Complex 1 also exhibited significant antimicrobial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC) values of 12.1 ± 3.9 μg mL−1) and both Candida glabrata and Candida albicans (MICs of 9.7 ± 2.7 and 19.9 ± 2.4 μg mL−1, respectively). In addition, all complexes displayed antiplasmodial activity against the Plasmodium berghei parasite liver stages, even exhibiting better results than the ones obtained using primaquine, an anti-malarial drug. Mechanistic studies support the idea that thioredoxin reductase, but not DNA, is a possible target of these complexes. Complex 1 is stable under biological conditions, which would be important if this compound i...

Virulence, Aug 21, 2016

During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces ... more During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to three different clones retrieved from three chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 years or the more recent isolation date after 12.5 years of chronic infection. The infection models proved us...

AMB Express, 2016

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important ... more Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from c...

Journal of Cystic Fibrosis, 2013

The protein Hfq is RNA chaperone plays an important role in riboregulation, promoting the interac... more The protein Hfq is RNA chaperone plays an important role in riboregulation, promoting the interaction of small non-coding regulatory RNAs (sRNAs) with their mRNA targets [1]. Previous work from our research group has shown that bacteria of the Burkholderia cepacia complex are among the few prokaryotes that encode two distinct and functional Hfq-like proteins in their genome sequences [2,3]. Although a few studies have identified putative sRNAs from strains of the Burkholderia cepacia complex, to the best of our knowledge the h2cR sRNA remains as the single sRNA from a Burkholderia cepacia complex organism that has been functionally characterized until now [4]. Our research group has initiated an experimental strategy envisaging the identification of sRNAs from B. cenocepacia J2315 based on co-precipitation experiments with the bacterium two Hfq-like proteins, Hfq and Hfq2 [3,4]. Details on the experimental approaches used, on the sRNAs identified and on their functional characterization will be presented.

Energy, 2011

The present work establishes intermittent operation of UASB reactors as a novel form of enhancing... more The present work establishes intermittent operation of UASB reactors as a novel form of enhancing the anaerobic degradation of complex wastewaters and its conversion to usable biogas. Results show that the average methane production rate is 25% higher with the ...

Cellular Microbiology, 2007

In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long... more In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long-term asymptomatic airway colonization, or severe lung infection leading to rapid pulmonary decline. To assess the virulence of Bcc strains, we established a lung infection model in mice with a null allele of the gene involved in X-linked chronic granulomatous disease (CGD). CGD mice, challenged intratracheally with 10 3 cells of the epidemic Burkholderia cenocepacia strain J2315, died within 3 days from sepsis after bacteria had multiplied to 3.3 ¥ 10 8 cells. Infected mice developed neutrophil-dominated lung abscesses. Other B. cenocepacia strains and a B. cepacia strain were less virulent and one B. multivorans and one B. vietnamensis CF isolate were both avirulent. Bcc mutants, defective in exopolysaccharide synthesis or quorum sensing revealed diminished or no abscess formation and mortality. Immunofluorescence staining of Bccinfected murine and CF lung tissues revealed colocalization of Bcc and neutrophils, suggesting Bcc persistence within neutrophils in CGD and CF. In vitro, Bcc cells were rapidly killed during aerobic neutrophil phagocytosis; however, the pathogens survived in neutrophils with blocked nicotinamide adenine dinucleotide phosphate oxidase activity and under anaerobic conditions. We conclude that the Bcc infection model in CGD mice is well suited for the assessment of Bcc virulence.

Vaccines

The discovery of antimicrobials is an outstanding achievement of mankind that led to the developm... more The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. Th...

1. Overview This chapter describes experimental and bioinformatics approaches used to identify po... more 1. Overview This chapter describes experimental and bioinformatics approaches used to identify potential targets for the rational design of antimicrobial agents to combat infections caused by the Burkholderia cepacia complex, a group of bacterial species that emerged in the 1980s as important pathogens among immunocompromised patients and patients suffering from the genetic disease Cystic Fibrosis. These bacteria were initially described as Pseudomonas cepacia but advances in taxonomy led to their inclusion into the genus Burkholderia, and to their grouping within a complex of species currently designated as the B. cepacia complex (Bcc). An important feature of these bacteria is their intrinsic resistance to most of the antibiotics clinically available. Many research groups have focused their efforts on the discovery of virulence determinants and mechanisms at the molecular level, envisaging the identification of potential targets for the rational design of novel antimicrobials. The...

Antibiotics

Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NM... more Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NMR, FTIR) and elemental analysis. The structural arrangement of three selected examples were computed by Density Functional Theory (DFT) showing that the complexes essentially keep the {AuI-CN} unit. The Minimum Inhibition Concentrations (MIC) were assessed for all complexes showing that they are active towards the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408 and the Gram-positive strain S. aureus Newman. The complexes display very high activity towards P. aeruginosa 477 and B. contaminans IST408 with selectivity towards B. contaminans. An inverse correlation between the MIC values and the gold content was found for B. contaminans and P. aeruginosa. However, plots of MIC values and Au content for P. aeruginosa 477 and B. contaminans IST408 follow distinct trends. No clear relationship could be established between the MIC values and the redox potent...

Vaccines

Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas... more Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas aeruginosa are of particular concern due to their severity, their multiple antibiotic resistance, and the limited eradication efficiency of the current available treatments. New therapeutic options have been pursued, being vaccination strategies to prevent or limit these infections as a rational approach to tackle these infections. In this review, immunization and immunotherapy approaches currently available and under study against these bacterial pathogens is reviewed. Ongoing active and passive immunization clinical trials against P. aeruginosa infections is also reviewed. Novel identified bacterial targets and their possible exploitation for the development of immunization and immunotherapy strategies against P. aeruginosa and B. cepacia complex and infections are also presented and discussed.

Progress in Understanding Cystic Fibrosis

The Burkholderia cepacia complex (Bcc) is a group of closely related bacterial species that emerg... more The Burkholderia cepacia complex (Bcc) is a group of closely related bacterial species that emerged in the 1980s as the etiological agents of severe and often lethal respiratory infections among cystic fibrosis (CF) patients.After several outbreaks in CF centers in Europe and North America, segregation measures were introduced to avoid patientto-patient transmission. Presently, the prevalence of Bcc infections among CF patients worldwideisbelow5%inthemajorityofCFcenters,althoughexceptionsareregistered in some European countries. Infections by these pathogens remain problematic due to the high resistance to antimicrobials, the easy patient-to-patient transmission, and the unpredictable outcome of infections that range from asymptomatic carriage to the cepaciasyndrome,afulminatingpneumoniaoftenassociatedwithsepticemiathatcan leadtothedeceaseofpatientswithinaperiodoftimeasshortas1week.Inthischapter, wereviewtheevolvingepidemiologyofBccinfectionsinCFpatients,thevirulencetraits and mechanisms used by these bacteria, and the recent developments in vaccine and vaccinecomponentsresearchtopreventBccinfections.

Nanomaterials

Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by... more Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by reducing HAuCl4 at different pHs (6.4–8.4) using ultrasonic irradiation: an easy, cheap, eco-friendly and green approach. Their antibacterial and anticancer activities were evaluated against Staphylococcus aureus and Escherichia coli, and A2780 ovarian cancer cells, respectively. The AuNPRn were characterized concerning their phytoconstituents (polyphenols, flavonoids and condensed tannins) and gold content. All of the nanoparticles were negatively charged. AuNPRn exhibited a hydrodynamic size distribution ranging from 32 nm to 280 nm, with the larger nanoparticles being obtained with an Au/root extract ratio of 0.56, pH 7 and 10 min of sonication (AuNPR1), whereas the smallest were obtained with an Au/root extract ratio of 0.24, pH 7.8 and 40 min of sonication (AuNPR4). The TEM/SEM images showed that the AuNPRn had different shapes. The ATR-FTIR indicated that AuNPRn interact mainly wi...

Antibiotics

Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analyti... more Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analytically and spectroscopically characterized, aiming to identify the key parameters that drive the antibacterial activity of the complexes with metal cores and imine substituents with distinct electronic and steric characteristics. The antimicrobial activity of all complexes was evaluated by determining their minimum inhibitory concentrations (MIC) against the Gram-negative Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, and Burkholderia contaminans IST408, and the Gram-positive Staphylococcus aureus Newman. Camphor imine complexes based on the hydroxyl silver center ({Ag(OH)}) typically perform better than those based on the nitrate silver center ({Ag(NO3)}), while ligands prone to establish hydrogen bonding facilitate interactions with the bacterial cell surface structures. A different trend is observed for the silver camphor sulfonimine complexes that are almost non-sensitive to ...

Vaccines

Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic ... more Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic fibrosis (CF) patients, and no effective eradication is available. A vaccine to protect patients against Bcc infections is a highly attractive therapeutic option, but none is available. A strategy combining the bioinformatics identification of putative surface-exposed proteins with an experimental approach encompassing the “shaving” of surface-exposed proteins with trypsin followed by peptide identification by liquid chromatography and mass spectrometry is here reported. The methodology allowed the bioinformatics identification of 263 potentially surface-exposed proteins, 16 of them also experimentally identified by the “shaving” approach. Of the proteins identified, 143 have a high probability of containing B-cell epitopes that are surface-exposed. The immunogenicity of three of these proteins was demonstrated using serum samples from Bcc-infected CF patients and Western blotting, valid...

Proceedings of 5th International Electronic Conference on Medicinal Chemistry

Cyclams are macrocyclic polyamines which medical interest was fueled by the therapeutic potential... more Cyclams are macrocyclic polyamines which medical interest was fueled by the therapeutic potential of a bicyclam derivative in HIV infection, inflammatory diseases, cancer and stem-cell mobilization.[1] Taking advantage of the biocompatibility, the high metal chelation stability constants and the possibility of N-functionalization of the cyclam backbone, a variety of compounds have been explored in a wide range of medicinal applications.[2] The use of cyclams and cyclam-based complexes as antimicrobial and antitumoral agents has been described in recent years. In particular, trans-disubstituted cyclam salts revealed to be active antibacterial agents against both Gram-positive and Gram-negative bacteria.[3,4] In the field of anticancer applications, several attempts are being made, mostly with CuII complexes, envisaging their use as 64/67Cu radionuclides.[5] Recently, we found that trans-disubstituted cyclam derivatives and their CuII and FeIII complexes display relevant antitumoral activity against HeLa cancer cell lines.[6] To the best of our knowledge, this is the first report on an iron-cyclam compound tested as anticancer agent. [1] De Clercq, E., Nat. Rev. Drug Discov. 2003, 2, 581–587 [2] Liang, X.; Sadler, P. J., Chem. Soc. Rev. 2004, 33, 246-266 [3] Yu, M.; Nagalingam, G.; Ellis, S.; Martinez, E.; Sintchenko, V.; Spain, M.; Rutledge, P. J.; Todd, M. H.; Triccas, J. A., J. Med. Chem. 2016, 59, 5917–5921 [4] Alves, L. G.; Pinheiro, P. F.; Feliciano, J. R.; Dâmaso, D. P.; Leitao, J. H.; Martins, A. M., Int. J. Antimicrob. Agents 2017, 49, 646-649 [5] Cai, Z.; Anderson, C. J., J. Label. Compd. Radiopharm. 2014, 57, 224–230 [6] Pilon, A.; Lorenzo, J.; Rodriguez-Calado, S.; Adao, P.; Martins, A. M.; Valente, A.; Alves, L. G., ChemMedChem, 2019, 14, 770-778

International Journal of Molecular Sciences

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ ... more The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)2) and the counter-ion (Ph4P+ or Et4N+). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC50 values ranged from 0.1–8 μM (A2780) and 0.8–29 μM (OVCAR8). The complexes with the Ph4P+ ([P]) counter-ion are in general more active than their Et4N+ ([N]) analogues, presenting IC50 values in...

Antibiotics, 2021

Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge b... more Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge burden to healthcare systems worldwide. The ongoing COVID-19 pandemic, with the raised hospitalization of patients and the increased use of antimicrobial agents, boosted the emergence of difficult-to-treat multidrug-resistant (MDR) bacteria in hospital settings. Therefore, current available antibiotic treatments often have limited or no efficacy against nosocomial bacterial infections, and novel therapeutic approaches need to be considered. In this review, we analyze current antibacterial alternatives under investigation, focusing on metal-based complexes, antimicrobial peptides, and antisense antimicrobial therapeutics. The association of new compounds with older, commercially available antibiotics and the repurposing of existing drugs are also revised in this work.

The reaction catalyzed by PMIs is the first step of the mannose pathway leading to the generation... more The reaction catalyzed by PMIs is the first step of the mannose pathway leading to the generation of guanosine diphosphate (GDP)-D-mannose (Fig. 2). In this pathway, M6P is subsequently converted into mannose-1-phosphate (M1P) by the phosphomannomutase (PMM, E.C.5.4.2.8) enzymatic activity, followed by the conversion of M1P into GDP-Dmannose by GDP-D-mannose pyrophosphorylase (GMP, E.C.2.7.7.22). GDP-D-mannose is an important precursor of many mannosylated structures such as glycoproteins, nucleotide sugars, glycolipids, cell wall components found in fungi, and bacterial polysaccharides (Dunwell et al., 2000). As shown in Fig. 2, GDP-D-mannose is also the precursor of the activated sugar nucleotides GDP-L-fucose, GDP-D-rhamnose, GDP-colitose, and GDPperosamine, that are required for the biosynthesis of several glycoconjugates, including lipopolysaccharide (LPS) O-antigens, exopolysaccharides (EPS) and glycoproteins (Richau et al., 2000b; Vinion-Dubiel and Goldberg, 2003, Fig. 2).

Metallomics, 2020

The anticancer, antimicrobial and antiplasmodial activities of six gold(iii) bis(dithiolene) comp... more The anticancer, antimicrobial and antiplasmodial activities of six gold(iii) bis(dithiolene) complexes were studied. Complexes 1–6 showed relevant anticancer properties against A2780/A2780cisR ovarian cancer cells (IC50 values of 0.08–2 μM), also being able to overcome cisplatin resistance in A2780cisR cells. Complex 1 also exhibited significant antimicrobial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC) values of 12.1 ± 3.9 μg mL−1) and both Candida glabrata and Candida albicans (MICs of 9.7 ± 2.7 and 19.9 ± 2.4 μg mL−1, respectively). In addition, all complexes displayed antiplasmodial activity against the Plasmodium berghei parasite liver stages, even exhibiting better results than the ones obtained using primaquine, an anti-malarial drug. Mechanistic studies support the idea that thioredoxin reductase, but not DNA, is a possible target of these complexes. Complex 1 is stable under biological conditions, which would be important if this compound i...

Virulence, Aug 21, 2016

During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces ... more During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to three different clones retrieved from three chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 years or the more recent isolation date after 12.5 years of chronic infection. The infection models proved us...

AMB Express, 2016

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important ... more Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among cystic fibrosis patients, highlighting the need for novel therapeutic strategies. In the present work we have studied the B. cenocepacia protein BCAL2958, a member of the OmpA-like family of proteins, demonstrated as highly immunogenic in other pathogens and capable of eliciting strong host immune responses. The encoding gene was cloned and the protein, produced as a 6× His-tagged derivative, was used to produce polyclonal antibodies. Bioinformatics analyses led to the identification of sequences encoding proteins with a similarity higher than 96 % to BCAL2958 in all the publicly available Bcc genomes. Furthermore, using the antibody it was experimentally demonstrated that this protein is produced by all the 12 analyzed strains from 7 Bcc species. In addition, results are also presented showing the presence of anti-BCAL2958 antibodies in sera from c...

Journal of Cystic Fibrosis, 2013

The protein Hfq is RNA chaperone plays an important role in riboregulation, promoting the interac... more The protein Hfq is RNA chaperone plays an important role in riboregulation, promoting the interaction of small non-coding regulatory RNAs (sRNAs) with their mRNA targets [1]. Previous work from our research group has shown that bacteria of the Burkholderia cepacia complex are among the few prokaryotes that encode two distinct and functional Hfq-like proteins in their genome sequences [2,3]. Although a few studies have identified putative sRNAs from strains of the Burkholderia cepacia complex, to the best of our knowledge the h2cR sRNA remains as the single sRNA from a Burkholderia cepacia complex organism that has been functionally characterized until now [4]. Our research group has initiated an experimental strategy envisaging the identification of sRNAs from B. cenocepacia J2315 based on co-precipitation experiments with the bacterium two Hfq-like proteins, Hfq and Hfq2 [3,4]. Details on the experimental approaches used, on the sRNAs identified and on their functional characterization will be presented.

Energy, 2011

The present work establishes intermittent operation of UASB reactors as a novel form of enhancing... more The present work establishes intermittent operation of UASB reactors as a novel form of enhancing the anaerobic degradation of complex wastewaters and its conversion to usable biogas. Results show that the average methane production rate is 25% higher with the ...

Cellular Microbiology, 2007

In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long... more In cystic fibrosis (CF), infection with Burkholderia cepacia complex (Bcc) strains may cause long-term asymptomatic airway colonization, or severe lung infection leading to rapid pulmonary decline. To assess the virulence of Bcc strains, we established a lung infection model in mice with a null allele of the gene involved in X-linked chronic granulomatous disease (CGD). CGD mice, challenged intratracheally with 10 3 cells of the epidemic Burkholderia cenocepacia strain J2315, died within 3 days from sepsis after bacteria had multiplied to 3.3 ¥ 10 8 cells. Infected mice developed neutrophil-dominated lung abscesses. Other B. cenocepacia strains and a B. cepacia strain were less virulent and one B. multivorans and one B. vietnamensis CF isolate were both avirulent. Bcc mutants, defective in exopolysaccharide synthesis or quorum sensing revealed diminished or no abscess formation and mortality. Immunofluorescence staining of Bccinfected murine and CF lung tissues revealed colocalization of Bcc and neutrophils, suggesting Bcc persistence within neutrophils in CGD and CF. In vitro, Bcc cells were rapidly killed during aerobic neutrophil phagocytosis; however, the pathogens survived in neutrophils with blocked nicotinamide adenine dinucleotide phosphate oxidase activity and under anaerobic conditions. We conclude that the Bcc infection model in CGD mice is well suited for the assessment of Bcc virulence.

Vaccines

The discovery of antimicrobials is an outstanding achievement of mankind that led to the developm... more The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. Th...

1. Overview This chapter describes experimental and bioinformatics approaches used to identify po... more 1. Overview This chapter describes experimental and bioinformatics approaches used to identify potential targets for the rational design of antimicrobial agents to combat infections caused by the Burkholderia cepacia complex, a group of bacterial species that emerged in the 1980s as important pathogens among immunocompromised patients and patients suffering from the genetic disease Cystic Fibrosis. These bacteria were initially described as Pseudomonas cepacia but advances in taxonomy led to their inclusion into the genus Burkholderia, and to their grouping within a complex of species currently designated as the B. cepacia complex (Bcc). An important feature of these bacteria is their intrinsic resistance to most of the antibiotics clinically available. Many research groups have focused their efforts on the discovery of virulence determinants and mechanisms at the molecular level, envisaging the identification of potential targets for the rational design of novel antimicrobials. The...

Antibiotics

Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NM... more Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NMR, FTIR) and elemental analysis. The structural arrangement of three selected examples were computed by Density Functional Theory (DFT) showing that the complexes essentially keep the {AuI-CN} unit. The Minimum Inhibition Concentrations (MIC) were assessed for all complexes showing that they are active towards the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408 and the Gram-positive strain S. aureus Newman. The complexes display very high activity towards P. aeruginosa 477 and B. contaminans IST408 with selectivity towards B. contaminans. An inverse correlation between the MIC values and the gold content was found for B. contaminans and P. aeruginosa. However, plots of MIC values and Au content for P. aeruginosa 477 and B. contaminans IST408 follow distinct trends. No clear relationship could be established between the MIC values and the redox potent...

Vaccines

Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas... more Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas aeruginosa are of particular concern due to their severity, their multiple antibiotic resistance, and the limited eradication efficiency of the current available treatments. New therapeutic options have been pursued, being vaccination strategies to prevent or limit these infections as a rational approach to tackle these infections. In this review, immunization and immunotherapy approaches currently available and under study against these bacterial pathogens is reviewed. Ongoing active and passive immunization clinical trials against P. aeruginosa infections is also reviewed. Novel identified bacterial targets and their possible exploitation for the development of immunization and immunotherapy strategies against P. aeruginosa and B. cepacia complex and infections are also presented and discussed.

Progress in Understanding Cystic Fibrosis

The Burkholderia cepacia complex (Bcc) is a group of closely related bacterial species that emerg... more The Burkholderia cepacia complex (Bcc) is a group of closely related bacterial species that emerged in the 1980s as the etiological agents of severe and often lethal respiratory infections among cystic fibrosis (CF) patients.After several outbreaks in CF centers in Europe and North America, segregation measures were introduced to avoid patientto-patient transmission. Presently, the prevalence of Bcc infections among CF patients worldwideisbelow5%inthemajorityofCFcenters,althoughexceptionsareregistered in some European countries. Infections by these pathogens remain problematic due to the high resistance to antimicrobials, the easy patient-to-patient transmission, and the unpredictable outcome of infections that range from asymptomatic carriage to the cepaciasyndrome,afulminatingpneumoniaoftenassociatedwithsepticemiathatcan leadtothedeceaseofpatientswithinaperiodoftimeasshortas1week.Inthischapter, wereviewtheevolvingepidemiologyofBccinfectionsinCFpatients,thevirulencetraits and mechanisms used by these bacteria, and the recent developments in vaccine and vaccinecomponentsresearchtopreventBccinfections.

Nanomaterials

Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by... more Root extracts from Danube Delta Nymphaea alba were used to prepare gold nanoparticles (AuNPRn) by reducing HAuCl4 at different pHs (6.4–8.4) using ultrasonic irradiation: an easy, cheap, eco-friendly and green approach. Their antibacterial and anticancer activities were evaluated against Staphylococcus aureus and Escherichia coli, and A2780 ovarian cancer cells, respectively. The AuNPRn were characterized concerning their phytoconstituents (polyphenols, flavonoids and condensed tannins) and gold content. All of the nanoparticles were negatively charged. AuNPRn exhibited a hydrodynamic size distribution ranging from 32 nm to 280 nm, with the larger nanoparticles being obtained with an Au/root extract ratio of 0.56, pH 7 and 10 min of sonication (AuNPR1), whereas the smallest were obtained with an Au/root extract ratio of 0.24, pH 7.8 and 40 min of sonication (AuNPR4). The TEM/SEM images showed that the AuNPRn had different shapes. The ATR-FTIR indicated that AuNPRn interact mainly wi...

Antibiotics

Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analyti... more Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analytically and spectroscopically characterized, aiming to identify the key parameters that drive the antibacterial activity of the complexes with metal cores and imine substituents with distinct electronic and steric characteristics. The antimicrobial activity of all complexes was evaluated by determining their minimum inhibitory concentrations (MIC) against the Gram-negative Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, and Burkholderia contaminans IST408, and the Gram-positive Staphylococcus aureus Newman. Camphor imine complexes based on the hydroxyl silver center ({Ag(OH)}) typically perform better than those based on the nitrate silver center ({Ag(NO3)}), while ligands prone to establish hydrogen bonding facilitate interactions with the bacterial cell surface structures. A different trend is observed for the silver camphor sulfonimine complexes that are almost non-sensitive to ...

Vaccines

Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic ... more Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic fibrosis (CF) patients, and no effective eradication is available. A vaccine to protect patients against Bcc infections is a highly attractive therapeutic option, but none is available. A strategy combining the bioinformatics identification of putative surface-exposed proteins with an experimental approach encompassing the “shaving” of surface-exposed proteins with trypsin followed by peptide identification by liquid chromatography and mass spectrometry is here reported. The methodology allowed the bioinformatics identification of 263 potentially surface-exposed proteins, 16 of them also experimentally identified by the “shaving” approach. Of the proteins identified, 143 have a high probability of containing B-cell epitopes that are surface-exposed. The immunogenicity of three of these proteins was demonstrated using serum samples from Bcc-infected CF patients and Western blotting, valid...

Proceedings of 5th International Electronic Conference on Medicinal Chemistry

Cyclams are macrocyclic polyamines which medical interest was fueled by the therapeutic potential... more Cyclams are macrocyclic polyamines which medical interest was fueled by the therapeutic potential of a bicyclam derivative in HIV infection, inflammatory diseases, cancer and stem-cell mobilization.[1] Taking advantage of the biocompatibility, the high metal chelation stability constants and the possibility of N-functionalization of the cyclam backbone, a variety of compounds have been explored in a wide range of medicinal applications.[2] The use of cyclams and cyclam-based complexes as antimicrobial and antitumoral agents has been described in recent years. In particular, trans-disubstituted cyclam salts revealed to be active antibacterial agents against both Gram-positive and Gram-negative bacteria.[3,4] In the field of anticancer applications, several attempts are being made, mostly with CuII complexes, envisaging their use as 64/67Cu radionuclides.[5] Recently, we found that trans-disubstituted cyclam derivatives and their CuII and FeIII complexes display relevant antitumoral activity against HeLa cancer cell lines.[6] To the best of our knowledge, this is the first report on an iron-cyclam compound tested as anticancer agent. [1] De Clercq, E., Nat. Rev. Drug Discov. 2003, 2, 581–587 [2] Liang, X.; Sadler, P. J., Chem. Soc. Rev. 2004, 33, 246-266 [3] Yu, M.; Nagalingam, G.; Ellis, S.; Martinez, E.; Sintchenko, V.; Spain, M.; Rutledge, P. J.; Todd, M. H.; Triccas, J. A., J. Med. Chem. 2016, 59, 5917–5921 [4] Alves, L. G.; Pinheiro, P. F.; Feliciano, J. R.; Dâmaso, D. P.; Leitao, J. H.; Martins, A. M., Int. J. Antimicrob. Agents 2017, 49, 646-649 [5] Cai, Z.; Anderson, C. J., J. Label. Compd. Radiopharm. 2014, 57, 224–230 [6] Pilon, A.; Lorenzo, J.; Rodriguez-Calado, S.; Adao, P.; Martins, A. M.; Valente, A.; Alves, L. G., ChemMedChem, 2019, 14, 770-778