Jorge H Leitão | Universidade de Lisboa (original) (raw)

Papers by Jorge H Leitão

Research paper thumbnail of Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections

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...

Research paper thumbnail of Broad Spectrum Functional Activity of Structurally Related Monoanionic Au(III) Bis(Dithiolene) Complexes

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...

Research paper thumbnail of Bacterial Nosocomial Infections: Multidrug Resistance as a Trigger for the Development of Novel Antimicrobials

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.

Research paper thumbnail of Optimizing Energy Production from Wastewater by Intermittent Operation of Anaerobic Reactors

This work shows the results of investigations performed with the objective of optimizing methane ... more This work shows the results of investigations performed with the objective of optimizing methane production from dairy wastewater in anaerobic reactors through the adaptation of microbial populations towards a more complete degradation of high methane yielding substrates. The innovative aspect of this work lies on the use of a novel operating mode – intermittent operation – that allows a raising of methane yield of over 20% in relation to what is obtained with conventional (continuous) systems with the same wastewater purification efficiencies. Results from molecular techniques confirm that this improvement in methane production is due to the development in the intermittent system of a microbial population highly adapted to the metabolization of lipids which are the substrates with higher methane yield potential in dairy wastewater.

Research paper thumbnail of Gold(iii) bis(dithiolene) complexes: from molecular conductors to prospective anticancer, antimicrobial and antiplasmodial agents

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...

Research paper thumbnail of {"__content__"=>"Postgenomic Approaches and Bioinformatics Tools to Advance the Development of Vaccines against Bacteria of the Complex.", "i"=>{"__content__"=>"Burkholderia cepacia"}}

Vaccines, Jan 8, 2018

Bacteria of the complex (Bcc) remain an important cause of morbidity and mortality among patients... more Bacteria of the complex (Bcc) remain an important cause of morbidity and mortality among patients suffering from cystic fibrosis. Eradication of these pathogens by antimicrobial therapy often fails, highlighting the need to develop novel strategies to eradicate infections. Vaccines are attractive since they can confer protection to particularly vulnerable patients, as is the case of cystic fibrosis patients. Several studies have identified specific virulence factors and proteins as potential subunit vaccine candidates. So far, no vaccine is available to protect from Bcc infections. In the present work, we review the most promising postgenomic approaches and selected web tools available to speed up the identification of immunogenic proteins with the potential of conferring protection against Bcc infections.

Research paper thumbnail of Microbial populations shifts as a tool for improving biogas production in anaerobic reactors

Research paper thumbnail of Leitão, J.H., Sousa S.A., Ramos C.G., Feliciano J.R., Grilo A.M. and DA COSTA, P.J.P

This chapter describes experimental and bioinformatics approaches used to identify potential targ... more This chapter describes experimental and bioinformatics approaches used to identify potential targets for the rationaldesign 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 most importa...

Research paper thumbnail of Retraction for Ramos et al., The Second RNA Chaperone, Hfq2, Is Also Required for Survival under Stress and Full Virulence of Burkholderia cenocepacia J2315

Journal of Bacteriology, 2014

Problems related to images published in this paper have been brought to our attention. Figure 8 c... more Problems related to images published in this paper have been brought to our attention. Figure 8 contains duplicated images as well as images previously published in articles in Microbiology and Microbial Pathogenesis, i.e., the following:

Research paper thumbnail of Burkholderia cepacia complex: Emerging multihost pathogens equipped with a wide range of virulence factors and determinants

International Journal of Microbiology, 2011

The Burkholderia cepacia complex (Bcc) comprises at least 17 closely-related species of the β-pro... more The Burkholderia cepacia complex (Bcc) comprises at least 17 closely-related species of the β-proteobacteria subdivision, widely distributed in natural and man-made inhabitats. Bcc bacteria are endowed with an extraordinary metabolic diversity and emerged in the 1980s as life-threatening and difficult-to-treat pathogens among patients suffering from cystic fibrosis. More recently, these bacteria became recognized as a threat to hospitalized patients suffering from other diseases, in particular oncological patients. In the present paper, we review these and other traits of Bcc bacteria, as well as some of the strategies used to identify and validate the virulence factors and determinants used by these bacteria. The identification and characterization of these virulence factors is expected to lead to the design of novel therapeutic strategies to fight the infections caused by these emergent multidrug resistant human pathogens.

Research paper thumbnail of Suitability of a Saccharomyces cerevisiae-based assay to assess the toxicity of pyrimethanil sprayed soils via surface runoff: Comparison with standard aquatic and soil toxicity assays

Science of the Total Environment 505:161-171, 2015

A yeast assay to assess pyrimethanil toxicity in soil surface runoff was evaluated. • Runoff was ... more A yeast assay to assess pyrimethanil toxicity in soil surface runoff was evaluated. • Runoff was simulated in soils sprayed with fungicide accidental spill doses. • Increased expression of pyrimethanil-responsive yeast genes was measured. • Toxicity to aquatic and soil standard organisms correlated with toxicity to yeast. • Yeast assay shown relevant for screening worst-cases of pyrimethanil contamination.

Research paper thumbnail of Identification and exploitation of Burkholderia cepacia complex virulence factors as potential antimicrobial targets

1st Portuguese Biomedical Engineering Meeting, 2011

Abstract A multidisciplinary approach is being used to identify and validate virulence factors an... more Abstract A multidisciplinary approach is being used to identify and validate virulence factors and determinants of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of problematic opportunistic pathogenic bacteria, particularly among cystic fibrosis patients, ...

Research paper thumbnail of A RNomics-based strategy identifies regulatory small RNAs in Burkholderia cepacia complex

1st Portuguese Biomedical Engineering Meeting, 2011

Research paper thumbnail of The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans

Microbiology, 2009

TheBurkholderia cepaciacomplex (Bcc) emerged as problematic opportunistic pathogens to cystic fib... more TheBurkholderia cepaciacomplex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of aB. cepaciaIST408 mutant containing a disruption in thehfqgene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). TheB. cepaciaHfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When providedin trans, theB. cepaciaIST408hfqgene complemented theEscherichia coli hfqmutant strain GS081. Our results also show that theB. cepacia hfqmutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In additio...

Research paper thumbnail of Burkholderia cenocepacia J2315 acyl carrier protein: A potential target for antimicrobials' development?

Microbial Pathogenesis, 2008

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant fr... more This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71 kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.

Research paper thumbnail of The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence

Microbial Pathogenesis, 2010

Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex,... more Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex, a group of bacteria that emerged as important pathogens, especially to cystic fibrosis (CF) patients. In this study, we report the identification and characterization of a mutant strain derived form the CF isolate Burkholderia cenocepacia K56-2, carrying a plasposon insertion in a gene, located in a 3516 bp chromosomal region with an atypical G þ C content, encoding a 80 amino acid putative regulatory protein named Pbr. Besides its inability to produce phenazines, the B. cenocepacia K56-2 pbr mutant exhibited a pleiotropic phenotype, including impaired survival to oxidative and osmotic stress, aromatic amino acid and prolonged nutrient starvation periods. In addition, the pbr mutant exhibited decreased virulence the nematode Caenorhabditis elegans. Altogether, our results demonstrate the involvement of Pbr on the regulation of phenazine biosynthesis, and an important role for this regulatory protein on several cellular processes related to stress resistance and virulence.

Research paper thumbnail of 109 The Burkholderia cepacia small colony variants (SCV) are a more pathogenic bacterial form that may facilitate persistent respiratory infections in CF patients

Journal of Cystic Fibrosis, 2013

Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse... more Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. Understanding the mechanisms involved in bacterial virulence and in genetic adaptation of these bacteria to the CF lung is crucial for an improved management of Bcc chronic infections [1]. A 2 decade-long systematic survey of Bcc respiratory infections in the major Portuguese CF Centre has allowed us to carry out a retrospective study of the adaptive clonal variation of these pathogens within the airways during lung function deterioration [1−4]. In this work, we assessed the virulence of 3 B. cenocepacia clonal isolates spanning a 3.5-year long chronic infection in a single CF patient, based on their ability to invade epithelial cells and open tight junctions. The isolates retrieved later before the patient's death with cepacia syndrome were found to be significantly more virulent than the one that initiated the infection. A genome-wide quantitative proteomics approach was exploited to identify differentially expressed proteins and infer mechanisms that could be associated with B. cenocepacia virulence and persistence in the lung (ref. [2], unpublished results). Overall, the results are suggestive of adaptation to microaerophilic conditions and iron availability, and highlight the important role of metabolic optimization in adaptation and increased pathogenic potential of B. cenocepacia during the course of a chronic CF infection.

Research paper thumbnail of 111 The Burkholderia cenocepacia J2315 small non-coding RNA MavA is required for fitness and virulence

Journal of Cystic Fibrosis, 2013

Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse... more Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. Understanding the mechanisms involved in bacterial virulence and in genetic adaptation of these bacteria to the CF lung is crucial for an improved management of Bcc chronic infections [1]. A 2 decade-long systematic survey of Bcc respiratory infections in the major Portuguese CF Centre has allowed us to carry out a retrospective study of the adaptive clonal variation of these pathogens within the airways during lung function deterioration [1−4]. In this work, we assessed the virulence of 3 B. cenocepacia clonal isolates spanning a 3.5-year long chronic infection in a single CF patient, based on their ability to invade epithelial cells and open tight junctions. The isolates retrieved later before the patient's death with cepacia syndrome were found to be significantly more virulent than the one that initiated the infection. A genome-wide quantitative proteomics approach was exploited to identify differentially expressed proteins and infer mechanisms that could be associated with B. cenocepacia virulence and persistence in the lung (ref. [2], unpublished results). Overall, the results are suggestive of adaptation to microaerophilic conditions and iron availability, and highlight the important role of metabolic optimization in adaptation and increased pathogenic potential of B. cenocepacia during the course of a chronic CF infection.

Research paper thumbnail of Variation of the antimicrobial susceptibility profiles of Burkholderia cepacia complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center

European Journal of Clinical Microbiology & Infectious Diseases, 2008

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia com... more The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.

Research paper thumbnail of Bioinformatics: A Molecular Microbiologist’s Perspective

Current Bioinformatics, 2014

Research activities in the area of biological sciences, and particularly molecular microbiology, ... more Research activities in the area of biological sciences, and particularly molecular microbiology, nowadays generate vast amounts of data. The development of faster and cheaper sequencing methods has definitively contributed to this huge amount of data, requiring the development of fast and reliable tools for their analysis. In the last years, many easy-to-use, reliable and powerful bioinformatics tools have been developed. In this work, we review the fundamentals of some of these tools, and describe how we have been using them to analyze data resulting from our research envisaging the identification and characterization of virulence factors and determinants from the human opportunistic pathogens of the Burkholderia cepacia complex. Examples given illustrate the user-friendly characteristic of these tools and their power both in analyzing information and in orientating future experimental work.

Research paper thumbnail of Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections

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...

Research paper thumbnail of Broad Spectrum Functional Activity of Structurally Related Monoanionic Au(III) Bis(Dithiolene) Complexes

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...

Research paper thumbnail of Bacterial Nosocomial Infections: Multidrug Resistance as a Trigger for the Development of Novel Antimicrobials

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.

Research paper thumbnail of Optimizing Energy Production from Wastewater by Intermittent Operation of Anaerobic Reactors

This work shows the results of investigations performed with the objective of optimizing methane ... more This work shows the results of investigations performed with the objective of optimizing methane production from dairy wastewater in anaerobic reactors through the adaptation of microbial populations towards a more complete degradation of high methane yielding substrates. The innovative aspect of this work lies on the use of a novel operating mode – intermittent operation – that allows a raising of methane yield of over 20% in relation to what is obtained with conventional (continuous) systems with the same wastewater purification efficiencies. Results from molecular techniques confirm that this improvement in methane production is due to the development in the intermittent system of a microbial population highly adapted to the metabolization of lipids which are the substrates with higher methane yield potential in dairy wastewater.

Research paper thumbnail of Gold(iii) bis(dithiolene) complexes: from molecular conductors to prospective anticancer, antimicrobial and antiplasmodial agents

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...

Research paper thumbnail of {"__content__"=>"Postgenomic Approaches and Bioinformatics Tools to Advance the Development of Vaccines against Bacteria of the Complex.", "i"=>{"__content__"=>"Burkholderia cepacia"}}

Vaccines, Jan 8, 2018

Bacteria of the complex (Bcc) remain an important cause of morbidity and mortality among patients... more Bacteria of the complex (Bcc) remain an important cause of morbidity and mortality among patients suffering from cystic fibrosis. Eradication of these pathogens by antimicrobial therapy often fails, highlighting the need to develop novel strategies to eradicate infections. Vaccines are attractive since they can confer protection to particularly vulnerable patients, as is the case of cystic fibrosis patients. Several studies have identified specific virulence factors and proteins as potential subunit vaccine candidates. So far, no vaccine is available to protect from Bcc infections. In the present work, we review the most promising postgenomic approaches and selected web tools available to speed up the identification of immunogenic proteins with the potential of conferring protection against Bcc infections.

Research paper thumbnail of Microbial populations shifts as a tool for improving biogas production in anaerobic reactors

Research paper thumbnail of Leitão, J.H., Sousa S.A., Ramos C.G., Feliciano J.R., Grilo A.M. and DA COSTA, P.J.P

This chapter describes experimental and bioinformatics approaches used to identify potential targ... more This chapter describes experimental and bioinformatics approaches used to identify potential targets for the rationaldesign 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 most importa...

Research paper thumbnail of Retraction for Ramos et al., The Second RNA Chaperone, Hfq2, Is Also Required for Survival under Stress and Full Virulence of Burkholderia cenocepacia J2315

Journal of Bacteriology, 2014

Problems related to images published in this paper have been brought to our attention. Figure 8 c... more Problems related to images published in this paper have been brought to our attention. Figure 8 contains duplicated images as well as images previously published in articles in Microbiology and Microbial Pathogenesis, i.e., the following:

Research paper thumbnail of Burkholderia cepacia complex: Emerging multihost pathogens equipped with a wide range of virulence factors and determinants

International Journal of Microbiology, 2011

The Burkholderia cepacia complex (Bcc) comprises at least 17 closely-related species of the β-pro... more The Burkholderia cepacia complex (Bcc) comprises at least 17 closely-related species of the β-proteobacteria subdivision, widely distributed in natural and man-made inhabitats. Bcc bacteria are endowed with an extraordinary metabolic diversity and emerged in the 1980s as life-threatening and difficult-to-treat pathogens among patients suffering from cystic fibrosis. More recently, these bacteria became recognized as a threat to hospitalized patients suffering from other diseases, in particular oncological patients. In the present paper, we review these and other traits of Bcc bacteria, as well as some of the strategies used to identify and validate the virulence factors and determinants used by these bacteria. The identification and characterization of these virulence factors is expected to lead to the design of novel therapeutic strategies to fight the infections caused by these emergent multidrug resistant human pathogens.

Research paper thumbnail of Suitability of a Saccharomyces cerevisiae-based assay to assess the toxicity of pyrimethanil sprayed soils via surface runoff: Comparison with standard aquatic and soil toxicity assays

Science of the Total Environment 505:161-171, 2015

A yeast assay to assess pyrimethanil toxicity in soil surface runoff was evaluated. • Runoff was ... more A yeast assay to assess pyrimethanil toxicity in soil surface runoff was evaluated. • Runoff was simulated in soils sprayed with fungicide accidental spill doses. • Increased expression of pyrimethanil-responsive yeast genes was measured. • Toxicity to aquatic and soil standard organisms correlated with toxicity to yeast. • Yeast assay shown relevant for screening worst-cases of pyrimethanil contamination.

Research paper thumbnail of Identification and exploitation of Burkholderia cepacia complex virulence factors as potential antimicrobial targets

1st Portuguese Biomedical Engineering Meeting, 2011

Abstract A multidisciplinary approach is being used to identify and validate virulence factors an... more Abstract A multidisciplinary approach is being used to identify and validate virulence factors and determinants of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of problematic opportunistic pathogenic bacteria, particularly among cystic fibrosis patients, ...

Research paper thumbnail of A RNomics-based strategy identifies regulatory small RNAs in Burkholderia cepacia complex

1st Portuguese Biomedical Engineering Meeting, 2011

Research paper thumbnail of The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans

Microbiology, 2009

TheBurkholderia cepaciacomplex (Bcc) emerged as problematic opportunistic pathogens to cystic fib... more TheBurkholderia cepaciacomplex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of aB. cepaciaIST408 mutant containing a disruption in thehfqgene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). TheB. cepaciaHfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When providedin trans, theB. cepaciaIST408hfqgene complemented theEscherichia coli hfqmutant strain GS081. Our results also show that theB. cepacia hfqmutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In additio...

Research paper thumbnail of Burkholderia cenocepacia J2315 acyl carrier protein: A potential target for antimicrobials' development?

Microbial Pathogenesis, 2008

This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant fr... more This work describes the isolation and characterization of an acyl carrier protein (ACP) mutant from Burkholderia cenocepacia J2315, a strain of the Burkholderia cepacia complex (Bcc). Bcc comprises at least 9 species that emerged as opportunistic pathogens able to cause life-threatening infections, particularly severe among cystic fibrosis patients. Bacterial ACPs are the donors of the acyl moiety involved in the biosynthesis of fatty acids, which play a central role in metabolism. The mutant was found to exhibit an increased ability to form biofilms in vitro, a more hydrophobic cell surface and reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as a model of infection. The B. cenocepacia J2315 ACP protein is composed of 79 amino acid residues, with a predicted molecular mass and pI of 8.71 kDa and 4.08, respectively. The ACP amino acid sequence was found to be 100% conserved within the genomes of the 52 Burkholderia strains sequenced so far. These data, together with results showing that the predicted structure of B. cenocepacia J2315 ACP is remarkably similar to the Escherichia coli AcpP, highlight its potential as a target to develop antibacterial agents to combat infections caused not only by Bcc species, but also by other Burkholderia species, especially B. pseudomallei and B. mallei.

Research paper thumbnail of The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence

Microbial Pathogenesis, 2010

Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex,... more Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex, a group of bacteria that emerged as important pathogens, especially to cystic fibrosis (CF) patients. In this study, we report the identification and characterization of a mutant strain derived form the CF isolate Burkholderia cenocepacia K56-2, carrying a plasposon insertion in a gene, located in a 3516 bp chromosomal region with an atypical G þ C content, encoding a 80 amino acid putative regulatory protein named Pbr. Besides its inability to produce phenazines, the B. cenocepacia K56-2 pbr mutant exhibited a pleiotropic phenotype, including impaired survival to oxidative and osmotic stress, aromatic amino acid and prolonged nutrient starvation periods. In addition, the pbr mutant exhibited decreased virulence the nematode Caenorhabditis elegans. Altogether, our results demonstrate the involvement of Pbr on the regulation of phenazine biosynthesis, and an important role for this regulatory protein on several cellular processes related to stress resistance and virulence.

Research paper thumbnail of 109 The Burkholderia cepacia small colony variants (SCV) are a more pathogenic bacterial form that may facilitate persistent respiratory infections in CF patients

Journal of Cystic Fibrosis, 2013

Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse... more Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. Understanding the mechanisms involved in bacterial virulence and in genetic adaptation of these bacteria to the CF lung is crucial for an improved management of Bcc chronic infections [1]. A 2 decade-long systematic survey of Bcc respiratory infections in the major Portuguese CF Centre has allowed us to carry out a retrospective study of the adaptive clonal variation of these pathogens within the airways during lung function deterioration [1−4]. In this work, we assessed the virulence of 3 B. cenocepacia clonal isolates spanning a 3.5-year long chronic infection in a single CF patient, based on their ability to invade epithelial cells and open tight junctions. The isolates retrieved later before the patient's death with cepacia syndrome were found to be significantly more virulent than the one that initiated the infection. A genome-wide quantitative proteomics approach was exploited to identify differentially expressed proteins and infer mechanisms that could be associated with B. cenocepacia virulence and persistence in the lung (ref. [2], unpublished results). Overall, the results are suggestive of adaptation to microaerophilic conditions and iron availability, and highlight the important role of metabolic optimization in adaptation and increased pathogenic potential of B. cenocepacia during the course of a chronic CF infection.

Research paper thumbnail of 111 The Burkholderia cenocepacia J2315 small non-coding RNA MavA is required for fitness and virulence

Journal of Cystic Fibrosis, 2013

Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse... more Burkholderia cepacia complex (Bcc) infections in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. Understanding the mechanisms involved in bacterial virulence and in genetic adaptation of these bacteria to the CF lung is crucial for an improved management of Bcc chronic infections [1]. A 2 decade-long systematic survey of Bcc respiratory infections in the major Portuguese CF Centre has allowed us to carry out a retrospective study of the adaptive clonal variation of these pathogens within the airways during lung function deterioration [1−4]. In this work, we assessed the virulence of 3 B. cenocepacia clonal isolates spanning a 3.5-year long chronic infection in a single CF patient, based on their ability to invade epithelial cells and open tight junctions. The isolates retrieved later before the patient's death with cepacia syndrome were found to be significantly more virulent than the one that initiated the infection. A genome-wide quantitative proteomics approach was exploited to identify differentially expressed proteins and infer mechanisms that could be associated with B. cenocepacia virulence and persistence in the lung (ref. [2], unpublished results). Overall, the results are suggestive of adaptation to microaerophilic conditions and iron availability, and highlight the important role of metabolic optimization in adaptation and increased pathogenic potential of B. cenocepacia during the course of a chronic CF infection.

Research paper thumbnail of Variation of the antimicrobial susceptibility profiles of Burkholderia cepacia complex clonal isolates obtained from chronically infected cystic fibrosis patients: a five-year survey in the major Portuguese treatment center

European Journal of Clinical Microbiology & Infectious Diseases, 2008

The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia com... more The treatment of cystic fibrosis (CF) patients chronically infected with Burkholderia cepacia complex (Bcc) bacteria requires extensive and aggressive antibiotics therapy, exposing these bacteria to prolonged antibiotics-selective pressure. In the present study, we have compared the susceptibility patterns to 13 antimicrobials of 94 Bcc isolates obtained from 15 Portuguese CF patients in the course of chronic infection during a five-year survey. These isolates were previously genotyped and represent 11 different strains of the species B. cenocepacia (subgroups A and B), B. cepacia, B. multivorans, and B. stabilis. The results are consistent with the notion that CF Bcc isolates are resistant to the most clinically relevant antimicrobials and suggest an uneven distribution of resistance rates among the different species, with B. cenocepacia subgroup A isolates being the most resistant. Phenotypic variants exhibiting differences in the antimicrobial susceptibility patterns were obtained from the sputum samples of clinically deteriorated CF patients during chronic lung infection. The isolation of resistant variants coincided with periods of pulmonary exacerbation and antibiotics therapy.

Research paper thumbnail of Bioinformatics: A Molecular Microbiologist’s Perspective

Current Bioinformatics, 2014

Research activities in the area of biological sciences, and particularly molecular microbiology, ... more Research activities in the area of biological sciences, and particularly molecular microbiology, nowadays generate vast amounts of data. The development of faster and cheaper sequencing methods has definitively contributed to this huge amount of data, requiring the development of fast and reliable tools for their analysis. In the last years, many easy-to-use, reliable and powerful bioinformatics tools have been developed. In this work, we review the fundamentals of some of these tools, and describe how we have been using them to analyze data resulting from our research envisaging the identification and characterization of virulence factors and determinants from the human opportunistic pathogens of the Burkholderia cepacia complex. Examples given illustrate the user-friendly characteristic of these tools and their power both in analyzing information and in orientating future experimental work.