Novel strategies to combat gram-negative bacterial pathogens using bacteriophage proteins

Dissertação de mestrado em Bioengenharia

Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections

Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics and nowadays, a common infection can be fatal. To revert this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since P. aeruginosa is one of the most common causes of healthcare-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review will further address the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Construction of an innocuous Salmonella phage

Dissertação de mestrado em Bioengenharia

Distance perception in immersive environments: the role of photorealism

Immersive environments (IE) are being increasingly used in order to perform psychophysical experiments. The versatility in terms of stimuli presentation and control and the less time-consuming procedures are their greatest strengths. However, to ensure that IE results can be generalized to real world scenarios we must first provide evidence that performance in IE is quantitatively indistinguishable from performance in real-world. Our goal was to perceptually validate distance perception for CAVE-like IEs. Participants performed a Frontal Matching Distance Task (Durgin & Li, 2011) in three different conditions: real-world scenario (RWS); photorealistic IE (IEPH) and non-photorealistic IE (IENPH). Underestimation of distance was found across all the conditions, with a significant difference between the three conditions (Wilks’ Lambda = .38, F(2,134)= 110.8, p<.01, significant pairwise differences with p<.01). We found a mean error of 2.3 meters for the RWS, 5 meters for the IEPH, and of 6 meters for the IENPH in a pooled data set of 5 participants. Results indicate that while having a photorealistic IE with perspective and stereoscopic depth cues might not be enough to elicit a real-world performance in distance judgment tasks, nevertheless this type of environment minimizes the discrepancy between simulation and real-world when compared with non-photorealistic IEs.

Engineering modular viral scaffolds for targeted bacterial population editing

Bacteria are central to human health and disease, but existing tools to edit microbial consortia are limited. For example, broad-spectrum antibiotics are unable to precisely manipulate bacterial communities. Bacteriophages can provide highly specific targeting of bacteria, but assembling well-defined phage cocktails solely with natural phages can be a time-, labor- and cost-intensive process. Here, we present a synthetic biology strategy to modulate phage host ranges by engineering phage genomes in Saccharomyces cerevisiae. We used this technology to redirect Escherichia coli phage scaffolds to target pathogenic Yersinia and Klebsiella bacteria, and conversely, Klebsiella phage scaffolds to target E. coli by modular swapping of phage tail components. The synthetic phages achieved efficient killing of their new target bacteria and were used to selectively remove bacteria from multi-species bacterial communities with cocktails based on common viral scaffolds. We envision this approach accelerating phage biology studies and enabling new technologies for bacterial population editing.

Curry-Howard for sequent calculus at last!

This paper tries to remove what seems to be the remaining stumbling blocks in the way to a full understanding of the Curry-Howard isomorphism for sequent calculus, namely the questions: What do variables in proof terms stand for? What is co-control and a co-continuation? How to define the dual of Parigot's mu-operator so that it is a co-control operator? Answering these questions leads to the interpretation that sequent calculus is a formal vector notation with first-class co-control. But this is just the "internal" interpretation, which has to be developed simultaneously with, and is justified by, an "external" one, offered by natural deduction: the sequent calculus corresponds to a bi-directional, agnostic (w.r.t. the call strategy), computational lambda-calculus. Next, the duality between control and co-control is studied and proved in the context of classical logic, where one discovers that the classical sequent calculus has a distortion towards control, and that sequent calculus is the de Morgan dual of natural deduction.

Strategies to combat infections of Acinetobacter baumannii biofilms

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r=0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

Bacteriophage-encoded depolymerases: their diversity and biotechnological applications

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

Blow-up and finite time extinction for p(x, t)-curl systems arising in electromagnetism

"Available online 22 March 2016"

Synthetic biology approaches to shape bacteriophages towards Pseudomonas aeruginosa biofilm control

Dissertation for Ph.D. degree in Biomedical Engineering.