Development of antimicrobial protein-based polymers for biomedical applications

Inspired in naturally occurring fibrous proteins and composed of amino acid building blocks commonly found in structural proteins, protein-based polymers (PBPs) are a group of materials with unique chemical, physical and biological properties. Coventional recombinant DNA technology allows the biological synthesis of recombinant protein-based polymers (rPBPs) with precise control over its size and composition and the incorporation of functional bioactive domains such as antimicrobial peptides (AMPs). Owing to the unique balance between their mechanical properties, biocompatibility, biodegradability and thermostability, elastin-like recombinamers (ELRs) and silk-elastin like proteins (SELPs) are two of the most remarkable families of rPBP for biotechnological applications. Here, we describe the functionalization of a SELP and an ELR with different antimicrobial peptides that showed promising results against several Gram-positive and Gram-negative bacterial strains. This will provide the basis for the development of advanced biomaterials processed into different types of structures (e.g. hydrogels, films, fibers, particles) suitable for biomedical applications

Protein-engineered polymers functionalized with antimicrobial peptides for the development of active surfaces

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.This work was supported by the “Contrato-Programa” UIDB/04050/2020, project FunBioPlas (ERA-IB-2-6/0004/2014) and project FUN2CYT (POCI-01-0145-FEDER-030568) funded by Portugal national funds through the Fundação para a Ciência e a Tecnologia (FCT I.P.). A.M.P. acknowledges the Doctoral Programme in Applied and Environmental Microbiology (DP_AEM) and FCT I.P. for the PD/BD/113811/2015 grant. R.M. acknowledges FCT I.P. for funding in the scope of the Scientific Employment Stimulus instrument (CEECIND/00526/2018)

Electrospun silk-elastin fibres functionalized with silver nanoparticles as antibacterial wound dressings

[Excerpt] Silk-elastin-like proteins (SELPs) are a class of bioinspired, genetically engineered block copolymers, composed of silk and elastin repeating units. As base materials for biomedical purposes, SELP nanofibre mats demonstrate potential to be applied as wound dressing materials [1]. The increasing antimicrobial resistance associated with the excessive and inappropriate use of antibiotics demands the research for new pathogen-free healthcare polymeric materials with enhanced biological performance. [...]This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01- 0145-FEDER- 007569) through FCT I.P. and by ERDF through COMPETE2020 - POCI. The authors are grateful for funding from FCT (project “FunBioPlas” ERA-IB-2-6/0004/2014) and a fellowship to RM (SFRH-BPD/86470/2012). The authors also thank support from the COST Action MP1206

Genetically engineered silk-based composite biomaterials functionalized with fibronectin type-II that promote cell adhesion

[Excerpt] Recombinant protein-based polymers (rPBPs) are an emerging class of biopolymers inspired by Nature and produced by synthetic protein biotechnology approaches. Due to their exceptional physical-chemical and biological characteristics, as well as their ability to be customized for specific applications, rPBPs have been explored for the development of advanced biomaterials [1]. Within rPBPs, silk-like polymers (SLP) are being utilized in a range of studies in materials science [2]. [...]This work was supported by FCT Funded Project “Chimera” (PTDC/EBB-EBI/109093/2008), by FCT/MEC through Portuguese funds (PIDDAC) – PEst-OE/BIA/UI4050/2014, by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). TC is thankful to the FCT for its support through Investigador FCT 2015. ARibeiro thanks FCT for the SFRH/BPD/98388/2013 grant. RMachado and AdaCosta acknowledge FCT for SFRH-BPD/86470/2012 and SFRH/BD/75882/2011 grants, respectively

Corrigendum to ‘‘Silk-based biomaterials functionalized with fibronectin type II promotes cell adhesion” [Acta Biomater. 47 (2017) 50–59]

The authors regret that Telma C. Bernardo was inadvertently omitted in the author line-up. The correct authorship order should be as follows: Ana Margarida Pereira, Raul Machado, André da Costa, Artur Ribeiro, Telma C. Bernardo, Tony Collins, Andreia C. Gomes, Isabel B. Leonor, David L. Kaplan, Rui L. Reis, Margarida Casal. Telma C. Bernardo participated in recombinant 6mer+FNII production and purification. The authors regret the error and would like to apologize for any inconvenience caused.- (undefined

Genetically engineered Silk-Elastin-Like Proteins as a versatile platform for the development of new biomaterials

[Excerpt] Throughout evolution nature created and refined proteins for a wide range of functions, working as structural components or as molecular motors. In this sense, the natural fibrous proteins represent the utmost case of function specialization and high performance materials. The remarkable mechanical properties of proteins like elastin or silk are founded on conservative blocks of amino acid sequences that propagate through the natural protein. These repetitive amino acid sequences are arranged in a way that creates flexible, rigid or tough domains, which are responsible for the physical and mechanical properties of the natural protein. Indeed, the recognition of the mechanics linking the nano- and micro-scale structure with the macromolecular assembly and organization, enabled molecular biologists to understand nature’s refined ways of creating high performance structural materials. Advances in synthetic protein biotechnology, emerging from the increase of knowledge in structural and molecular biology, combined with the use of recombinant DNA technology and biotechnology processes, made possible the advent of a new class of artificial biomacromolecules, the recombinant Protein-Based Polymers (rPBPs). This new class of protein-based materials, inspired in nature and with precisely controlled amino acid sequences, mimic the properties of their natural counterparts but can also combine in the same polypeptide chain the properties of two or more different proteins, creating copolymers with distinct properties from their native equivalents. Indeed, by recombinant DNA technology, it is possible to design and produce tailored synthetic genes, allowing for the creation of multifunctional complex PBPs with absolute control of its composition, structure and molecular weight. [...]This work is supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by the project EcoAgriFood (NORTE-01-0145-FEDER-000009), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It is also supported by FCT within the ERA-NET IB, project FunBioPlas with grant number ERA-IB-15-089 and FCT reference ERA-IB-2-6/0004/2014. AMPereira acknowledges DP_AEM and FCT for the PD/BD/113811/2015 grant

Advanced silk-based genetic polymers with improved cell adhesion properties

[Excerpt] Recombinant protein-based polymers (rPBPs) are an emerging class of genetic polymers inspired by Nature and produced by synthetic protein biotechnology approaches. Due to their exceptional physical-chemical and biological characteristics, as well as their ability to be customized for specific applications, rPBPs have been explored for the development of advanced biomaterials [1]. Most of the polymers used as biomaterials thus far have been chemically synthesized, originating random copolymers with diverse and uncontrolled distribution of molecular weight (MW) and composition. However, advances in recombinant DNA technology allow the biological synthesis of fine-tuned rPBPs with precise control of their composition, polymer size and structure [2]. Furthermore, with the development of recombinant protein engineering and biotechnology, it is now possible to design new bioactive rPBPs by combining active peptides/domains from different natural proteins in the same fusion protein. [...

Perceções sobre o mecanismo de atividade antimicrobiana e sua exploração para o desenvolvimento de novas terapias contra bactérias clinicamente relevantes

Programa Doutoral em Biologia (Especialidade em Biotecnologia Molecular)No último século, o uso excessivo e incorreto de antibióticos conduziu ao desenvolvimento de bactérias multirresistentes, dando origem a uma crise de resistência bacteriana a antibióticos, representando uma ameaça à saúde pública global. Assim, é crucial o desenvolvimento de novas abordagens terapêuticas contra infeções bacterianas. Ubíquos na natureza, os péptidos antimicrobianos (AMPs) são moléculas pequenas, anfipáticas e com carga positiva, cujo modo de ação melhor descrito se baseia na interação eletrostática entre os péptidos catiónicos e os componentes aniónicos das membranas celulares. O rompimento destas estruturas fundamentais e a interação dos AMPs com múltiplos alvos intracelulares, dificultam o desenvolvimento de mecanismos de resistência bacteriana aos AMPs. Além disso, a seletividade dos AMPs para bactérias em relação a células de mamífero acentua o seu potencial terapêutico. A morosidade e o baixo custo-benefício da síntese química e extração de fontes naturais faz com que a produção de AMPs recombinantes seja uma alternativa vantajosa. No entanto, os AMPs são suscetíveis a degradação proteolítica e podem ser tóxicos para os seus hospedeiros de expressão. Estas limitações podem ser ultrapassadas pela expressão dos AMPs fundidos com outras proteínas, nomeadamente polímeros recombinantes de base proteica (rPBP), que atuam como tags de produção e purificação, protegendo os AMPs de potencial proteólise. Adicionalmente, esta estratégia permite o desenvolvimento de biopolímeros com atividade antimicrobiana e seu processamento em diferentes materiais antimicrobianos. Dado o equilíbrio único entre suas propriedades mecânicas, de biocompatibilidade e de biodegradabilidade, polímeros recombinantes semelhantes à elastina (ELRs) e as proteínas com base na seda e na elastina (SELPs) são duas das famílias mais extraordinárias de rPBPs para aplicações biotecnológicas. Nesta tese é descrita a funcionalização de um SELP e um ELR com diferentes AMPs, a sua produção biológica em Escherichia coli e o processamento dos polímeros híbridos em diferentes estruturas. Estes polímeros mostraram atividade antimicrobiana contra bactérias Gram-positivas e Gram-negativas. Além disso, foi explorado o uso do ELR como parceiro de fusão para a produção e isolamento de AMPs bioativos. O trabalho desenvolvido contribui para o desenvolvimento de biomateriais avançados adequados para aplicações biomédicas e para o desenvolvimento de uma plataforma biotecnológica para a produção e purificação de AMPs.Over the last century, the overuse and misuse of antibiotics has led to the development of bacteria resistant to these antimicrobial agents, prompting an antimicrobial resistance (AMR) crisis that continues to emerge as a global public health threat. For this reason, it is of foremost importance to develop new therapeutic approaches against bacterial infections. Ubiquitous in nature, antimicrobial peptides (AMPs) are small, amphipathic and positively charged molecules, with the best described mode of action based on the electrostatic interaction between the cationic peptides and the negatively charged components on bacterial cell membranes. The disruption of these fundamental structures and their interaction with multiple targets within the cell turns unlikely the development of bacterial resistance mechanisms against AMPs. In addition, selectivity of AMPs towards bacteria over mammalian cells further increases their therapeutic potential. Chemical synthesis of AMPs or extraction from natural sources are often time-consuming and low cost-effective procedures. The heterologous production of AMPs represents an advantageous alternative, allowing the implementation of scalable and efficient processes. Albeit the benefits of recombinant production, AMPs are susceptible to proteolysis and can be toxic for their production hosts. Nevertheless, these limitations can be overcome through the expression of AMPs fused with other proteins, namely protein-based recombinant polymers (rPBP), that act as production and purification tags while protecting AMPs from proteolytic degradation. In addition, this strategy also allows the development of biopolymers with antimicrobial activity and their processing into different antimicrobial materials. Owing to the unique balance between their mechanical properties, biocompatibility and biodegradability, elastin-like recombinamers (ELRs) and silk-elastin-like proteins (SELPs) are two of the most remarkable families of rPBPs for biotechnological applications. In this thesis, we report the functionalization of a SELP and an ELR with different AMPs, their biological production using Escherichia coli as the expression system and the processing of the hybrid polymers into different types of structures. These polymers showed antimicrobial activity against both Gram-positive and Gram-negative bacteria. In addition, we explored the use the ELR as fusion partner for the production and isolation of bioactive AMPs. The work developed provides the basis for the development of advanced biomaterials suitable for biomedical applications and the development of a biotechnological platform for the production and purification of AMPs.This work was supported by the project FunBioPlas ERA-IB-2-6/0004/2014 funded by national funds through FCT I.P., the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - POCI, and the project EcoAgriFood (NORTE01-0145-FEDER-000009), supported by the NORTE 2020 under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). AMP is a student of the Doctoral Programme in Applied and Environmental Microbiology (DP_AEM) and FCT grantee PD/BD/113811/2015. We thank Prof. Simoni Campos-Dias for the contribution to Chapters VII and VIII

Structural and functional measures of leaf-associated invertebrates and fungi as predictors of stream eutrophication

Eutrophication is a major threat to freshwater ecosystems worldwide that affects aquatic biota and compromises ecosystem functioning. In this study, we assessed the potential use of leaf decomposition and associated decomposer communities to predict stream eutrophication. Because leaf quality is expected to affect leaf decomposition, we used five leaf species, differing in their initial nitrogen concentration. Leaves of alder, chestnut, plane, oak and eucalyptus were placed in coarse-mesh bags and immersed in six streams along an eutrophication gradient to assess leaf decomposition and the structure of associated decomposer communities. A hump-shaped relationship was established between leaf decomposition and the eutrophication gradient for all leaf species, except for eucalyptus. Invertebrate biomass and density as well as fungal biomass and sporulation were lowest at the extremes of the gradient. Leaf-associated invertebrate and fungal assemblages were mainly structured by stream eutrophication. The percentage of shredders on leaves decreased, whereas the percentage of oligochaeta increased along the eutrophication gradient. The Iberian Biological Monitoring Working Party Index (IBMWP) applied to benthic invertebrates increased from oligotrophic to moderately eutrophic streams and then dropped sharply at highly and hypertrophic streams. Overall, leaf decomposition was a valuable tool to assess changes in stream water quality, and it allowed the discrimination of sites classified by the IBMWP within class I and class IV. Moreover, decomposition of most leaf species responded in a similar way to eutrophication when decomposition was normalized by the quality of leaves. (C) 2016 Elsevier Ltd. All rights reserved.FEDER-POFC-COMPETE and the Portuguese Foundation for Science and Technology (FCT) supported this study (PTDC/AAC-AMB/117068/2010, UID/BIA/04050/2013, and PEst-OE/BIA/UI4050/2014). Financial support given by FCT to IF (SFRH/BPD/97656/2013) and PG (SFRH/BD/75516/2010) is also acknowledged.info:eu-repo/semantics/publishedVersio

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved