Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum

The cathelicidin derived human peptide LL37 has a broad spectrum of antimicrobial and immunomodulatory activities. The large variety of biological activities makes LL37 a very promising candidate for clinical applications. The production of biologically active LL37 in large amounts with reduced costs can only be achieved using recombinant techniques. In this work, LL37 has been cloned to the N- and C-termini of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; both constructions (LL37-LK-CBM3 and LK-CBM3-LL37) were cloned into the pET-21a vector. A formic acid recognition site was introduced between the two modules, allowing the isolation of LL37 after chemical cleavage. The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and solubilized with Triton X-100. The purification was achieved using cellulose CF11 fibers, taking advantage of the CBM3 specific affinity for cellulose; after hydrolysis with formic acid, LL37 was further purified by reverse-phase HPLC, as confirmed by MALDI-TOF mass spectrometry. The production and purification methodology developed in this work compares advantageously to other protocols previously described, having fewer purification steps. Only the recombinant LL37 obtained from the C-terminally fused protein (LK-CBM3-LL37) showed antibacterial activity against E. coli K12, with a MIC of 180 μg/ml.University of Porto. Institute of Molecular Pathology and Immunology. Proteomics Unit (IPATIMUP)Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/27404/200

Destabilization of α-helical structure in solution improves bactericidal activity of antimicrobial peptides: Opposing effects on bacterial and viral targets

We have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. Here we show that the formulation of peptides LL37 and magainin-2B amide in polysorbate 20 (Tween-20™) results in greater reductions in virus titre than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high ionic strength buffer. In contrast, formulation with polysorbate 20 or high ionic strength buffer has the opposite effect on bactericidal activity of both peptides, resulting in lesser reductions in titre for both gram-positive and gram-negative bacteria. Circular dichroism spectroscopy shows that the differential action of polysorbate 20 and salt on the virucidal and bactericidal activities correlates with the α-helical content of peptide secondary structure in solution, suggesting that the virucidal and bactericidal activities are mediated through distinct mechanisms. The correlation of a defined structural feature with differential activity against a host-derived viral membrane and the membranes of both gram-positive and gram-negative bacteria suggests that overall helical content in solution under physiological conditions is an important feature for consideration in the design and development of candidate peptide-based antimicrobial compounds

LL37, a human antimicrobial peptide with immunomodulatory properties

Cationic antimicrobial peptides (AMPs) represent the first line of defense against many invading pathogens. These small amphipathic peptides are part of the innate immune system and have a broad-spectrum activity against bacteria, fungi and viruses. In mammals, at least two distinct groups of AMPs are found. Defensins are the more representatives and cathelicidins form the second group. The hCAP18/LL37 is the only known human cathelicidin. The antimicrobial peptide is referred to as LL37, since it has a 37 amino acids sequence starting with two leucines. It is a 4.5 kDa, cationic (+6), amphipathic -helical peptide, with a broad spectrum of antimicrobial activity. Besides its antimicrobial properties LL37 plays a central role in innate immune responses and inflammation. It has been identified as a potent chemoattractant for mast cells, monocytes, T lymphocytes and neutrophils using formyl-peptide receptorlike 1 (FPRL1). LL37 also promotes wound healing, angiogenesis and arteriogenesis and acts as immune adjuvant.y FEDER funds through the Programa Operacional Factores de Competitividade – COMPETE and by national funds through the project PTDC/EDB/EBI/112170/2009 from the Fundação para a Ciência e Tecn

The oral microbiome of denture wearers is influenced by natural dentition

Objectives: The composition of dental plaque has been well defined, whereas currently there is limited understanding of the composition of denture plaque and how it directly influences denture related stomatitis (DS). The aims of this study were to compare the microbiomes of denture wearers, and to understand the implications of these towards inter-kingdom and host-pathogen interactions within the oral cavity. Methods: Swab samples were obtained from 123 participants wearing either a complete or partial denture; the bacterial composition of each sample was determined using bar-coded illumina MiSeq sequencing of the bacterial hypervariable V4 region of 16S rDNA. Sequencing data processing was undertaken using QIIME, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. The dentures were sonicated to remove the microbial flora residing on the prosthesis, sonicate was then cultured using diagnostic colorex Candida media. Samples of unstimulated saliva were obtained and antimicrobial peptides (AMP) levels were measured by ELISA. Results: We have shown that dental and denture plaques are significantly distinct both in composition and diversity and that the oral microbiome composition of a denture wearer is variable and is influenced by the location within the mouth. Dentures and mucosa were predominantly made up of Bacilli and Actinobacteria. Moreover, the presence of natural teeth has a significant impact on the overall microbial composition, when compared to the fully edentulous. Furthermore, increasing levels of Candida spp. positively correlate with Lactobacillus spp. AMPs were quantified, though showed no specific correlations. Conclusions: This is the first study to provide a detailed understanding of the oral microbiome of denture wearers and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS, though we were unable to show a defined role for AMPs

Direct thrust measurement of a 30-cm ion thruster

A direct thrust measurement of a 30-cm diameter ion thruster was accomplished by means of a laser interferometer thrust stand. The thruster was supported in a pendulum manner by three 3.65-m long wires. Electrical power was provided by means of 18 mercury filled pots. A movable 23-button planar probe rake was used to determine thrust loss due to ion beam divergence. Values of thrust, thrust loss due to ion beam divergence, and thrust loss due to multiple ionization were measured for ion beam currents ranging from 0.5 A to 2.5 A. Measured thrust values indicate an accuracy of approximately 1% and are in good agreement with thrust values calculated by indirect measurements

Антимикробные пептиды в патогенезе инфекционных осложнений в акушерстве и гинекологии

Описаны основные виды антимикробных пептидов и особенности их экспрессии в органах репродуктивного тракта женщин вне и во время беременност

Antimicrobial peptides as novel anti-tuberculosis therapeutics

"Available online 24 May 2016"Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, has recently joined HIV/AIDS as the world's deadliest infectious disease, affecting around 9.6 million people worldwide in 2014. Of those, about 1.2 million died from the disease. Resistance acquisition to existing antibiotics, with the subsequent emergence of Multi-Drug Resistant mycobacteria strains, together with an increasing economic burden, has urged the development of new anti-TB drugs. In this scope, antimicrobial peptides (AMPs), which are small, cationic and amphipathic peptides that make part of the innate immune system, now arise as promising candidates for TB treatment. In this review, we analyze the potential of AMPs for this application. We address the mechanisms of action, advantages and disadvantages over conventional antibiotics and how problems associated with its use may be overcome to boost their therapeutic potential. Additionally, we address the challenges of translational development from benchside to bedside, evaluate the current development pipeline and analyze the expected global impact from a socio-economic standpoint. The quest for more efficient and more compliant anti-TB drugs, associated with the great therapeutic potential of emerging AMPs and the rising peptide market, provide an optimal environment for the emergence of AMPs as promising therapies. Still, their pharmacological properties need to be enhanced and manufacturing-associated issues need to be addressed.Portuguese Foundation for Science and Technology (FCT) - UID/ BIO/04469/2013 unit ; COMPETE 2020 (POCI-01-0145-FEDER- 006684) ; SFRH/BPD/64958/2010Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462

Role of Pattern Recognition Receptors in the Modulation of Antimicrobial Peptide Expression in the Corneal Epithelial Innate Response to F. solani

Purpose: Fusarium solani (F. solani) keratitis is a potentially sight-threatening fungal infection of the cornea. Antimicrobial peptides (AMPs), such as human β-defensins (hBDs) and cathelicidins, essential components of the immune system, likely have a protective role against F. solani keratitis. We examined the role of pattern recognition receptors (PRRs), Dectin-1, and TLR2 in F. solani–induced modulation of AMP expression in vitro. Methods: Human corneal epithelial cells (HCECs) were exposed to heat-inactivated F. solani or pathogen-associated molecular patterns (PAMPs) of F. solani (Zymosan or Zymosan Depleted) for 6, 12, or 24 hours following which AMP mRNA and protein levels were determined. Involvement of TLR2 and Dectin-1 was confirmed by using siRNA knock-down (TLR2 and Dectin-1) or chemical inhibitor BAY 61-3606 (Dectin-1). The functional significance of AMP upregulation was tested using culture supernatant from F. solani or PAMP-treated HCECs against F. solani in the presence of hBD2 or LL37 neutralizing antibody. Results: We confirm that HCECs express Dectin-1 and TLR2. HCECs demonstrated upregulation of AMPs hBD2 and cathelicidin LL37 following exposure to heat-inactivated F. solani or PAMPs. TLR2 and Dectin-1 knockdown and BAY 61-3606 treatment decreased AMP mRNA upregulation confirming PRR involvement. The culture supernatant from F. solani or PAMP-treated HCECs showed substantial killing of F. solani and hBD2 or LL37 neutralizing antibody significantly decreased this effect implicating involvement of these AMPs. Conclusions: These findings demonstrate that Dectin-1 and TLR2 have an important role in regulating F. solani-induced AMP expression in corneal epithelial cells