Albert Van Dijk - Academia.edu (original) (raw)

Papers by Albert Van Dijk

PLOS ONE, Apr 22, 2014

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate ... more The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial-and immunomodulatory activity of PR-39, and N-and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-a production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.

PLOS ONE, Feb 5, 2016

Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodul... more Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that fulllength CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives.

Peptide science, May 31, 2021

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis v... more Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), a coronavirus of domestic fowl. IB is a major concern in the poultry industry, causing worldwide economic losses through decreased egg production and quality and by increasing the chicken's susceptibility for secondary bacterial infections, particularly Escherichia coli. In this study, the anti-IBV activity of cathelicidins, small antimicrobial peptides of the innate immune system was investigated. The cell culture adapted (nonvirulent) IBV strain Beaudette was effectively inhibited by the human cathelicidin LL-37 in bovine hamster kidney-21 cells at nontoxic concentrations. The peptide needed to be present during virus inoculation to effectively inhibit the infection of IBV-Beaudette, indicating that LL-37 likely bound viral particles. However, no clear morphological changes in the IBV virion upon binding were observed by electron microscopy. In this cell culture model, chicken cathelicidins (CATH1-3) were inactive against IBV-Beaudette. In contrast, in multicellular infection models using the virulent IBV-M41 strain the activities of human and chicken cathelicidins were different. In particular, upon inoculation of 10-day-old embryonic eggs with IBV-M41, CATH-2 reduced the viral load to a higher extend than LL-37. Similarly, viral infection of chicken tracheal organ cultures with IBV-M41 was significantly reduced in the presence of CATH-2 but not LL-37. These results indicate a potential antiviral role for CATH-2 upon IBV infection in vivo. K E Y W O R D S coronavirus, host defense peptides, innate immunity 1 | INTRODUCTION Host defense peptides (HDPs) are small cationic peptides that play an important role in the innate host defense against pathogens. [1] These peptides are produced as prepropeptides by epithelial and innate immune cells. [2] Upon release of the HDPs the prepro sequence is cleaved releasing the active peptides locally where they exert their antimicrobial and immunomodulatory activities. While HDPs of the

Antimicrobial Agents and Chemotherapy, Mar 1, 2007

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and ... more Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of ␤-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken ␤-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 g/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken ␤-defensin gallinacin-6 plays an important role in chicken innate host defense.

Veterinary Immunology and Immunopathology, Jun 1, 2018

Background: Macrophages play an important role in the innate immune system as part of the mononuc... more Background: Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Methods: Porcine monocytes or mononuclear bone marrow cells were used to culture M1-and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Results: Porcine M1-and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1-and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Conclusion: Porcine M1-and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research.

Biochimica et Biophysica Acta, 2017

Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunit... more Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 βlike protein. Methods Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. Results GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is Highlights A new human cryptic peptide, named GVF27, has been identified in 11-hydroxysteroid dehydrogenase-1 β-like GVF27 is unstructured in aqueous buffer but assumes a well defined conformation in mimic membrane environments GVF27 exerts anti-biofilm actions and broad spectrum antimicrobial activities GVF27 binds bacterial endotoxins as LPS and LTA GVF27 induces mitigation of pro-inflammatory mediators production in LPS induced murine macrophages

Biochimica Et Biophysica Acta - General Subjects, Sep 1, 2021

BACKGROUND Antimicrobial peptides are considered potential alternatives for antibiotics. Here we ... more BACKGROUND Antimicrobial peptides are considered potential alternatives for antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. METHODS Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of Pepbiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. RESULTS A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using isothermal titration calorimetry and 31P solid state NMR. CONCLUSIONS PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. GENERAL SIGNIFICANCE These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

mSphere, Aug 25, 2021

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of ... more Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs. KEYWORDS Gram-negative bacteria, antibiotic resistance, antimicrobial peptides, host defense peptides, outer membrane vesicles W hen pathogens enter a host, they enter a hostile environment. Host species have developed many measures to eliminate and remove pathogens; however, pathogens have been evolving simultaneously. Well known is the development of antibiotic resistance, but pathogens also have found ingenious mechanisms to evade the host's intrinsic immune system (1). One of the first innate defense molecules pathogens will encounter are host defense peptides (HDPs). HDPs are small, cationic molecules and have antibacterial activities against a broad range of pathogens. They are amphipathic,

Biochimica Et Biophysica Acta - General Subjects, Sep 1, 2017

Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunit... more Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 βlike protein. Methods Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. Results GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is Highlights A new human cryptic peptide, named GVF27, has been identified in 11-hydroxysteroid dehydrogenase-1 β-like GVF27 is unstructured in aqueous buffer but assumes a well defined conformation in mimic membrane environments GVF27 exerts anti-biofilm actions and broad spectrum antimicrobial activities GVF27 binds bacterial endotoxins as LPS and LTA GVF27 induces mitigation of pro-inflammatory mediators production in LPS induced murine macrophages

Veterinary Microbiology, Dec 1, 2012

Campylobacter jejuni, a small Gram-negative, spiral shaped bacterium, is a major cause of bacteri... more Campylobacter jejuni, a small Gram-negative, spiral shaped bacterium, is a major cause of bacterial food-borne disease, responsible for approximately 400 million cases of enterocolitis per year world wide (Girard et al., 2006). Wild and domesticated birds act as natural reservoirs for Campylobacter with chicken being the most important source of human Campylobacteriosis (Dasti et al., 2010). The interactions between Campylobacter and the avian innate immune system are poorly understood. Although the adaptive immune system is not yet fully developed after hatching (Bar-Shira and Friedman, 2006), intestinal colonization by the ubiquitously present Campylobacter

International Journal of Antimicrobial Agents, Sep 1, 2010

Molecular Immunology, Aug 1, 2009

Canine atopic dermatitis (AD) is a chronic allergic skin disorder with an immunopathogenesis comp... more Canine atopic dermatitis (AD) is a chronic allergic skin disorder with an immunopathogenesis comparable to that in humans with AD. The high frequency of recurrent infections with Staphylococcus pseudo intermedius and Malassezia pachydermatis may indicate a defective innate immune response in the skin of atopic dogs. Production of ␤-defensins constitutes an important role in skin defense but information on canine ␤-defensin localization and regulation is scarce. We conducted a gene-expression study of 16 canine ␤-defensins (cBDs) in 11 tissues of healthy dogs, which revealed a variable expression of cBDs in different organ systems of the dog. In skin, three ␤-defensins, cBD1, cBD103 and cBD107, were extensively expressed, while inconsistent expression of five other ␤-defensins was detected. Using immunohistochemistry abundant expression of cBD103 peptide was detected in the epidermis, hair follicles and sebaceous glands, comparable to hBD3 expression in human skin. To examine the gene-expression of ␤-defensins in atopic dogs, full thickness skin biopsy specimens (non-lesional and lesional) of 10 atopic dogs and 7 healthy dogs were examined with real-time PCR. A significant 12-fold increased expression of cBD1 was detected in lesional atopic skin compared to healthy skin, while non-lesional skin showed a 5-fold increase. Contrary to cBD1, expression of cBD103 was slightly (2-fold) downregulated in skin of atopic dogs. Gene-expression levels of S100A8, a marker for atopic dermatitis, were also highly upregulated in skin of atopic dogs, confirming the diagnostics of the skin biopsies. Taken together these results provide new evidence for a possible defect in the innate immune response of dogs with atopic dermatitis, and indicate the potential of the dog as a model for human AD.

Scientific Reports, Mar 18, 2019

Host defense peptides (HDPs) play a pivotal role in innate immunity and have, in addition to anti... more Host defense peptides (HDPs) play a pivotal role in innate immunity and have, in addition to antimicrobial activity, also important immunomodulatory functions. Bacteria are less likely to develop resistance against HDPs because these peptides target and kill bacteria in multiple ways, as well as modulate the immune system. Therefore, HDPs, and derivatives thereof, are promising alternatives to traditional antibiotics. Hardly anything is known about the immunomodulatory functions of porcine cathelicidin PMAP-36. In this study, we aimed to determine both antibacterial and immunomodulatory activities of PMAP-36 comparing the properties of PMAP-36 analogs with two well-studied peptides, human LL-37 and chicken CATH-2. Transmission electron microscopy revealed different killing mechanisms of E. coli for PMAP-36, CATH-2 and LL-37. LL-37 binds LPS very weakly in contrast to PMAP-36, but it inhibits LPS activation of macrophages the strongest. The first 11 amino acids of the N-terminal side of PMAP-36 are dispensable for E. coli killing, LPS-neutralization and binding. Deletion of four additional amino acids resulted in a strong decrease in activity. The activity of full length PMAP-36 was not affected by monomerization, whereas the shorter analogs require dimerization for proper immunomodulatory activity but not for their antibacterial activity. Cathelicidins constitute a family of host defense peptides (HDPs) and play an important role during the innate immune response 1. They consist of a highly conserved N-terminal region, containing a signal peptide and a cathelin domain, while the C-terminal region represents the highly variable domain of the active peptide 2-4. Although the sequences of cathelicidins are highly variable, almost all cathelicidins show, in simple media, direct antimicrobial activity against many different bacteria 3,5-7 , viruses 8-10 , fungi 11 , and parasites 12,13. Besides their direct antimicrobial activities, cathelicidins can also modulate the immune response. These peptides can induce chemotaxis directly or indirectly by inducing chemokine release 3,14,15. In addition, cathelicidins have been shown to be involved in phagocytosis 3,16-18 , neutralization of LPS or LTA during TLR stimulation 3,15,19,20 or enhancement of DNA uptake and subsequent TLR-9 activation 3,5,21,22 and they can skew macrophage differentiation towards a pro-inflammatory phenotype 23. Bacteria are less likely to develop resistance against HDPs because they do not only target and kill bacteria in multiple ways but also modulate the immune system. This dual function makes HDPs promising alternatives to antibiotics. In humans only one cathelicidin has been identified, LL-37 2 , while in chicken four cathelicidins have been identified, CATH-B1 and CATH-1-3 24. The pig has an even larger arsenal of 11 cathelicidins: protegrin-1-5, prophenin-1-2, and pig myeloid antibacterial peptide (PMAP)-23,-36, and-37 4. Human LL-37 is a 37 amino acid cationic (6+) peptide and has been widely studied. LL-37 penetrates the bacterial membrane and forms pores in the membrane. LL-37 adopts an α-helical structure 25 , which resembles the structure of PMAP-36. In addition, many different immunomodulatory functions have been described for LL-37 26-28. Chicken CATH-2 is a 26 amino acid cationic (11+) arginine-lysine-rich peptide, consisting of two α-helical regions with a proline induced hinge region 29. CATH-2 displays strong antimicrobial activities against many different pathogens, e.g. Gram-positive and Gram-negative bacteria 29-32 and fungi 32,33. In addition, CATH-2 has been shown to have immunomodulatory capacities 29,30,34 .

Molecular Immunology, Aug 1, 2009

Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomo... more Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two ␣-helices connected by a hinge region. Here we report the biological properties of the separate ␣-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNF␣, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.

Peptides, May 1, 2011

A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broa... more A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F(2,5,12)W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with D-amino acids and head-to-tail cyclization. Both cyclic and D-amino acid variants showed enhanced stability in human serum compared to C1-15 and F(2,5,12)W. The D-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F(2,5,12)W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 D-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency.

Molecular Immunology, Apr 1, 2009

The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins hav... more The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins have shown to possess potent broad-range antimicrobial activity as well as immunomodulatory activities. Therefore, we investigated the microbicidal activities and localization of Cathelicidin-2 in non-infected and Salmonellachallenged broiler chickens. Using immunohistochemistry, Cathelicidin-2 was shown to be abundantly present in heterophils, localized in the large rod-shaped granules, but absent in other peripheral blood cells and intestinal epithelial cells. Cathelicidin-2 synthesis was observed to be initiated at the early promyelocyte stage. Considerable infiltration of Cathelicidin-2 containing heterophils was observed in the jejunum of Salmonella enteritidis-challenged broilers within 8 h post-infection. Heterophils were shown to release mature Cathelicidin-2 peptide upon stimulation with Salmonella-derived LPS in a time-dependent way. Processing of the Cathelicidin-2 precursor was mediated by serine proteases with a divalent cation dependency. Cathelicidin-2 peptide showed potent bactericidal and fungicidal activity against all tested microorganisms, including chicken-specific Salmonella isolates. These results underscore the importance of avian heterophils as a first line of defence against invading pathogens and implicate that via heterophilmediated release, cathelicidins may greatly contribute to avian innate immunity.

Journal of Global Antimicrobial Resistance

Developmental & Comparative Immunology

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of ... more The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP-D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SPA like gene. Here we investigated the evolution of the SPA and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SPA analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SPA. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SPA. In fact, the CRD of SPA is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

Protein & Peptide Letters, 2019

Background: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important ro... more Background: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. Objective: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. Method: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. Results: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, marker...

mSphere, 2021

Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2... more Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development.

PLOS ONE, Apr 22, 2014

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate ... more The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial-and immunomodulatory activity of PR-39, and N-and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-a production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics.

PLOS ONE, Feb 5, 2016

Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodul... more Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that fulllength CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives.

Peptide science, May 31, 2021

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis v... more Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV), a coronavirus of domestic fowl. IB is a major concern in the poultry industry, causing worldwide economic losses through decreased egg production and quality and by increasing the chicken's susceptibility for secondary bacterial infections, particularly Escherichia coli. In this study, the anti-IBV activity of cathelicidins, small antimicrobial peptides of the innate immune system was investigated. The cell culture adapted (nonvirulent) IBV strain Beaudette was effectively inhibited by the human cathelicidin LL-37 in bovine hamster kidney-21 cells at nontoxic concentrations. The peptide needed to be present during virus inoculation to effectively inhibit the infection of IBV-Beaudette, indicating that LL-37 likely bound viral particles. However, no clear morphological changes in the IBV virion upon binding were observed by electron microscopy. In this cell culture model, chicken cathelicidins (CATH1-3) were inactive against IBV-Beaudette. In contrast, in multicellular infection models using the virulent IBV-M41 strain the activities of human and chicken cathelicidins were different. In particular, upon inoculation of 10-day-old embryonic eggs with IBV-M41, CATH-2 reduced the viral load to a higher extend than LL-37. Similarly, viral infection of chicken tracheal organ cultures with IBV-M41 was significantly reduced in the presence of CATH-2 but not LL-37. These results indicate a potential antiviral role for CATH-2 upon IBV infection in vivo. K E Y W O R D S coronavirus, host defense peptides, innate immunity 1 | INTRODUCTION Host defense peptides (HDPs) are small cationic peptides that play an important role in the innate host defense against pathogens. [1] These peptides are produced as prepropeptides by epithelial and innate immune cells. [2] Upon release of the HDPs the prepro sequence is cleaved releasing the active peptides locally where they exert their antimicrobial and immunomodulatory activities. While HDPs of the

Antimicrobial Agents and Chemotherapy, Mar 1, 2007

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and ... more Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of ␤-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken ␤-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 g/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken ␤-defensin gallinacin-6 plays an important role in chicken innate host defense.

Veterinary Immunology and Immunopathology, Jun 1, 2018

Background: Macrophages play an important role in the innate immune system as part of the mononuc... more Background: Macrophages play an important role in the innate immune system as part of the mononuclear phagocyte system (MPS). They have a pro-inflammatory signature (M1-polarized macrophages) or anti-inflammatory signature (M2-polarized macrophages) based on expression of surface receptors and secretion of cytokines. However, very little is known about the culture of macrophages from pigs and more specific about the M1 and M2 polarization in vitro. Methods: Porcine monocytes or mononuclear bone marrow cells were used to culture M1-and M2-polarized macrophages in the presence of GM-CSF and M-CSF, respectively. Surface receptor expression was measured with flow cytometry and ELISA was used to quantify cytokine secretion in response to LPS and PAM 3 CSK 4 stimulation. Human monocyte-derived macrophages were used as control. Results: Porcine M1-and M2-polarized macrophages were cultured best using porcine GM-CSF and murine M-CSF, respectively. Cultures from bone marrow cells resulted in a higher yield M1-and M2-polarized macrophages which were better comparable to human monocyte-derived macrophages than cultures from porcine monocytes. Porcine M1-polarized macrophages displayed the characteristic fried egg shape morphology, lower CD163 expression and low IL-10 production. Porcine M2-polarized macrophages contained the spindle-like morphology, higher CD163 expression and high IL-10 production. Conclusion: Porcine M1-and M2-polarized macrophages can be most efficiently cultured from mononuclear bone marrow cells using porcine GM-CSF and murine M-CSF. The new culture method facilitates more refined studies of porcine macrophages in vitro, important for both porcine and human health since pigs are increasingly used as model for translational research.

Biochimica et Biophysica Acta, 2017

Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunit... more Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 βlike protein. Methods Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. Results GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is Highlights A new human cryptic peptide, named GVF27, has been identified in 11-hydroxysteroid dehydrogenase-1 β-like GVF27 is unstructured in aqueous buffer but assumes a well defined conformation in mimic membrane environments GVF27 exerts anti-biofilm actions and broad spectrum antimicrobial activities GVF27 binds bacterial endotoxins as LPS and LTA GVF27 induces mitigation of pro-inflammatory mediators production in LPS induced murine macrophages

Biochimica Et Biophysica Acta - General Subjects, Sep 1, 2021

BACKGROUND Antimicrobial peptides are considered potential alternatives for antibiotics. Here we ... more BACKGROUND Antimicrobial peptides are considered potential alternatives for antibiotics. Here we describe the antibacterial properties of a family of novel cathelicidin-related (CR-) peptides, which we named PepBiotics, against bacteria typically present in cystic fibrosis (CF) patients. METHODS Broth dilution assays were used to determine antibacterial activity of PepBiotics under physiological conditions, as well as development of bacterial resistance against these peptides. Toxicity was tested in mice and cell cultures while molecular interactions of Pepbiotics with bacterial membrane components was determined using CD, ITC and LPS/LTA induced macrophage studies. RESULTS A relatively small number of PepBiotics remained highly antibacterial against CF-related respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus, at high ionic strength and low pH. Interestingly, these PepBiotics also prevented LPS/LTA induced activation of macrophages and was shown to be non-toxic to primary human nasal epithelial cells. Furthermore, both P. aeruginosa and S. aureus were unable to induce resistance against CR-163 and CR-172, two PepBiotics selected for their excellent antimicrobial and immunomodulatory properties. Toxicity studies in mice indicated that intratracheal administration of CR-163 was well tolerated in vivo. Finally, interaction of CR-163 with bacterial-type anionic membranes but not with mammalian-type (zwitterionic lipid) membranes was confirmed using isothermal titration calorimetry and 31P solid state NMR. CONCLUSIONS PepBiotics are a promising novel class of highly active antimicrobial peptides, of which CR-163 showed the most potential for treatment of clinically relevant (CF-) pathogens in physiological conditions. GENERAL SIGNIFICANCE These observations emphasize the therapeutic potential of PepBiotics against CF-related bacterial respiratory infections.

mSphere, Aug 25, 2021

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of ... more Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs. KEYWORDS Gram-negative bacteria, antibiotic resistance, antimicrobial peptides, host defense peptides, outer membrane vesicles W hen pathogens enter a host, they enter a hostile environment. Host species have developed many measures to eliminate and remove pathogens; however, pathogens have been evolving simultaneously. Well known is the development of antibiotic resistance, but pathogens also have found ingenious mechanisms to evade the host's intrinsic immune system (1). One of the first innate defense molecules pathogens will encounter are host defense peptides (HDPs). HDPs are small, cationic molecules and have antibacterial activities against a broad range of pathogens. They are amphipathic,

Biochimica Et Biophysica Acta - General Subjects, Sep 1, 2017

Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunit... more Background Host Defence Peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 βlike protein. Methods Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. Results GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is Highlights A new human cryptic peptide, named GVF27, has been identified in 11-hydroxysteroid dehydrogenase-1 β-like GVF27 is unstructured in aqueous buffer but assumes a well defined conformation in mimic membrane environments GVF27 exerts anti-biofilm actions and broad spectrum antimicrobial activities GVF27 binds bacterial endotoxins as LPS and LTA GVF27 induces mitigation of pro-inflammatory mediators production in LPS induced murine macrophages

Veterinary Microbiology, Dec 1, 2012

Campylobacter jejuni, a small Gram-negative, spiral shaped bacterium, is a major cause of bacteri... more Campylobacter jejuni, a small Gram-negative, spiral shaped bacterium, is a major cause of bacterial food-borne disease, responsible for approximately 400 million cases of enterocolitis per year world wide (Girard et al., 2006). Wild and domesticated birds act as natural reservoirs for Campylobacter with chicken being the most important source of human Campylobacteriosis (Dasti et al., 2010). The interactions between Campylobacter and the avian innate immune system are poorly understood. Although the adaptive immune system is not yet fully developed after hatching (Bar-Shira and Friedman, 2006), intestinal colonization by the ubiquitously present Campylobacter

International Journal of Antimicrobial Agents, Sep 1, 2010

Molecular Immunology, Aug 1, 2009

Canine atopic dermatitis (AD) is a chronic allergic skin disorder with an immunopathogenesis comp... more Canine atopic dermatitis (AD) is a chronic allergic skin disorder with an immunopathogenesis comparable to that in humans with AD. The high frequency of recurrent infections with Staphylococcus pseudo intermedius and Malassezia pachydermatis may indicate a defective innate immune response in the skin of atopic dogs. Production of ␤-defensins constitutes an important role in skin defense but information on canine ␤-defensin localization and regulation is scarce. We conducted a gene-expression study of 16 canine ␤-defensins (cBDs) in 11 tissues of healthy dogs, which revealed a variable expression of cBDs in different organ systems of the dog. In skin, three ␤-defensins, cBD1, cBD103 and cBD107, were extensively expressed, while inconsistent expression of five other ␤-defensins was detected. Using immunohistochemistry abundant expression of cBD103 peptide was detected in the epidermis, hair follicles and sebaceous glands, comparable to hBD3 expression in human skin. To examine the gene-expression of ␤-defensins in atopic dogs, full thickness skin biopsy specimens (non-lesional and lesional) of 10 atopic dogs and 7 healthy dogs were examined with real-time PCR. A significant 12-fold increased expression of cBD1 was detected in lesional atopic skin compared to healthy skin, while non-lesional skin showed a 5-fold increase. Contrary to cBD1, expression of cBD103 was slightly (2-fold) downregulated in skin of atopic dogs. Gene-expression levels of S100A8, a marker for atopic dermatitis, were also highly upregulated in skin of atopic dogs, confirming the diagnostics of the skin biopsies. Taken together these results provide new evidence for a possible defect in the innate immune response of dogs with atopic dermatitis, and indicate the potential of the dog as a model for human AD.

Scientific Reports, Mar 18, 2019

Host defense peptides (HDPs) play a pivotal role in innate immunity and have, in addition to anti... more Host defense peptides (HDPs) play a pivotal role in innate immunity and have, in addition to antimicrobial activity, also important immunomodulatory functions. Bacteria are less likely to develop resistance against HDPs because these peptides target and kill bacteria in multiple ways, as well as modulate the immune system. Therefore, HDPs, and derivatives thereof, are promising alternatives to traditional antibiotics. Hardly anything is known about the immunomodulatory functions of porcine cathelicidin PMAP-36. In this study, we aimed to determine both antibacterial and immunomodulatory activities of PMAP-36 comparing the properties of PMAP-36 analogs with two well-studied peptides, human LL-37 and chicken CATH-2. Transmission electron microscopy revealed different killing mechanisms of E. coli for PMAP-36, CATH-2 and LL-37. LL-37 binds LPS very weakly in contrast to PMAP-36, but it inhibits LPS activation of macrophages the strongest. The first 11 amino acids of the N-terminal side of PMAP-36 are dispensable for E. coli killing, LPS-neutralization and binding. Deletion of four additional amino acids resulted in a strong decrease in activity. The activity of full length PMAP-36 was not affected by monomerization, whereas the shorter analogs require dimerization for proper immunomodulatory activity but not for their antibacterial activity. Cathelicidins constitute a family of host defense peptides (HDPs) and play an important role during the innate immune response 1. They consist of a highly conserved N-terminal region, containing a signal peptide and a cathelin domain, while the C-terminal region represents the highly variable domain of the active peptide 2-4. Although the sequences of cathelicidins are highly variable, almost all cathelicidins show, in simple media, direct antimicrobial activity against many different bacteria 3,5-7 , viruses 8-10 , fungi 11 , and parasites 12,13. Besides their direct antimicrobial activities, cathelicidins can also modulate the immune response. These peptides can induce chemotaxis directly or indirectly by inducing chemokine release 3,14,15. In addition, cathelicidins have been shown to be involved in phagocytosis 3,16-18 , neutralization of LPS or LTA during TLR stimulation 3,15,19,20 or enhancement of DNA uptake and subsequent TLR-9 activation 3,5,21,22 and they can skew macrophage differentiation towards a pro-inflammatory phenotype 23. Bacteria are less likely to develop resistance against HDPs because they do not only target and kill bacteria in multiple ways but also modulate the immune system. This dual function makes HDPs promising alternatives to antibiotics. In humans only one cathelicidin has been identified, LL-37 2 , while in chicken four cathelicidins have been identified, CATH-B1 and CATH-1-3 24. The pig has an even larger arsenal of 11 cathelicidins: protegrin-1-5, prophenin-1-2, and pig myeloid antibacterial peptide (PMAP)-23,-36, and-37 4. Human LL-37 is a 37 amino acid cationic (6+) peptide and has been widely studied. LL-37 penetrates the bacterial membrane and forms pores in the membrane. LL-37 adopts an α-helical structure 25 , which resembles the structure of PMAP-36. In addition, many different immunomodulatory functions have been described for LL-37 26-28. Chicken CATH-2 is a 26 amino acid cationic (11+) arginine-lysine-rich peptide, consisting of two α-helical regions with a proline induced hinge region 29. CATH-2 displays strong antimicrobial activities against many different pathogens, e.g. Gram-positive and Gram-negative bacteria 29-32 and fungi 32,33. In addition, CATH-2 has been shown to have immunomodulatory capacities 29,30,34 .

Molecular Immunology, Aug 1, 2009

Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomo... more Chicken host defense peptide cathelicidin-2 (CATH-2) is known to exert antimicrobial and immunomodulatory activities and consists of two ␣-helices connected by a hinge region. Here we report the biological properties of the separate ␣-helical segments and the importance of the proline residue in the hinge region. Substitution of proline-14 in the CATH-2 hinge region by leucine, but not by glycine, strongly reduced antibacterial and hemolytic activity. Furthermore, substitution by leucine strongly reduced the neutralization of LPS-induced cytokine production and peptide-induced monocyte chemotactic protein-1 (MCP-1) production by human peripheral blood mononuclear cells (PBMCs). This indicates that the hinge region is important for rapid penetration of the bacterial membrane as well as indirect and direct immunomodulatory activities. The highly cationic and amphipathic N-terminal segment (C1-15) exhibited very potent antibacterial activity and fast killing kinetics, while displaying low cytotoxicity towards chicken erythrocytes and PBMCs. The N-terminal and, to a lesser extent, the C-terminal helical regions potently neutralized LPS-induced release of TNF␣, IL-6 and IL-10 by PBMCs, while IL-8 production was only moderately affected. These results indicate that core elements within mature CATH-2 can be identified that are linked to antibacterial and/or immunomodulatory activities. Further studies may lead to the development of peptide antibiotics with specific properties that can be used for prophylactic and/or therapeutic applications.

Peptides, May 1, 2011

A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broa... more A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F(2,5,12)W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with D-amino acids and head-to-tail cyclization. Both cyclic and D-amino acid variants showed enhanced stability in human serum compared to C1-15 and F(2,5,12)W. The D-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F(2,5,12)W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 D-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency.

Molecular Immunology, Apr 1, 2009

The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins hav... more The biological functions of avian cathelicidins are poorly defined. In mammals, cathelicidins have shown to possess potent broad-range antimicrobial activity as well as immunomodulatory activities. Therefore, we investigated the microbicidal activities and localization of Cathelicidin-2 in non-infected and Salmonellachallenged broiler chickens. Using immunohistochemistry, Cathelicidin-2 was shown to be abundantly present in heterophils, localized in the large rod-shaped granules, but absent in other peripheral blood cells and intestinal epithelial cells. Cathelicidin-2 synthesis was observed to be initiated at the early promyelocyte stage. Considerable infiltration of Cathelicidin-2 containing heterophils was observed in the jejunum of Salmonella enteritidis-challenged broilers within 8 h post-infection. Heterophils were shown to release mature Cathelicidin-2 peptide upon stimulation with Salmonella-derived LPS in a time-dependent way. Processing of the Cathelicidin-2 precursor was mediated by serine proteases with a divalent cation dependency. Cathelicidin-2 peptide showed potent bactericidal and fungicidal activity against all tested microorganisms, including chicken-specific Salmonella isolates. These results underscore the importance of avian heterophils as a first line of defence against invading pathogens and implicate that via heterophilmediated release, cathelicidins may greatly contribute to avian innate immunity.

Journal of Global Antimicrobial Resistance

Developmental & Comparative Immunology

The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of ... more The air-liquid interface of the mammalian lung is lined with pulmonary surfactants, a mixture of specific proteins and lipids that serve a dual purpose-enabling air-breathing and protection against pathogens. In mammals, surfactant proteins A (SP-A) and D (SP-D) are involved in innate defence of the lung. Birds seem to lack the SP-D gene, but possess SP-A2, an additional SPA like gene. Here we investigated the evolution of the SPA and SP-D genes using computational gene prediction, homology, simulation modelling and phylogeny with published avian and other vertebrate genomes. PCR was used to confirm the identity and expression of SPA analogues in various tissue homogenates of zebra finch and turkey. In silico analysis confirmed the absence of SP-D-like genes in all 47 published avian genomes. Zebra finch and turkey SP-A1 and SP-A2 sequences, confirmed by PCR of lung homogenates, were compared with sequenced and in silico predicted vertebrate homologs to construct a phylogenetic tree. The collagen domain of avian SP-A1, especially that of zebra finch, was dramatically shorter than that of mammalian SPA. Amphibian and reptilian genomes also contain avian-like SP-A2 protein sequences with a collagen domain. NCBI Gnomon-predicted avian and alligator SP-A2 proteins all lacked the collagen domain completely. Both avian SP-A1 and SP-A2 sequences form separate clades, which are most closely related to their closest relatives, the alligators. The C-terminal carbohydrate recognition domain (CRD) of zebra finch SP-A1 was structurally almost identical to that of rat SPA. In fact, the CRD of SPA is highly conserved among all the vertebrates. Birds retained a truncated version of mammalian type SP-A1 as well as a non-collagenous C-type lectin, designated SP-A2, while losing the large collagenous SP-D lectin, reflecting their evolutionary trajectory towards a unidirectional respiratory system. In the context of zoonotic infections, how these evolutionary changes affect avian pulmonary surface protection is not clear.

Protein & Peptide Letters, 2019

Background: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important ro... more Background: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. Objective: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. Method: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. Results: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, marker...

mSphere, 2021

Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2... more Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development.