Linköping University | Department of Physics, Chemistry and Biology Bachelor thesis, 16 hp | Biology programme: Physics, Chemistry and Biology Spring term 2019 | LITH-IFM-x-EX--19/3696--SE
A Versatile Group of
Molecules, Can Defensins
Make an Impact in Medicine?
Filip Fors
Examinator, Matthias Laska, IFM Biologi, Linköpings universitet Tutor, Jordi Altimiras, IFM Biologi, Linköpings universitet
Datum
Date
6/6-19
Avdelning, institution Division, Department
Department of Physics, Chemistry and Biology Linköping University
URL för elektronisk version
ISBN
ISRN: LITH-IFM-x-EX--19/3696--SE
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Serietitel och serienummer ISSN
Title of series, numbering ______________________________
Språk Language Svenska/Swedish Engelska/English ________________ Rapporttypi Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title
A Versatile Group of Molecules, Can Defensins Make an Impact in Medicine?
Författare
Author Filip Fors
Nyckelord
Keyword
Defensin, Adjuvant, Biomarker, Virus inhibitor, Application, Potential
Sammanfattning
Abstract
Antimicrobial peptides are an ancient form of innate defense and is present in all ways of life. In humans they are present as cathelicidins and defensins. Both are important for the immune system and they exhibit activity against viruses, bacteria and fungi. Defensins exhibit less cytotoxicity and are better characterized and are thus more easily developed as therapeutic tools. Defensins are apt at doing a multitude of things, from inhibiting Herpes simplex virus replication and preventing anthrax’ lethality to helping with wound closure and acting as biomarkers for a variety of ailments. Defensins have consistently shown good results in a laboratory setting but have less than exemplary in vivo results. Defensins’ multifunctionality as well as the complex environment in living organisms makes characterizing why defensins are not performing as well in vivo difficult. They can also exhibit negative side-effects such as increasing the infectivity of the HIV and inhibiting anti-viral molecules of the innate immune system. Nevertheless, they exhibit big potential as complementary drugs, adjuvants, biomarkers, wound treatment and much more. Further characterization and development is absolutely necessary in these times of increasing antibiotic resistance.
Table of Contents
1 Abstract ... 1
2 Introduction ... 2
3 Structure and mechanism of interaction ... 3
4 Defensins in various therapeutical areas ... 4
4.1 Defensin usage as biomarkers and cancer suppressors ... 5
4.2 Defensins as antiviral agents ... 6
4.3 Defensins aiding in wound healing ... 6
4.4 Defensins in vaccination ... 7
5 Potential negative effects ... 8
6 Conclusion ... 8
7 Societal relevance ... 9
1
1 Abstract
Antimicrobial peptides are an ancient form of innate defense and is present in all ways of life. In humans they are present as cathelicidins and defensins. Both are important for the immune system and they exhibit activity against viruses, bacteria and fungi. Defensins exhibit less cytotoxicity and are better characterized and are thus more easily developed as therapeutic tools. Defensins are apt at doing a multitude of things, from inhibiting Herpes simplex virus replication and preventing anthrax’ lethality to helping with wound closure and acting as biomarkers for a variety of ailments. Defensins have consistently shown good results in a laboratory setting but have less than exemplary in vivo results. Defensins’ multifunctionality as well as the complex environment in living organisms makes characterizing why defensins are not performing as well in vivo difficult. They can also exhibit negative side-effects such as increasing the infectivity of the HIV and inhibiting anti-viral molecules of the innate immune system. Nevertheless, they exhibit big potential as complementary drugs, adjuvants, biomarkers, wound treatment and much more. Further
characterization and development is absolutely necessary in these times of increasing antibiotic resistance.
2
2 Introduction
Large organisms defend themselves from small organism-based threats in many ways. One of the oldest defences against microbes is the production of antimicrobial
peptides (AMPs) which are present in all lifeforms1. There are two main groups of
AMPs in humans, cathelicidins and defensins. This review will deal with defensins and their potential use in a therapeutical setting.
Defensins are known to have antibacterial, antiviral and antifungal properties, as well as the ability to modulate the innate and adaptive immune systems in various
organisms2. Structurally all defensins are small cationic peptides with many
conserved cysteine residues. These cysteine residues form a so called “defensin fold” by stabilizing a Beta-sheet through disulphide bonds. Other than the cysteines there is great sequence diversity amongst defensins3.
Defensins have been found in animals, fungi and plants. It has been suggested that all defensins evolved from a singular progenitor, but the exact evolutionary relationship is uncertain4. In insects there is a single defensin group, conveniently called insect defensins. They are used as an acute phase reaction to infectious threat and are secreted into the blood stream, in contrast to mammalian defensins that fulfil a more static role5. In mammals there are three distinct defensin families that all evolved from the same precursor gene, they are called Alpha-, beta- and theta-defensins6.
Alpha and beta defensins are the main groups and are present in almost all mammals. Theta defensins were first isolated in rhesus macaques and are found in non-human primates7,8. The gene for theta-defensin has a stop-codon within the signal sequence
in humans, rendering it inert8. There is great variety between mammal species as to what defensin exists where. In humans, there are six different alpha defensins. Four of them (HNP1-4) are primarily found in neutrophil granules and the two other types (HD5 & 6) are produced and distributed by Paneth cells in the gut. Beta-defensins are present in epithelial tissues and secretions all over the body9.
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3 Structure and mechanism of interaction
All defensins are classified as small amphipathic, cationic peptides of roughly 29-42 amino acids in length. They have conserved cysteine residues that stabilizes a Beta-sheet through three disulphide bonds3,10. This basic structure is the layout for alpha
and beta defensins, the difference between them being the order of cysteine linkages. Theta-defensins have a different layout compared to the others, with the cystine linkages stabilizing a circular structure7. Insect defensins differ from mammalian defensins by containing a beta-sheet disulphide-linked with an alpha-helix and having different cysteine links3.
Figure 1. A 3D rendering of human beta defensin 2 (hBD2) with the characteristic defensin fold. By Emw - Own work, CC BY-SA 3.0,
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Defensins’ direct activity includes forming pores in negatively charged lipid bilayers. This makes them active against bacteria’s and viruses’ anionic membranes, but not against the hosts own cells11. This was discovered in tests with human alpha-defensin 1 (HNP-1) where it was found to permeabilize the outer and inner membrane of E.
coli, leading to cell death12. Bacteria can acquire resistance to the defensin mechanism
of lipid interaction, it is however limited to specific strains of bacteria13.
HNP1-3, HD5 and human beta defensin 3 are lectins, which makes them capable of binding to glycoproteins and glycolipids found on the surface of pathogenic
organisms and initiating immune responses14,15,16.
Because defensins are cationic and amphipathic they can also interact with DNA and other proteins through charge-charge or hydrophobic interactions10,17,18.
Defensins can be found as oligomers or multimers which has important, but very poorly understood, implications for their varied capabilities10,19,20
4 Defensins in various therapeutical areas
Human beta-defensin (hBD) 1 and 2 in combination with each other has been shown to reduce the growth of Salmonella typhimurium by 85-96% in vitro. The survival rate of mice treated with hBD-1 and -2 was roughly 50% at 206 hours after inoculation. Compared to the 0% survival rate at 24 hours after inoculation for the Salmonella infected control this is a marked improvement21.
Defensins are potent against pathogenic organisms but they can also perform other functions, in HNP-1’s case it can inhibit anthrax toxin. Tests on mice revealed that the lethal toxin produced by Bacillus anthracis, which causes anthrax, was neutralized effectively by human alpha-defensin 1. Anthrax is hard to treat with antibiotics if treatment is not initiated promptly after inhalation of B. anthracis due to the secreted toxin left in the victim. HNP-1 could effectively be used to complement the antibiotic treatment as a toxin inhibitor. When there is such a potent inhibitor of the toxin
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present in our bodies, the reason why B. anthracis toxin is toxic to humans is unclear. It is theorized that the endogenous concentration of HNP-1 is not sufficient to reach the neutralizing threshold. Alternatively, B. anthracis could have a suppressive effect on human leukocytes’ secretion of defensins22.
These kinds of results indicate the potential of defensins in a therapeutical setting and the supposed applications are many and diverse. Some of the most interesting ones are outlined below.
4.1 Defensin usage as biomarkers and cancer suppressors
Measuring the concentration of defensins in circulation or relevant tissues can indicate infectious activity indirectly with the body upregulating defensin production when it is facing infectious threat. Using this principle, defensins have been suggested to work as biomarkers for diseases such as inflammatory bowel disease23, cirrhosis24 and idiopathic pulmonary fibrosis25.
Patient infected by HIV receive antiretroviral therapy (ART) to prevent the infection reaching AIDS-status. The same patients are more susceptible to opportunistic infection and by extension the development of cancer in the oral cavity26. hBD-2 concentration correlates directly with development of oral squamous cell carcinoma (OSCC) and can thus be used as a biomarker for malignant transformation27.
Monitoring patients receiving long-term ART for changes in hBD-2 concentration could be a useful strategy in discovering OSCC as early as possible28. Defensins have
been suggested to work very well as biomarkers for colorectal cancer, with different expression levels both in tissue and plasma being indicative of different stages in cancer progression29,30,31. Beta-defensins have been used in fusion proteins that targets the epidermal growth factor receptor (EGFR) which is overexpressed in a lot of cancers. In vivo tests have shown that this type of fusion protein suppresses cancer cell proliferation and induces mitochondrial-mediated apoptosis of the cancer cells32.
Alfa-defensins are useful as a biomarker when testing for periprosthetic joint
infection. Using a simple commercially available test-kit that measures alpha-defensin concentrations, a specific and sensitive result could be produced. This method is more
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accurate than current diagnostic methods which do not use alpha-defensins as markers33,34.
Psoriasis is an autoimmune disease that lacks good quantitative markers for disease activity. hBD-2 concentration in serum is directly correlated with the disease activity of psoriasis, enabling monitoring of the disease activity over time and treatment35.
4.2 Defensins as antiviral agents
It has been shown that all three families of defensins have antiviral activity in vitro. Theta- and beta-defensins erect networks of crosslinked surface glycoproteins to block membrane fusion by viruses. They are effective in stopping viral entry even if the virus has started to fuse with the host membrane16. Alpha-defensins also inhibit viral entry at multiple steps but the mechanism is not known. Both alpha and beta defensins interact with the herpes simplex virus at multiple steps in pre- and post-entry, inhibiting its life cycle18,36. All types of defensins also interact with influenza virus A to aggregate the virus and promote uptake by neutrophils37.
In vivo testing has revealed that HNP-1 plays an important part in antiviral immunity
that is not related to their direct antiviral activity in vitro38. HNP-1 has also proved to inhibit influenza virus replication post-infection through cell-mediated pathways, inhibiting protein kinase C in the process which suggests PKC pathway
involvement39.
HNP-1 has the ability to inhibit HIV-1 infection in many ways, this ability is however diminished greatly in human and bovine serum. The proposed reason for this is that the oligomeric form of defensins is the main contributor to the antiviral activity, and this form is disrupted in serum40.
4.3 Defensins aiding in wound healing
Maggot therapy has been used worldwide to treat wounds for a very long time, and it has been seeing a surge of use in modern times41. It works by debridement (removal of dead tissue), disinfection and an accelerated rate of wound healing42. It is useful in treating non-healing wounds and has been used to treat infections by methicillin-resistant Staphylococcus aureus (MRSA)43. The insect defensin lucifensin is the main
7
disinfecting component found in maggots from the green bottle fly (Lucilia sericata) which is the most common larvae used44,45. This was discovered as recently as 2010,
which means that defensins have been used therapeutically as alternatives to antibiotics without knowing of their involvement44.
Coprisin is a defensin-like AMP discovered in the dung beetle46. It has potent antimicrobial activity against both gram-negative and gram-positive species of bacteria and uses the same mechanism as defensins and other AMPs, disrupting the bacterial membrane through a strong net positive charge47. Coprisin has a similar effect to the antibiotic ampicillin when treating wounds infected by Staphylococcus
aureus and promotes reepithelization and neovascularisation of the wound tissues.
Compared to controls, coprisin- and ampicillin-treated wounds have more leukocytes and fibroblasts which indicates an accelerated rate of healing48.
Similarly to coprisin, hBD-3 has a positive effect on wound closure. It also inhibits bacterial growth of S. aureus in infected diabetic wounds in preclinical tests on pigs as well as on epithelial skin grafts on nude rats49.
Bee defensin-1, a peptide found in royal jelly, has demonstrated an ability to promote reepithelization and wound closure in uninfected excision wounds. It does so by promoting MMP-9 secretion from keratinocytes and increasing keratinocyte migration50.
4.4 Defensins in vaccination
Beta-defensins show potential as viral vaccine adjuvants51. Murine beta-defensin 2 (mBD2) increases specific immune response in the host when used as an adjuvant in a DNA-construct vaccine. The vaccines using mBD2-antigen DNA constructs were more successful at eliciting an immune response than those with just the antigen52.
Bacillus Calmette-Guérin (BCG) is the standard vaccine for tuberculosis (TB) currently in use. An issue with the BCG vaccine is that the protection against TB is very inconsistent, made even more problematic by the fact that revaccination does not increase the protection. This makes it hard to gain consistent protection against
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Revaccination has therefore been discontinued in many developed countries53,54. It has been shown that when combined with different antigens, two mBD2 DNA vaccines can provide similar protection against tuberculosis infection as BCG. The DNA-vaccines when used together with the BCG vaccine offers a greater protection against tuberculosis infection than just BCG alone. This dual vaccination strategy could prove a very effective way of boosting the immunization against TB52.
5 Potential negative effects
A major consideration when designing therapy with the respiratory tract in mind is Surfactant protein (SP)-D. It is an important antiviral protein of the innate immune system, unsurprisingly found in lung surfactant. Alpha defensins bind to SP-D which reduces D’s antiviral properties substantially. Beta defensins do not bind into SP-D with the same affinity, however they have much lower antiviral activity compared to alpha defensins. Theta defensins bind into SP-D with strong affinity, however they do not reduce its antiviral activity55.
Alpha-defensins promote epithelial cell proliferation at low concentrations and conversely are cytotoxic at higher concentrations. Note that a relatively high concentration of defensins are required for their antimicrobial effect. Thus, alpha defensins are poor candidates for antimicrobial therapy. However, beta-defensins do not affect epithelial cells at any concentration, making them a better option against bacteria56. Theta-defensins are not cytotoxic or hemolytic. Acyclic porcine
protegrins are however, as a representative of the cathelicidin family of AMPs, toxic to human fibroblasts and leads to lysis of erythrocytes57. This makes defensins a far
easier AMP to work with in therapeutical development.
6 Conclusion
It seems clear that defensins have the potential to work in a multitude of different therapeutical applications, while already working effectively in maggot therapy. However, in vivo testing is still lacking good data and clinical trials have shown poor results58.
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Alpha-defensins have in vitro properties that allow it to inactivate several different viruses, these are however lost in vivo59. There is also conflicting data on whether
HD5 and HD6 increases the infectivity of HIV in physiological conditions60,61.
Because of the complex nature of the in vivo environment it is not yet fully know why the results are poor. It is partly because of weak binding to the host cells which
reduces the effectiveness of pathogen-defensin interaction. It is theorized that finding or synthesizing defensins that have lost the ability to interact with the host cells would result in minimal loss of antipathogen activity in vivo. This would be a crucial step towards making defensins viable in a therapeutical setting62.
Given that defensins play a large role in a lot of processes, from regulating the microbiotic homeostasis in the small intestine63, to preventing HIV-1 entry or even enhancing HIV infectivity, the in vivo implications for therapy are extensive and needs to be characterized properly before effective treatment options can become available. This also means that defensins have the potential to be very versatile therapeutic tools that can be used in a wide variety of situations. Because of their ancient origin and widespread usage in the tree of life as well as their obvious
usefulness it would be foolish not to try to apply them therapeutically as treatment to various ailments. Will they reach the point where they can be clinically relevant? Only time will tell.
7 Societal relevance
With antibiotic resistance ever increasing, more methods are required to stem the flood of infectious threats. These methods could be alternatives to the currently overused antibiotics, or they could take part in the antibiotic process and minimize resistance development. Defensins could work as both alternatives to antibiotics as well as a method of helping the process. And defensins could do much more, showing potential in wound healing and combating viruses among other things. The only thing seemingly preventing them from working therapeutically is poor characterization. With an increased knowledge of defensins and the way they work inside our bodies comes a great therapeutical value. This knowledge could lead to a vast array of new methods to not only combat infection but also control regulation of the immune
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system and bodily microbial homeostasis. This is in turn can lead to advances in treatment of autoimmune diseases and allergies among other things.
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