Antimicrobial peptides in the first line defence of human colon mucosa
Introduction
The human colon harbours over 2 kg of bacteria, which are present at approximately one-tenth of the theoretical cell packing limit [24]. Hence the colon mucosa is exposed to more bacteria than any other site in our body. Symbiosis between the host and the microbial flora is important, and our commensal microbes do not normally cause any intestinal dysfunction. However, this balance can be disturbed, and even our highly adapted commensal bacteria can cause disease [24].
To avoid infection from the normal flora, an unbroken mucosal epithelium is a prerequisite. Epithelial cells constitute a mechanical barrier but participate also in immune regulation and generation of biologically active mediators. The colonic epithelial cells produce proinflammatory immune mediators including cytokines, chemokines, prostaglandins and nitric oxide [22]. These substances have the potential to regulate a coordinated mucosal immune response. Antimicrobial peptides have been highlighted in this setting since they are expressed at epithelial surfaces and are able to destroy invading micro-organisms. They have been found in almost all higher eukaryotic organisms [5] and the major families of mammalian antimicrobial peptides are the defensins and the cathelicidins. The defensins are cationic with three intrachain disulphide bonds. They can be divided into α- and β-defensins, which share a similar 3D-structure but differ in primary structure and disulphide patterns [28], and the recently described θ-defensins [40], which are not known to be expressed in humans. Six human α-defensins have been characterised. Human neutrophil peptide 1–4 (HNP1–4) are expressed by circulating neutrophils [27], while human defensins 5–6 (HD 5–6) are expressed by Paneth cells in the small intestine [20], [21]. Four different β-defensins (HBD1–4) [4], [10], [13], [14] have been characterised and are mainly expressed by epithelial cells. Recently, detailed genome analyses showed 28 additional β-defensin genes and 12 are most likely transcribed [39]. Cathelicidins are a diverse group of antimicrobial peptides, derived from a preproprotein, sharing a conserved N-terminal proform and a variable antimicrobial C-terminal domain [48]. The only cathelicidin-derived peptide in the human is LL-37, which forms an amphipathic α-helical structure in physiological salt medium [1] and is mainly localized in neutrophils [6], [12]. LL-37 is also expressed by epithelial cells [9], is induced in human keratinocytes during inflammatory disorders [8], and has been shown to be downregulated in colon epithelial cells during Shigella infection [18].
The antimicrobial peptides known to be produced by colon epithelia are LL-37 [16], [18] and the β-defensins HBD-1 and HBD-2 [31]. Both LL-37 and HBD-1 are constitutively expressed, while HBD-2 is induced in inflammation by proinflammatory stimuli that activate the transcription factor NF-κB [31].
In this study with the aim of identifying antimicrobial components, we have isolated and characterised 10 antimicrobial proteins/peptides from human colon mucosa, 5 of which were isolated and properly characterised. This strongly indicates that the chemical mucosal barrier in the large bowel is mediated by polypeptides that prevent microbial invasion. Besides killing bacteria, some of these peptides are chemoattractants for immune cells and can thereby participate in orchestrating the total immune response.
Section snippets
Materials
With approval by the Ethics Committee at Karolinska Hospital, human colon mucosa was obtained from a patient colectomised because of carcinoma. The material used was taken from the non-neoplastic part of the colon and was macroscopically normal, that is, no polyps and no inflammation. In the microscope, the histology also was quite normal with preserved architecture of the mucosa, no signs of inflammation or dysplasia. The mucosa was stripped with a knife from the underlying tissue, frozen in
Antimicrobial activity in protein extracts from colon mucosa
Proteins/peptides were extracted from human colon mucosa by 60% acetonitrile in 1% TFA and concentrated on an OASIS™ column. After lyophilization, 75 μg of this material was analysed for antimicrobial activity using an inhibition zone assay [11]. The test microbes used were the Gram-positive B. megaterium strain Bm11, the Gram-negative E. coli strain D21, and the fungus C. albicans. The highest activity was recorded against B. megaterium with a zone of 19.5 mm. The activity against E. coli and C.
Discussion
In this study, we find that the human colon mucosa harbours components with high activity against Gram-positive bacteria, Gram-negative bacteria and fungi. Several of these components have now been identified.
Ubiquicidin is one of the characterised polypeptides and this active polypeptide has now been isolated from human tissue. Ubiquicidin is identical to the 40S ribosomal protein S30 [17] and is the C-terminal cleavage product of the ubiquitin-like S30 fusion protein that is encoded by the fau
Acknowledgements
We thank Mona Bajaj Elliot for kindly providing us with HBD-1 antibody and Carina Palmberg and Monica Lindh for technical assistance. This study was supported by grants from the Swedish Research Council, Petrus and Augusta Hedlund’s foundation, Magnus Bergvall’s Foundation, Ruth and Richard Julin’s Foundation and Prof. Nanna Svartz’ Foundation.
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