Cyanobacterial lipopeptides puwainaphycins and minutissamides induce disruptive and pro-inflammatory processes in Caco-2 human intestinal barrier model

Highlights

• Puwainaphycins and minutissamides are cytotoxic for intestinal epithelial cells.

• In nontoxic concentrations they induce production of pro-inflammatory interleukin 8.

• They also induce increase in claudin 4 expression known from various carcinomas.

Abstract

Puwainaphycins (PUW) and minutissamides (MIN) are cyanobacterial lipopeptides found in various cyanobacterial species. The first possible target of human exposure to them is intestinal epithelium but effect of PUW/MIN on enterocytes is not known at all. Using differentiated Caco-2 cells, PUW F was found to be cytotoxic from 5 µM concentration based on lactate dehydrogenase release assay and total protein concentration. However, it is also able to induce production of interleukin 8 in non-cytotoxic concentrations 1 and 2.5 µM detected by ELISA. Effects of MIN A and C were similar but less pronounced compared to PUW F. On the other hand, MIN D was the least toxic compound with no significant pro-inflammatory effects. Surprisingly, pro-inflammatory activation of the cells by PUW F and MIN C resulted in an increase in tight junction (TJ) protein claudin 4 expression determined by western blot analysis and confirmed by confocal microscopy. Furthermore, decrease in expression of zonula occludens 3, another TJ protein, was observed after the exposure to PUW F. Taken together, these cytotoxic lipopeptides, especially PUW F, are to be studied more deeply due to their capability to activate and/or deregulate human enterocytes in low concentrations.

Introduction

Adverse effects of cyanobacterial toxins on human health are well documented and recognized as an emerging environmental health issue due to increasing presence of toxic cyanobacteria in different ecosystems worldwide (Huisman et al., 2018). Along with well-recognized and studied cyanobacterial toxins, such as microcystins (MC) or cylindrospermopsin (CYN), cyanobacteria are known to produce tremendous diversity of other chemical structures. It is estimated that chemical structure of only about 20% of cyanobacterial compounds have been described to date (Berdy, 2005; Hrouzek et al., 2011; Welker and von Dohren, 2006). Moreover, only in few of the known metabolites the molecular mechanisms of their biological activity has been identified (Janssen, 2019). Some cyanobacterial metabolites are cytotoxic to various human cell models (Buratti et al., 2017; Janssen, 2019; Maru et al., 2010; Moon et al., 1992), but for most of them, the possible toxic effects are unknown (Maru et al., 2010; Moon et al., 1992).

One of such groups are cyanobacterial cyclic lipopeptides. Hundreds of such structures are described from various cyanobacterial strains and genome surveys are estimating that their occurrence might be quite frequent especially in soil or benthic cyanobacteria (Galica et al., 2017) but they were documented also in planktonic strains (Vestola et al., 2014). Puwainaphycins (PUW) A−G and minutissamides (MIN) A-L are structurally analogous cyclic decapeptides containing β-amino fatty acids and so far they have been isolated from soil and benthic cyanobacteria of genera Anabaena, Cylindrospermum and Symplocastrum (Gregson et al., 1992; Hrouzek et al., 2012; Kang et al., 2011, 2012; Mares et al., 2019; Moore et al., 1989). However, there are also indications that these compounds are produced by the planktonic cyanobacterial species forming cyanobacterial water blooms such as Microcystis and Sphaerospermopsis (Pancrace et al., 2019; Zapomelova et al., 2009). They share a common biosynthetic origin and their structures can vary especially concerning the length and substitution of the fatty acid moiety (Mares et al., 2019). PUW/MIN affect cell proliferation and viability of various human cell lines in low micromolar range (Hrouzek et al., 2012; Kang et al., 2011, 2012). Specifically, PUW F induces calcium influx into the HeLa cells (Hrouzek et al., 2012) and PUW C has a strong positive ionotropic effect on isolated mouse atria (Moore et al., 1989). Except for these observations, little is known about their interactions with specific cell types or tissues and overall potential bioactivity of PUW/MIN in mammals.

The most probable route of human exposure to PUW/MIN is oral consumption of cyanobacteria-contaminated soil, food and/or water. Thus the primary target of their biological activity would be the intestinal epithelium (Kubickova et al., 2019). Its selective permeability is determined mainly by tight junctions (TJ) (Turner, 2009). TJ are formed by transmembrane proteins, e.g. claudins and occludin. In vitro studies show that switching of the different claudins expression could be the mechanism beyond increased intestinal permeability (Luissint et al., 2016). These changes are also seen in vivo in patients with inflammatory bowel diseases or with epithelial cancers (Gunzel and Fromm, 2012). Transmembrane proteins bind to scaffolding/adapter proteins linking them to cytoskeleton. The most well-known are zonula occludens (ZO) 1, 2 and 3 (Lee, 2015). Overall, a modulation of TJ protein expressions and interactions in intestinal epithelial cells can have detrimental pathological consequences for bowel physiological functions.

In order to identify potential human health hazards of these understudied cyanobacterial metabolites - PUW/MIN, their toxicity and ability to affect intestinal epithelial barrier functions were studied employing differentiated Caco-2 cell model.