Investigation of potential antibiofilm properties of Antimicrobial Peptide (AMP) from Linckia laevigata against Candida albicans: An in vitro and in vivo study

Highlights

• In silico prediction of AMP from L. laevigata.

• In vitro and in vivo evaluation for biofilm and toxicity using zebrafish embryos.

• Peptides were represented as promising candidate to treat C. albicans biofilm.

Abstract

Biofilm-associated Candida albicans infection remains to be challenging, though there are several investigations and developments in the medical therapy. Antimicrobial peptides are promising candidates due to their efficacy to target pathogens and cancer cells. In the present study, LLpepII identified from Linckia laevigata (star fish), has been predicted as ɑ-helical, amphipathic antimicrobial peptide. LLpepII was investigated for their potency to inhibit C. albicans biofilm formation using crystal violet. LLpepII shows antibiofilm activity which inhibits only the biofilm formation and without inhibiting the growth of the yeast cells. This biofilm specific activity of LLpepII was confirmed through SEM and XTT assay. Cell viability assay revealed that LLpepII significantly inhibited the proliferation of human cervical (HeLa) cell lines. Morphological changes in HeLa cells, apoptosis and cell death was studied using AO/EB. It is also confirmed that LLpepII can prevent infection and it is non toxic using zebrafish egg infection model. These results suggest that amphipathic LLpepII as promising candidate with dual property can be explored further as antibiofilm and antiproliferate candidate for therapeutic applications.

Introduction

Candida albicans is a common opportunistic fungal pathogen, found in almost all the major parts of the human body. Any slight alterations in the host immune response or shift in microbiota or nutritional content can enable C. albicans to overgrow and cause infection [1]. C. albicans can cause cutaneous skin infections to superficial mucosal infections which lead to considerable mortality rates [2]. C. albicans establish well structured biofilms which includes different cell types (i) yeast cells that are round;(ii) budding yeast cells ovoid in shape;(iii) elongated- pseudohyphal cells and (iv) highly elongated tube like-hyphal cells. These cell types of C. albicans are encased in an extracellular polymeric matrix especially that render them highly tolerant to conventional antibiotics and host immune response [3]. This leads to serious infections in immunocompromised individuals and healthy individuals with implanted medical devices [1]. Hence, C. albicans biofilm-associated infections represent one of the major threats of modern medicine. In this regard, there is a pressing demand of new drugs active against microbial biofilms [4].

Antimicrobial peptides (AMPs) represent an option increasingly taken into consideration [5]. AMP over the past two decades was focused as an alternative for antibiotics due to antimicrobial drug resistance [6]. In recent years, due to their pleiotropic function, AMPs has gained focused as important therapeutic tools against fungal and viral [7] pathogens. The most defined feature of AMPs is their rapid response during bacterial, viral, fungal and protozoa infections [8]. AMPs also have promising role to control host physiological functions such as regulation of inflammation, angiogenesis, modulating signal transduction, and chemokine production and release [9]. Till date, 3122 AMPs were predicted from different organisms and their list is available from AP database (http://aps.unmc.edu/AP/main.php). Majority of AMPs share common feature such as short length of amino acids (12–50), cationic, hydrophobic and amphipathic molecules capable of perturbing the bacterial membrane [10]. AMPs also show low specificity to molecular target and thereby exhibit a broad-spectrum of activity. Studies have shown that AMPs can act against microbial biofilms, during early phases of biofilm development [11]. Later, other investigations have shown that AMPs can reduce the tendency to induce resistance and obtain high potential to target dormant cells that are found in the elevated levels of the biofilms [12]. Hence this new paradigm of antibiofilm peptide (ABP) which holds substantial promise as novel antibiofilm agents has been represented as important area for research to be explored.

In general, echinoderms lack vertebrate-type adaptive immune system which produces AMPs as their primary immune effector molecules in coelomocytes and coelomic fluid. Expression of AMPs in coelomocytes or coelomic fluid is either constitutive or induced on exposure to pathogens or other harmful external factors. They not only inactive the effect of the pathogen but also protect the host organism by modulating the immune system [13]. In this study echinoderm coelomic fluid (ECF) of starfish was investigated to identify ABPs. The identified ABP from ECF was explored in silico, in vitro and in vivo for their biological activity.