Release of biologically active kinin peptides, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin from human kininogens by two major secreted aspartic proteases of Candida parapsilosis

Highlights

• Candida parapsilosis secrete two aspartic proteases which cleave human kininogens.

• These proteases release two kinins, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin.

• Met-Lys-bradykinin and Leu-Met-Lys-bradykinin interact with kinin receptors B2.

• These kinins stimulate endothelial cells to the secretion of interleukins 1β and 6.

• In plasma, these kinins are converted into bradykinin and its des-Arg derivative.

Abstract

In terms of infection incidence, the yeast Candida parapsilosis is the second after Candida albicans as causative agent of candidiases in humans. The major virulence factors of C. parapsilosis are secreted aspartic proteases (SAPPs) which help the pathogen to disseminate, acquire nutrients and dysregulate the mechanisms of innate immunity of the host. In the current work we characterized the action of two major extracellular proteases of C. parapsilosis, SAPP1 and SAPP2, on human kininogens, proteinaceous precursors of vasoactive and proinflammatory bradykinin-related peptides, collectively called the kinins. The kininogens, preferably the form with lower molecular mass, were effectively cleaved by SAPPs, with the release of two uncommon kinins, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin. While optimal at acidic pH (4–5), the kinin release yield was only 2–3-fold lower at neutral pH. These peptides were able to interact with cellular kinin receptors of B2 subtype and to stimulate the human endothelial cells HMEC-1 to increased secretion of proinflammatory interleukins (ILs), IL-1β and IL-6. The analysis of the stability of SAPP-generated kinins in plasma suggested that they are biologically equivalent to bradykinin, the best agonist of B2 receptor subtype and can be quickly converted to des-Arg⁹-bradykinin, the agonist of inflammation-inducible B1 receptors.

Introduction

Yeasts of the genus Candida are nowadays the most prevalent causative agents of fungal diseases in humans [33]. Candida albicans and several “non-albicans” Candida species (NAC) contribute to the normal endogenous microflora of healthy hosts but under some circumstances, particularly in individuals whose immunity has been weakened, can cause infectious diseases (candidiases), ranging from relatively mild superficial mycoses of skin, hair, nails and mucosal membranes in the oral cavity, gastrointestinal tract and vagina, to life-threatening systemic diseases involving major body organs [37]. In general, more than 60% cases of candidiasis are diagnosed to be caused by C. albicans but in the last few decades the incidence of NAC-dependent infections, particularly deep-seated and systemic candidiases has significantly increased [23], [33], [41]. In terms of diagnosis frequency, a predominant NAC is Candida parapsilosis [41], [42] which is widely recognized as a cause of bloodstream infection among hospitalized patients, especially in neonates, but also in transplant recipients and patients receiving parenteral nutrition [26], [42]. Although C. parapsilosis exhibits a lower ability to adhere to epithelial cells than other Candida species, it adheres more efficiently to foreign materials, such as venous catheters and implants [22], a feature which explains the group risk and overall increasing incidences of C. parapsilosis-dependent candidiases. The rate of mortality in C. parapsilosis fungemia (approaching 40%), the progressively increasing number of infections caused by this species and more frequent problems with treatment of C. parapsilosis-dependent infections makes C. parapsilosis the important emerging fungal pathogen to humans, with the pathogenicity profile different with many respects from that of C. albicans [42].

Similarly to other Candida species, C. parapsilosis exploits multiple virulence mechanisms, including the morphological dimorphism, the binding to host cells and proteins via fungal cell wall adhesins and the secretion of a variety of hydrolytic enzymes [41]. Of the latter, the most important are secreted aspartic proteases (SAPs) which not only digest the host tissues to provide the pathogen with nutrients and to enable the colonization of the host organism but also, to enable the immune evasion by the pathogen, cleave proteins of the major host regulatory systems such as the coagulation pathway [38], the complement system [13] and the kallikrein–kinin system [4], [18], [36].

The kallikrein–kinin system, also known as the contact system, has a variety of functions in the human organism, the major being the production of vasoactive and proinflammatory bradykinin-related peptides, called the kinins (for recent reviews see [5], [24]). Kinins are universal mediators of inflammation [12] and, hence, their increased production has been reported to occur during bacterial [14], [34] and fungal [28], [36] infections. Although the enhanced release of kinins in response to infection should primarily be considered as an important part of the antimicrobial defense of the host, some of the kinin-mediated effects can be “beneficial” to pathogens, such as the vascular permeability enhancement, which causes the inflow of plasma, thereby increasing the availability of nutrients, and is also helpful for pathogen dissemination.

The effective release of kinins from human kininogens, i.e., the proteinaceous precursors of kinins, by the mixtures of proteases secreted into the culture medium by several Candida species was reported before [36], with the highest kinin-producing activity assigned to C. albicans and C. parapsilosis. The mechanism of kinin release by the major SAP (SAP2) of C. albicans was recently characterized in detail [4]. C. parapsilosis possesses three SAP genes (SAPP1, SAPP2, SAPP3) but only proteins encoded by two of theses genes, SAPP1 and SAPP2 have been isolated and characterized in terms of structural and enzymatic properties [8], [15]. In the current work, we showed that SAPP1 and SAPP2 could cleave human kininogens, identified the released peptides, and presented their kinin-like biological activities.