Neutrophils, the first line of the host’s defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

One of defense mechanisms of the human immune system to counteract infection by the opportunistic fungal pathogen Candida albicans is the recruitment of neutrophils to the site of invasion, and the subsequent production of neutrophil extracellular traps (NETs) that efficiently capture and kill the invader cells. In the current study, we demonstrate that within these structures composed of chromatin and proteins, the latter play a pivotal role in the entrapment of the fungal pathogen. The proteinous components of NETs, such as the granular enzymes elastase, myeloperoxidase and lactotransferrin, as well as histones and cathelicidin-derived peptide LL-37, are involved in contact with the surface of C. albicans cells. The fungal partners in these interactions are a typical adhesin of the agglutinin-like sequence protein family Als3, and several atypical surface-exposed proteins of cytoplasmic origin, including enolase, triosephosphate isomerase and phosphoglycerate mutase. Importantly, the adhesion of both the elastase itself and the mixture of proteins originating from NETs on the C. albicans cell surface considerably increased the pathogen potency of human epithelial cell destruction compared with fungal cells without human proteins attached. Such an implementation of adsorbed NET-derived proteins by invading C. albicans cells might alter the effectiveness of the fungal pathogen entrapment and affect the further host colonization.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the “red complex” species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic “moonlighting” proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Biofilm is a compact coating formed on various artificial and physiologic surfaces by a population of microorganisms which in this habitat establish a close cooperation, exploiting both the physical interactions that stabilize the community and chemical cooperation, engaging numerous agents to modify the environment, i.e., to influence the acidity, nutrient acquisition, or oxygen availability. Microorganisms can also communicate using quorum-sensing molecules carrying specific messages. Some microbes temporarily dominate, while others are constantly replaced by different community members. But these co-operations or competitions have a deep sense-they serve to protect the whole community against the defense system of the host to assure survival. The oral cavity is inhabited by diverse microorganisms, including bacteria, but also yeast-like fungi from the genus Candida that stay under a tight control of the host as long as its immune system is not weakened; then these relatively mild commensals convert to dangerous pathogens that start the invasion often in collaboration with other microbes. Elongated hyphal forms of fungal cells favor the biofilm type of growth and communication with other microbes supporting immune resistance of the biofilm. In this chapter, we discuss the mechanisms of interactions between bacteria and C. albicans in the oral cavity, their communication, host responses, and possible strategies of anti-biofilm treatment.

Neutrophils represent the first line of innate host defense. The ability to inhibit the development of infections is associated with the involvement of several fighting strategies. The still poorly understood mechanism is netosis, involving the release of Extracellular Neutrophil Traps (NETs). NETs are complexes of chromosomal DNA and granule content. Such a web-like structure inhibits the spread of invaders. Netosis plays a significant role in combating Candida albicans infections. It has been shown that several factors, composing C. albicans cell surface mediate NETs production. However, the development of difficult to eradicate fungal infection is associated with the formation of the biofilm structure, which partially protects the pathogen cells from contact with the host’s immune system. One of the reasons for the creation of a such protective environment is the production of the extracellular matrix (ECM). The major components of the C. albicans ECM layer are lipids, proteins, carbohydrates but also extracellular nucleic acids, among which we observed a significant RNA content. Considering that the ECM consisting of RNA molecules is one of the first lines of contact between biofilms and neutrophils, our current studies aimed to assess the potential role of extracellular RNA in the triggering of the netosis process by human neutrophils in vitro. We showed that RNA purified from C. albicans biofilm structure and the whole cells have the capability to induction of ROS-dependent netosis pathway. Additionally, cell migration analysis indicate that RNA molecules may also be an effective chemotactic agent. This work was supported by NCN (2019/33/B/NZ6/02284).