Eukaryotic Organisms



Fungi are eukaryotic organisms which are found everywhere on the earth in every environmental conditions including temperate to tropical areas. There are about two million kinds of living organisms on the earth, of which fungi constitute about a hundred thousand species and many more await discovery. Of those described, nearly 150 species are generally recognized as primary pathogens of man and animals. They may cause a variety of infections, ranging from systemic and potentially fatal diseases to localized cutaneous, subcutaneous or mucosal infections and the fungi causing these infections are termed as opportunistic pathogens. The most common fungi those act as causal agents are the species of Aspergillus and Candida. Candida species are frequently found in the normal microbiota of humans, which facilitates their encounter with surfaces of immunocompromised host and most implanted biomaterials.
The majority of microorganisms in their natural habitats grow as structured biofilm communities attached to the surfaces rather than individually in suspension. It is known that about 65% of all human microbial infections involve biofilms (Potera 1999). In biofilms, cells are encapsulated within a matrix of protective extracellular material and display altered character in comparison to planktonic counterpart. The biofilms are significantly less susceptible to antimicrobial agents and display unique characteristics that confer survival advantages against host immunity (O'Toole et al., 2000; Mah et al., 2001 Ramage et al., 2006).
Candida albicans is also a fungal species that remains most commonly associated with biofilm formation (Douglas, 2002; Douglas, 2003; Kumamoto, 2002), and the increase in Candida infections in the last decade has increased proportionately to the widespread use of a broad range of medical implant devices, mainly in persons with immunosuppresed host defenses. Strikingly, fungi (mainly C. albicans) are the third leading cause of catheter-related infections, representing the second highest colonization-to-infection rate and the overall highest crude mortality (Crump et al., 2000). The formation of Candida biofilms carries important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Also, biofilm formation on medical devices can have a negative impact on the host by causing failure of the device and by serving as a reservoir or source for future continuing infections (Douglas, 2002; Kojic et al., 2004). Antifungal therapy alone is insufficient for cure and the affected devices generally need to be removed (Mermel et al., 2001; Rex et al., 2000). Removal of these devices has serious implications in the setting of heart valves, joint prostheses, and central nervous system shunts. Until recently, the reason for the need for device removal has been a mystery.

C. albicans has also evolved various mechanisms to counter the front line antifungal drugs available for the treatment of candidiasis. Relatively few classes of antifungal drugs are available in the market and the development of resistance enlarges the problem. The antifungal drugs that are in clinical use or under advance stages of clinical evaluation are polyenes (amphotericin B, nystatin), azoles (clotrimazole, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, oxiconazole and ravuconazole), 5-flucytosine, allylamines (terbinafine), echinocandins (caspofungin, anidulafungin, FK463) (Carrillo-Munoz et al., 2006). Perhaps resistance to polyenes is a rare phenomenon, but drugs of this class can impose significant side effects.
Candida albicans has been reported to be responsible for the release of arachidonic acid (AA) from the host cells during infections (Castro et al., 1994, Deva et al., 2000) which may modulate the cell growth, morphogenesis and invasiveness of causal agent by several modes. AA is a precursor for the production of eicosanoids which play an important role in morphogenesis and biofilm formation. Prostaglandin E2 (PGE2) is a primary product of arachidonic acid metabolism in most of the eukaryotic cells that has also been reported in pathogenic fungi as well (Lamacka et al., 1995, Lodewyk et al., 1997). Enhanced prostaglandin production during fungal infections could be one of the important factors in promoting colonization as well as chronic infections. The shift in host immune response towards increased colonization and chronic infections is due to PGE2 which has ability to elicit both pro and anti inflammatory responses depending upon the host cells (Mairi et al., 2001; Mairi et al., 2002). Exogenous AA has been reported to increase PGE2 level significantly in C. albicans whereas the behavior of Candida non-albicans species and resistant strains in presence of AA is not much studied till now (Mairi et al., 2001; Mairi et al., 2002; Mohammed et al., 2005). The studies related to determining level of PGE2 in C. albicans and non-albicans species in presence of AA may help in understanding the biofilm forming capacity.
The content of biofilms and the properties such as reduced susceptibility to antimicrobial agents make them difficult to remove and, in the case of biofilms associated with disease, a therapeutic dilemma. The cell surface is also the site that mediates adherence with itself, other microbes in biofilms and host surfaces, binds host ligands and interacts with innate and acquired host defenses. The cell wall structure of C. albicans biofilm, its proteins, and their role has proven a key factor of the relationship of C. albicans and the host (Costerton et al., 1999). Generally cell wall of C. albicans consists of polysaccharides meshwork, primarily ??- 1, 3-glucan, and ??-1, 6-glucan with the some chitin. C. albicans cell wall has two major classes of proteins (CWPs) based on whether they are covalently attached to glucan. The most abundant attached proteins are linked to ??- 1, 6-glucan through a glycophosphatidylinositol (GPI) remnant (GPI-CWP), whereas the least abundant are attached via an alkali labile linkage. The nonglucan-attached proteins may belong to one of the two classes with the one class having the signal for conventional secretion whereas the most abundant class lacks this signal (Costerton et al., 1999; Jenkinson et al., 2002; Douglas, 2003). Several studies have focused on identifying the cell wall proteome including both covalent and non-covalent attached proteins (Douglas 2003, Kumamoto, 2002). The cell wall of this dimorphic organism is a robust but dynamic structure that protects organisms from environmental insults and adjusts in response to the environment (Chaffin, 2008). The cell surface is also the site that mediates adherence with itself, other microbes in biofilms and host surfaces, bind host ligands and interact with innate and acquired host defenses. The study of cell wall structure, its proteins, and their functions has proven a key for approaching the relationship of C. albicans and the host in health and disease (Chaffin, 2008).
There are other proteins that are not covalently attached and found at the cell surface (Nombela et al., 2006). Several studies have employed proteomic approaches to identify proteins of the cell wall sub proteome for both covalent and non-covalent protein species (Pitarch et al., 2006; Saville et al., 2006; Urban et al., 2003). Candida biofilm formation is the major threat for immunocompromised patient as well as patient with medical implants. The important issue is the diagnosis of systemic infection of Candida particularly in patients who are under medical treatment of transplantation. Rapid diagnosis of fungi may be helpful in reducing the use of inappropriate antifungal compounds to treat Candida spp. that are resistant to a particular agent (Ghannoum et al.,1999). Diagnosis of invasive Candida infections may be difficult due to the variability and lack of specificity of clinical presentations and also the symptoms (eg. fever) of Candida infections are not very specific (Larriba et al., 2000). A definitive diagnosis is not reached until late in the infection, with subsequent delays in the initiation of therapy that may result in substantial morbidity and mortality (Hernando et al., 2006). Laboratory diagnosis of Candida infections includes microscopic examination of smear from cutaneous, mucosal, oesophageal and vaginal lesions, culture of sputum, bronchoalveolar lavage, oesophageal brushings, urine, stool and surgical drains. These methods are time consuming, tedious and are not fool proof. Biopsy may be required in some cases of deep-seated candidiasis. Novel methods of diagnosis include PCR based amplification of the infectious agents DNA. This method is sensitive, rapid and less cumbersome but is limited due to false positive results arising from the non-specific contamination with other microorganisms sharing the same ecological niche and also the technique is not available commercially (Bille, 2005). Antibody based detection techniques for immuno-diagnosis of systemic candidiasis include latex agglutination (Dee et al., 1981), counter immuno-electrophoresis , indirect immuno-fluorescence and enzyme linked immunoassay (Kostiala & Kostiala, 1981, Quindos et al., 1987). The antibody-based approaches for diagnosis of Candida infections are rapid and sensitive but often the sensitivity decreases considerably when it comes to discriminating between superficial and disseminated candidiasis (Martinez et al., 1998). This is because of the poly-specific nature of the serum antibodies that sometimes may also lead to false positive results. Thus there is an urgent need to develop new techniques for rapid and accurate diagnosis of these infections.
Monoclonal antibodies (MAbs) are antibody molecules produced by the cells resulting from a single clone and thus are specific to a single epitope of a protein. Diagnostic techniques based on MAbs are rapid, sensitive and very specific (Marcilla et al., 1999). The technique can be pathogen specific, species specific and even stage specific for the same pathogen. Identification of stage specific molecules and development of monoclonal antibodies against them is a promising method of diagnosis (Marot-Leblond et al.,2000). Many protein molecules are constitutively expressed by C. albicans biofilm. These molecules are attractive targets for diagnosis as they are expressed at all times particularly during adherence or biofilm formation and thus offer a reliable diagnostic method decreasing the percentage of false positive results (Fradin et al., 2003). Many monoclonal antibodies have been produced against C. albicans that have therapeutic value (Han et al., 1998 & 2000) but against biofilm cell surface adhesive proteins is rare. Thus identification of new target molecules that are stage specific or constitutively expressed can open new avenues for the development of diagnostic as well as therapeutic monoclonal antibodies against the C. albicans biofilm. Also many molecules identified may be targeted for the development of new antifungal compounds.

There are many proteins that are either cell associated or secreted depending on growth condition. Many enzymes on the other hand are present on the cell surface and cell wall of Candida biofilm (Pugh & Cawson,1975). Biofilm formation on SE disks/ polystyrene cell culture plates are offered a novel approach to study cell surface as well as cell wall proteins that are responsible for adhesion during biofilm formation and also in virulence factor (Pardo et al., 1999). Several workers utilized biofilm to study the cell wall and secretory proteins of C. albicans and there are well-established proteome maps available for yeast form (Rico et al., 1997; Kapteyn et al., 1995).
The objective of the thesis work was to generate monoclonal antibodies against cell surface/cell wall proteins of C. albicans biofilm and to evaluate their therapeutic and/or diagnostic value for Candida infection in the form of biofilm. A total of six strains of Candida albicans were used in the present study to evaluate their biofilm formation. In this piece of work, biofilm formation of different Candida species was compared under different growth conditions supplemented with arachidonic acid and subinhibitory concentration of two antifungals. Along with this study, effect of arachidonic acid and subinhibitory concentration of two antifungals was also evaluated on Candida biofilm and prostaglandin production. Cell surface/ cell wall proteins of C. albicans biofilm were isolated to generation of poyclonal and monoclonal antibodies. The cell surface proteins of C. albicans biofilm were characterized by peptide mass fingerprinting using MALDI-TOF-MS technique. As C. albicans biofilm cell surface/cell wall proteins consist of largest fraction in adhesive proteins, C. albicans biofilm cell surface/cell wall proteins were used for generation and evaluation of monoclonal antibodies for their therapeutic and diagnostic potential. Paratope derived peptides were designed from the sequences obtained by reverse transcription and cDNA sequencing of hybridoma line showing the most effective response.

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