Shanshan Luo

Immune evasion of the human pathogenic yeast Candida albicans: Pra1 is a Factor H, FHL-1 and plasminogen binding surface protein

The pathogenic yeast Candida albicans utilizes human complement regulators, like Factor H and Factor H like protein-1 (FHL-1) for immune evasion. By screening a C. albicans cDNA expression library, we identified the pH-regulated antigen 1 (Pra1) as a novel Factor H and FHL-1 binding protein. Consequently Pra1 was recombinantly expressed in Pichia pastoris and purified from culture supernatant. Recombinant Pra1 binds Factor H, FHL-1 and also plasminogen. Attached to Pra1, the three human proteins are functionally active. Factor H and FHL-1 inactivate complement and plasminogen can be activated to plasmin which then degrades the extra-cellular matrix component fibrinogen. Polyclonal Pra1 anti-serum was generated and used to localize Pra1 on the surface and also in the culture supernatant of both yeast cells and the hyphal form of C. albicans. Furthermore Pra1 expression was up-regulated upon induction of hyphal growth. Pra1, released by Candida cells binds back to the surface of Candida hyphae and in addition enhances the complement regulatory activity of Factor H in the fluid phase. A Pra1 overexpression strain, with about twofold higher levels of Pra1 on the surface binds more Factor H, and plasminogen. In summary, C. albicans Pra1 is a yeast immune evasion protein that binds host immune regulators and acts at different sites. As a surface protein, Pra1 acquires the two human complement regulators Factor H, FHL-1 and plasminogen, mediates complement evasion, as well as extra-cellular matrix interaction and/or degradation. As a released protein, Pra1 enhances complement control in direct vicinity of the yeast and thus generates an additional protective layer which controls host complement attack.

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8–11 and SCR19–20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19–20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.

Immune escape of the human facultative pathogenic yeast Candida albicans: the many faces of the Candida Pra1 protein.

Infectious diseases caused by human pathogenic fungi represent a major and global health problem. Based on the limited efficacy of existing drugs and the increasing resistance to antifungal compounds, new strategies are urgently needed to fight such fungal infections. The medically important pathogen Candida albicans can exist as an opportunistic yeast, but can also cause severe diseases, septicaemia, and death. In order to establish new strategies to fight Candida infections and to interfere with survival of the pathogen, it is highly relevant to understand the molecular and immunology interactions between the pathogen C. albicans and the human host. This immune cross talk has moved into the focus of infectious disease research. In this review, we summarize the diverse and multiple levels of the immune cross talk between the fungal pathogen C. albicans and the human host. In particular, we define how one single fungal protein Pra1 (pH-regulated antigen 1) interferes and controls host immune attack at multiple levels and thus contributes to fungal immune escape. Candida Pra1 represents a promising candidate for immune interference.

The pH-regulated Antigen 1 of Candida albicans Binds the Human Complement Inhibitor C4b-binding Protein and Mediates Fungal Complement Evasion

Candida albicans binds and utilizes human complement inhibitors, such as C4b-binding protein (C4BP), Factor H, and FHL-1 for immune evasion. Here, we identify Candida pH-regulated antigen 1 (Pra1) as the first fungal C4BP-binding protein. Recombinant Pra1 binds C4BP, as shown by ELISA and isothermal titration calorimetry, and the Pra1-C4BP interaction is ionic in nature. The Pra1 binding domains within C4BP were localized to the complement control protein domain 4 (CCP4), CCP7, and CCP8. C4BP bound to Pra1 maintains complement-inhibitory activity. C4BP and Factor H bind simultaneously to Candida Pra1 and do not compete for binding at physiological levels. A Pra1-overexpressing C. albicans strain, which had about 2-fold Pra1 levels at the surface acquired also about 2-fold C4BP to the surface, compared with the wild type strain CAI4. A Pra1 knock-out strain showed ∼22% reduced C4BP binding. C4BP captured by C. albicans from human serum inhibits C4b and C3b surface deposition and also maintains cofactor activity. In summary, Candida Pra1 represents the first fungal C4BP-binding surface protein. Pra1, via binding to C4BP, mediates human complement control, thereby favoring the immune and complement evasion of C. albicans.

Complement and innate immune evasion strategies of the human pathogenic fungus Candida albicans

Candida albicans is a medically important fungus that can cause a wide range of diseases ranging from superficial infections to disseminated disease, which manifests primarily in immuno-compromised individuals. Despite the currently applied anti-fungal therapies, both mortality and morbidity caused by this human pathogenic fungus are still unacceptably high. Therefore new prophylactic and therapeutic strategies are urgently needed to prevent fungal infection. In order to define new targets for combating fungal disease, there is a need to understand the immune evasion strategies of C. albicans in detail. In this review, we summarize different sophisticated immune evasion strategies that are utilized by C. albicans. The description of the molecular mechanisms used for immune evasion does on one hand help to understand the infection process, and on the other hand provides valuable information to define new strategies and diagnostic approaches to fight and interfere with Candida infections.

Secreted pH-Regulated Antigen 1 of Candida albicans Blocks Activation and Conversion of Complement C3

The complement system forms the first defense line of innate immunity and is activated within seconds upon infection by human pathogenic yeast Candida albicans. In this study, we identified a new complement evasion strategy used by C. albicans. The fungus secretes a potent complement inhibitor, pH-regulated Ag 1 (Pra1), which in the direct surrounding of the pathogen binds to fluid-phase C3 and blocks cleavage of C3 to C3a and C3b, as shown by ELISA, native gel electrophoresis, and Western blotting. Consequently, complement activation via the alternative and classical pathways is inhibited. In addition, the release of the anaphylatoxins C3a and C5a, as well as C3b/iC3b surface deposition, is reduced, as demonstrated by Western blotting, ELISA, confocal microscopy, and flow cytometry. By reducing C3b/iC3b levels at the yeast surface, Pra1 decreases complement-mediated adhesion, as well as uptake of C. albicans by human macrophages, as shown by flow cytometry. Thus, Pra1 is, to our knowledge, the first potent fungal complement inhibitor that favors C. albicans immune escape by inactivating and controlling host complement attack at the level of C3.

Glycerol-3-phosphate dehydrogenase 2 is a novel factor H-, factor H-like protein 1-, and plasminogen-binding surface protein of Candida albicans.

Candida albicans uses human complement regulators such as factor H and factor H-like protein 1 (FHL-1) for immune evasion. To define the whole panel of fungal complement-evasion molecules, C. albicans cell extract was absorbed to a factor H-coupled matrix. One 52-kDa protein was eluted and identified by mass spectrometry as glycerol-3-phosphate dehydrogenase 2 (Gpd2). Consequently, Gpd2 was recombinantly expressed and purified. Recombinant Gpd2 binds factor Hand and FHL-1, mainly via short consensus repeat 7; and binds plasminogen, via lysine residues. The 3 human complement regulators, when attached to candida Gpd2, became functionally active, and the attached host proteins assist in inactivation of the complement cascade or cleave fibrinogen in the extracellular matrix component fibrinogen. Polyclonal Gpd2 antiserum was generated and localized Gpd2 at the surface of C. albicans. In addition, candida Gpd2 bound to human nonphagocytic cells but not to phagocytic U937 cells. Thus, candida Gpd2 is a novel fungal immune evasion protein that binds several human complement regulators and, in addition, binds human cells.

Sequence Variations and Protein Expression Levels of the Two Immune Evasion Proteins Gpm1 and Pra1 Influence Virulence of Clinical Candida albicans Isolates

Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence.

The secreted Candida albicans protein Pra1 disrupts host defense by broadly targeting and blocking complement C3 and C3 activation fragments

Graphical abstract The secreted C. albicans protein Pra1 disrupts complement by targeting C3 in multiple manners. 1) Secreted Pra1 directly cleaves C3 and generates C3aL and C3bL fragments. 2) Pra1 further degrades the C3aL activation fragment. 3) C3bL is degraded by human protease Factor I assisted by the cofactor Factor H. In addition, Pra1 binds to the activation fragments C3a and C3b which are generated by the human convertase and blocks their effector functions. 4) Pra1 blocks antifungal activity of C3a and 5) inhibits C3b opsonization. Figure. No Caption available. HighlightsCandida Pra1 is fungal protease that cleaves human C3.Pra1 cleaves C3 and generates C3a like and C3b like fragments.Pra1 further cleaves c3aL and blocks the antifungal acitivty.Pra1 binds to C3a and C3b generated by the human C3 convertase and blocks their effector functions. Abstract Candida albicans the most frequently isolated clinical fungal pathogen can cause local as well as systemic and life‐threatening infections particularly in immune‐compromised individuals. A better and more detailed understanding how C. albicans evades human immune attack is therefore needed for identifying fungal immune‐evasive proteins and develop new therapies. Here, we identified Pra1, the pH‐regulated C. albicans antigen as a hierarchical complement inhibitor that targets C3, the central human complement component. Pra1 cleaved C3 at a unique site and further inhibited effector function of the activation fragments. The newly formed C3a‐like peptide lacked the C‐terminal arginine residue needed for C3a‐receptor binding and activation. Moreover, Pra1 also blocked C3a‐like antifungal activity as shown in survival assays, and the C3b‐like molecule formed by Pra1 was degraded by the host protease Factor I. Pra1 also bound to C3a and C3b generated by human convertases and blocked their effector functions, like C3a antifungal activity shown by fungal survival, blocked C3a binding to human C3a receptor‐expressing HEK cells, activation of Fura2‐AM loaded cells, intracellular Ca2+ signaling, IL‐8 release, C3b deposition, as well as opsonophagocytosis and killing by human neutrophils. Thus, upon infection C. albicans uses Pra1 to destroy C3 and to disrupt host complement attack. In conclusion, candida Pra1 represents the first fungal C3‐cleaving protease identified and functions as a fungal master regulator of innate immunity and as a central fungal immune‐escape protein.

Kallikrein Cleaves C3 and Activates Complement

The human plasma contact system is an immune surveillance system activated by the negatively charged surfaces of bacteria and fungi and includes the kallikrein-kinin, the coagulation, and the fibrinolytic systems. Previous work shows that the contact system also activates complement, and that plasma enzymes like kallikrein, plasmin, thrombin, and FXII are involved in the activation process. Here, we show for the first time that kallikrein cleaves the central complement component C3 directly to yield active components C3b and C3a. The cleavage site within C3 is identical to that recognized by the C3 convertase. Also, kallikrein-generated C3b forms C3 convertases, which trigger the C3 amplification loop. Since kallikrein also cleaves factor B to yield Bb and Ba, kallikrein alone can trigger complement activation. Kallikrein-generated C3 convertases are inhibited by factor H; thus, the kallikrein activation pathway merges with the amplification loop of the alternative pathway. Taken together, these data suggest that activation of the contact system locally enhances complement activation on cell surfaces. The human pathogenic microbe Candida albicans activates the contact system in normal human serum. However, C. albicans immediately recruits factor H to the surface, thereby evading the alternative and likely kallikrein-mediated complement pathways.