Microbial-Arms Race: How does Cryptococcusneoformans evade the innate immune system?
Introduction
Humans possess two main forms of immunity, our adaptive and innate immunity. Innate immunity is extremely crucial for recognition of various antigens on viruses, bacterial and fungal pathogens. This recognition is important as it is followed by an immune response against the pathogen. Various microbes have adopted strategies of immune evasion to ensure microbial survival, host infection and replication. This article will provide mechanistic insight into the specific strategies used by the major opportunistic fungal pathogen Cryptococcus neoformans to evade recognition and elimination by the innate immune system. C. neoformans is categorised under the World Health Organisation’s (WHO) critical priority group concerning fungal pathogens (WHO, 2022). Subsequently, this article will delve deep into the molecular mechanisms used by the fungi before stressing the importance of understanding immune evasion in regards to developing a greater appreciation of the challenges faced in medical mycology and producing effective novel therapeutics for clinical use.
Innate immune cells recognise a variety of PAMPs on the cell wall of fungal pathogens
Cryptococcus and other pathogenic fungal species contain similar pathogen associated molecular patterns (PAMPs) on their cell walls which are recognised by specific pattern recognition receptors (PRRs) of the innate immune cells (Table 1). Dectin-1 receptors and Toll-like receptors (TLRs) are dominant PRRs present on all three innate immune cells (macrophages, monocytes and neutrophils). Mannose receptors (MR) are specific to monocytes and macrophages. Dectin-1 and TLR2 have crucial roles in phagocytosis leading to robust immune responses (Netea et al., 2008, Bojang et al., 2021).
Table 1: Specific PRRs are responsible for the recognition of key PAMPs which include B-1,3 glucan, N and O-mannan proteins and chitins. More than one PRR could recognise the same PAMP. Dectin-1, MR and DC-SIGN are characterised as C-type lectin receptors whilst TLR2 and 4 are in their own class of toll-like receptors. Although chitin is a well-studied component of the cell wall, PRRs recognising chitin are currently not well characterised.
PAMP recognition is followed by varied host immune responses which include phagosomal engulfment and elimination, cytokine production, neutrophil entrapment and antigen presentation by dendritic cells (which would lead to intervention of T-lymphocytes of the adaptive immune system) (Bojang et al., 2021). C. neoformans has developed immune evasion approaches such as the formation of titan cells (Okagaki et al., 2010, Zaragoza et al., 2010) and specialised mechanisms for macrophage escape. Cryptococcal cells are able to escape but not destroy macrophages via induced vomocytosis (Ma et al., 2006). Lastly, consistent with other fungal species, C. neoformans is also able to mask their PAMPs from innate immune recognition (O’Meara & Alspaugh, 2012). It is important to note that these immune evasion strategies often contribute to fungal pathogen virulence.
The glucuronoxylomannan polysaccharide (GXM) capsule protects C. neoformans from microbial stresses and is subject to antigenic variation
C. neoformans is known to employ capsule-dependent and capsule independent mechanisms for immune evasion. Capsule-dependent mechanisms, such as the formation of the GXM capsule, aims to prevent internalisation of Cryptococcal cells by macrophages (O’Meara & Alspaugh, 2012). The GXM capsule is primarily composed of glucuronoxylomannan polysaccharides (90%). Galactomannan polysaccharides and mannoproteins make up a minority composition of 10% and < 1% respectively (Kuttel et al., 2020). Deletion of the GXM capsule to form acapsular mutants will deem the fungi avirulent and unable to evade host immune defenses (Chang and Kwon-Chung, 1994). This demonstrates that the GXM capsule is vital for C. neoformans immune evasion and virulence.
Interestingly, the GXM capsule can bind to TLR2 and 4, preventing nuclear translocation of transcription factor NF-kB and inhibiting secretion of interferon TNF-alpha by macrophages. This results in deficient macrophage activation and therefore, reduced phagosomal uptake (Shoham et al., 2001). The capsule can bind to glycolytic enzymes such as phosphofructokinase-1 (PK1), interfering with macrophage metabolism (Garcia-Rodas and Zaragoza, 2012). The GXM capsule is also subject to antigenic variation, in which structure and composition can be modified to avoid recognition by immune cells. Enzymes like glucanases and α-1,3 mannosyltransferases are responsible for Beta-glucan and mannan epitope modification (Doering, 1999, McFadden et al., 2007). This allows for added protection against environmental stresses and antifungals. This emphasises that the GXM capsule is a key C. neoformans virulence factor for immune evasion, intracellular survival and replication.
Morphogenic switching into Cryptococcal titan cells is governed by cAMP signal transduction
Cryptococcus are able to undergo morphogenesis into titan cells to evade phagocytosis due to its gigantic size and is a major contributor of disease progression and antifungal resistance. This phenomenon was recently discovered through a simultaneous publication by two competing labs in 2010! (Okagaki et al., 2010, Zaragoza et al., 2010).
Yeast-to-Titan transition is induced by aforementioned environmental stresses. Titanisation is a macrophage-induced process and cell size ranges from around 10 - 100 µm! A normal-sized Cryptococcal cell ranges from 5 - 10 µm. This shows that titan cells are 10 times larger than normal cells (Crabtree et al., 2012). Titanisation is triggered by external signals, such as alkaline pH and is driven by cAMP/protein kinase A (PKA) signalling involving upregulation of transcription factor Rim101 (Figure 2) (Okagaki et al., 2011, Ost et al., 2015, Dambuza et al., 2018). Rim101 is responsible for activation of genes controlling cell wall remodelling, capsule biosynthesis, titanisation and melanisation. This allows C. neoformans to alter changes in virulence factors such as the GXM capsule and cell wall chitin content to ensure resistance to stress and phagocytosis.
Interestingly, Zaragoza et al reported high polyploidy in titan cells as high DNA content was observed through flow cytometry experiments. This is evident of polyploidy due to the presence of an extra chromosome. This implies that Cryptococcus achieves titanisation through continued DNA replication with no fission (Okagaki et al., 2010, Zaragoza et al., 2010). Subsequently, titan mother cells produce aneuploid progeny through gene duplication. Aneuploidy cells lacking a chromosome are a key driver of antifungal resistance. Resistance arose via gene duplication of chromosomes encoding for ERG11 (ergosterol in the fungal cell wall) and AFR1 (azole antifungal transport) (Sionov et al., 2010). This emphasises that titanisation is vital for immune evasion and antifungal resistance development.
Figure 2: Rim101 pathway governs Cryptococcus neoformans titanisation. The pathway is stimulated by environmental signals such as alkaline pH, leading to a cascade of events which ultimately activates Rim101-T (transcription factor) via proteolytic cleavage. Rim101-T transcribes genes required for titanisation in the nucleus. Modified and adapted from: Ost et al., 2015.
C. neoformans can survive intracellularly and then escape from macrophages through a phenomenon termed vomocytosis
Cryptococcus neoformans can evade immune defences via survival within the phagolysosome and subsequent exit from macrophages. The Cryptococcus capsule is subject to enlargement and contains antioxidants which decreases C. neoformans susceptibility to free radicals and antimicrobial peptides, permitting survival and nutrient uptake within macrophages. Melanin is one such antioxidant produced by Cryptococcus to aid survival within phagolysosomes (Nicola et al., 2011). The fungi then secretes laccase enzymes responsible for catalysis of melanin formation in which melanisation confers protection from stressful environments in the macrophage. Survival within phagolysosomes is termed as the Trojan horse mechanism (Figure 3) (Botts and Hull, 2010).
On rare occasions, C. neoformans induces vomocytosis whereby Cryptococcal cells will be spewed out from the macrophage, leaving the immune cells undamaged in the process (Figure 3). Vomocytosis was first described in 2006, also through a simultaneous publication and has garnered worldwide attention as the mechanism of action is currently not well understood (Alvarez and Casadevall 2006, Ma et al., 2006). Both Casadevall and May labs believe that the vomocytosis phenomenon is an instantaneous process induced by phagosomal pH acidification and only occurs with live Cryptococcal cells. Actin rearrangements in the cytoskeleton mediated by the Wiskott-Aldrich syndrome protein (WASP)-Arp 2/3 complex in vomocytosis have been reported to be vital for the occurrence of the phenomena (Alvarez and Casadevall 2006, Ma et al., 2006, Johnston and May, 2010, Guera et al., 2014). From these reports, it can be deduced that the vomocytosis plays a key role in the promotion of pathogen survival and evasion of the innate immune system through remodelling of cytoskeletal proteins involved in phagocytosis (Cruz-Acuña et al., 2019). It is crucial to understand that little is known regarding the relationship between actin polymerisation and vomocytosis.
Figure 3: A summary of various immune evasion strategies employed by C. neoformans including vomocytosis, titanisation and Trojan horse. Key strategies that have been discussed are highlighted in blue boxes. Modified and adapted from: Wang et al., 2022.
Conclusion: a cautionary 21st century tale!
To summarise, this article has underlined an appreciation of a series of immune evasion strategies adopted by Cryptococcus neoformans to evade innate immunity. C. neoformans ability to undergo a morphogenic switch controlled by conserved transcription factor Rim101. It is also able to mask itself by epitope shaving. C. neoformans also possess protective capsules which protect them from stressful conditions and permit intracellular survival.
Finally, Cryptococcal cells are able to escape macrophages without damage through the vomocytosis phenomenon. C. neoformans heavily contributes to an underlying fungal burden, especially in immunocompromised individuals. Fungal infections, evasion strategies and resistance to antifungals are increasing daily. Improving our understanding and appreciation for immune evasion methods is instrumental for novel therapeutic development. These efforts would also prevent extensively drug resistant fungal strains from arising, which would lengthen our ensuing battle with pathogenic fungi.
Article prepared by: Eldrian Tho Jiat Yang, MBIOS 23/24 Advisor, MBIOS 22/23 President, & Research and Development Director 21/22
If you enjoyed this article, do sign up to become a part of our MBIOS family and receive our monthly newsletter along with many more resources in the link below.
Glossary
References
Bojang, E., Ghuman, H., Kumwenda, P. and Hall, R.A., 2021. Immune sensing of Candida albicans. Journal of Fungi, 7(2), p.119.
Botts, M.R. and Hull, C.M., 2010. Dueling in the lung: how Cryptococcus spores race the host for survival. Current opinion in microbiology, 13(4), pp.437-442.
Childers, D.S., Avelar, G.M., Bain, J.M., Pradhan, A., Larcombe, D.E., Netea, M.G., Erwig, L.P., Gow, N.A. and Brown, A.J., 2020. Epitope shaving promotes fungal immune evasion. MBio, 11(4), pp.10-1128.
Cruz-Acuña, M., Pacifici, N. and Lewis, J.S., 2019. Vomocytosis: too much booze, base, or calcium?. Mbio, 10(6), pp.10-1128.
Dambuza, I.M., Drake, T., Chapuis, A., Zhou, X., Correia, J., Taylor-Smith, L., LeGrave, N., Rasmussen, T., Fisher, M.C., Bicanic, T. and Harrison, T.S., 2018. The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLoS pathogens, 14(5), p.e1006978.
Doering, T.L., 1999. A unique α-1, 3 mannosyltransferase of the pathogenic fungus Cryptococcus neoformans. Journal of bacteriology, 181(17), pp.5482-5488.
Garcia-Rodas, R. and Zaragoza, O., 2012. Catch me if you can: phagocytosis and killing avoidance by Cryptococcus neoformans. FEMS Immunology & Medical Microbiology, 64(2), pp.147-161.
Guerra, C.R., Seabra, S.H., de Souza, W. and Rozental, S., 2014. Cryptococcus neoformans is internalized by receptor-mediated or ‘triggered’phagocytosis, dependent on actin recruitment. PloS one, 9(2), p.e89250.
Johnston, S.A. and May, R.C., 2010. The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation. PLoS pathogens, 6(8), p.e1001041.
Kuttel, M.M., Casadevall, A. and Oscarson, S., 2020. Cryptococcus neoformans capsular GXM conformation and epitope presentation: a molecular modelling study. Molecules, 25(11), p.2651.
Ma, H., Croudace, J.E., Lammas, D.A. and May, R.C., 2006. Expulsion of live pathogenic yeast by macrophages. Current Biology, 16(21), pp.2156-2160.
McFadden, D.C., Fries, B.C., Wang, F. and Casadevall, A., 2007. Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans. Eukaryotic Cell, 6(8), pp.1464-1473.
Nicola AM, Robertson EJ, Albuquerque P, Derengowski LD, Casadevall A. Nonlytic exocytosis of Cryptococcus neoformans from macrophages occurs in vivo and is influenced by phagosomal pH. MBio. 2011 Sep 1;2(4):10-128.
Nobile, C.J., Solis, N., Myers, C.L., Fay, A.J., Deneault, J.S., Nantel, A., Mitchell, A.P. and Filler, S.G., 2008. Candida albicans transcription factor Rim101 mediates pathogenic interactions through cell wall functions. Cellular microbiology, 10(11), pp.2180-2196.
Okagaki, L.H., Strain, A.K., Nielsen, J.N., Charlier, C., Baltes, N.J., Chrétien, F., Heitman, J., Dromer, F. and Nielsen, K., 2010. Cryptococcal cell morphology affects host cell interactions and pathogenicity. PLoS pathogens, 6(6), p.e1000953.
Okagaki, L.H., Wang, Y., Ballou, E.R., O'Meara, T.R., Bahn, Y.S., Alspaugh, J.A., Xue, C. and Nielsen, K., 2011. Cryptococcal titan cell formation is regulated by G-protein signaling in response to multiple stimuli. Eukaryotic cell, 10(10), pp.1306-1316.
O'Meara, T.R. and Alspaugh, J.A., 2012. The Cryptococcus neoformans capsule: a sword and a shield. Clinical microbiology reviews, 25(3), pp.387-408.
Ost, K.S., O’Meara, T.R., Huda, N., Esher, S.K. and Alspaugh, J.A., 2015. The Cryptococcus neoformans alkaline response pathway: identification of a novel rim pathway activator. Plos genetics, 11(4), p.e1005159.
Phan, Q.T., Myers, C.L., Fu, Y., Sheppard, D.C., Yeaman, M.R., Welch, W.H., Ibrahim, A.S., Edwards Jr, J.E. and Filler, S.G., 2007. Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. PLoS biology, 5(3), p.e64.
Sionov, E., Lee, H., Chang, Y.C. and Kwon-Chung, K.J., 2010. Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes. PLoS pathogens, 6(4), p.e1000848.
Wang, Y., Pawar, S., Dutta, O., Wang, K., Rivera, A. and Xue, C., 2022. Macrophage mediated immunomodulation during cryptococcus pulmonary infection. Frontiers in Cellular and Infection Microbiology, 12, p.859049.
World Health Organisation (WHO)., 2022. WHO fungal priority pathogens list to guide research, development and public health action. Taken from https://www.who.int/publications/i/item/9789240060241 ., Last accessed: 15th December 2023.
Zaragoza, O., Garcia-Rodas, R., Nosanchuk, J.D., Cuenca-Estrella, M., Rodríguez-Tudela, J.L. and Casadevall, A., 2010. Fungal cell gigantism during mammalian infection. PLoS pathogens, 6(6), p.e1000945.