Cryptococcus neoformans – a major human fungal pathogen

The basidiomycete C. neoformans infects the central nervous system of mainly immunocompromised patients causing a severe meningoencephalitis. In previous classic studies the virulence of C. neoformans has been linked first to its ability to produce a characteristic polysaccharide capsule that inhibits phagocytosis and promotes survival in macrophages. A second specialized virulence factor involves the synthesis of the pigment melanin by the enzyme laccase. Melanin may serve as an antioxidant to protect fungal cells from oxidative products of macrophages. Other factors linked to virulence include the ability to grow at 37°C, prototrophy, and the production of the enzymes urease and phospholipase B. In addition, the MATα mating type has been linked to prevalence in the environment and virulence of C. neoformans. The majority of strains isolated from the environment are of the MATα mating type, and virtually all clinical isolates are MATα. In addition, MATα strains are more virulent than MATa strains in animal models. It was shown that in a pair of congenic serotype D MATa/MATα strains infection with MATα strains resulted in a more rapid progression to lethal infection than congenic MATa strains .

Capsule: Probably the most striking feature of C. neoformans that distinguishes this organism from other pathogenic yeasts is the large polysaccharide capsule that regularly surrounds the organism (Fig. 1). The capsule is essential for virulence, and all acapsular mutants studied have been found to be avirulent.

The majority of the polysaccharide capsule (90%) is glucurono-xylo-mannan (GXM), a linear chain of α-1,3-linked mannose residues that may be substituted with singular xylose or β-glucuronyl sidegroups and can be O-acetylated. Besides GXM, smaller amounts of galacto-xylo-mannan (GalXM) and mannoproteins are present. The capsular polysaccharides form a complex network of fibrillar structures that surrounds the cell but, surprisingly, does not appear to be covalently linked to the cell wall. Several environmental factors have been identified that regulate capsule production. Whereas high salt concentrations (~1 M) prevent capsule production, low levels of glucose, iron deprivation, or carbon dioxide concentrations similar to host physiological levels dramatically increase the production of capsular material. 

Cell mediated immunity is thought to be the most important defense mechanism for a mammalian host infected with C. neoformans. In early stages of infection neutrophils are the primary line of defense. In later stages, monocytes and monocyte-derived macrophages are more important. These phagocytotic cells engulf cryptococcal cells and kill them through oxidative burst. The polysaccharide capsule is thought to protect the yeast cells from phagocytosis by activated immune cells and to promote intracellular survival by inhibiting digestion in the phagolysosomes. Acapsular and hypocapsular strains are phagocytized by macrophages more rapidly then fully encapsulated strains and are then destroyed inside the phagolysosome.

Capsular polysaccharides are a major virulence factor in C. neoformans: acapsular mutants are generally completely avirulent. Wild-type C. neoformans (Cap⁺) and a hypocapsular mutant (gpa1; Cap^-) were grown under capsule inducing conditions and cells were stained with India ink. Capsular polysaccharides surrounding the cells exclude the colloidal stain resulting in clear halos surrounding the wild-type cells (Cap⁺), and considerably smaller halos with the hypocapsular mutant strain (Cap^-). Magnification: 200x.

Melanin: A second characteristic feature of C. neoformans that is correlated with virulence is the ability to produce a dark pigment called melanin (Fig. 2). C. neoformans melanin is a very heterogeneous, high molecular weight molecule that is synthesized from various phenol-based precursors, such as L-dopamine (DOPA), caffeic acid or chlorogenic acid. In contrast to other melanin producing organisms such as mammals or several mushrooms, C. neoformans is unable to produce melanin de novo from L-tyrosine and therefore is completely dependent on precursors provided by environmental sources.

Genetic analysis of Mel^- strains thus far identified seven complementation groups involved in pigment production. One of them encodes an enzyme that catalyses the rate limiting step in the biosynthesis of melanin and was found to be a copper containing phenoloxidase called laccase. Transcript levels of the corresponding gene, CnLAC1, are increased by various signals: low glucose levels, low temperature, the presence of phenolic precursors, and stationary growth phase. On the other hand, high temperature (37^oC) reduces CnLAC1 transcript levels to some extent, but simultaneously it was found that laccase enzyme activity increases at elevated temperatures. 

Mel +	Mel-

Figure 2: Melanin production by C. neoformans

A second important virulence factor of C. neoformans is the ability to produce melanin. If provided with diphenolic compounds such as L-dopamine, which are present in bird seed extract (Guizotia abyssinica), wild-type C. neoformans cells produce the dark brown pigment melanin (Mel⁺). Strains defective in melanin production on the other hand show an albino-like appearance (pka1, Mel^-).

The biological function of melanin in C. neoformans in its environmental niche is not known. Besides a possible role in cell integrity, protective functions against extreme temperature or oxidative damage induced by UV light have been proposed due to the reduced susceptibility of melanized C. neoformans cells to UV irradiation and heat or cold. In addition, laccase may enable the organism to degrade lignin and use it as a carbon and nitrogen source.  In mammalian hosts melanin most likely protects C. neoformans from oxidative damage by neutrophils and macrophages (oxidative burst), as has been demonstrated in several in vitro studies. In general, strains lacking melanin are much more susceptible to oxygen- or nitrogen-derived oxidants. Melanin might also contribute to virulence by altering the net charge of the cell wall to be more negatively charged. Finally, deposition of melanin in the cell wall may confer increased resistance to the antimycoticum amphotericin B.

Extracellular enzymes: A series of secreted enzymes, including proteinases, esterases, and lipases have been identified in C. neoformans. Many of these enzymes have been associated with virulence in other pathogens. Thus, extracellular protease activity has been shown to contribute to virulence by degrading host tissue or destroying immunologically important proteins at the site of infection. Urease is a nickel metalloenzyme that converts urea into ammonia and carbamate to result in a local increase in pH. Urease is associated with virulence in other pathogens and C. neoformans urease-negative strains have only rarely been isolated from infected hosts. The gene encoding urease has recently been cloned and urease deleted strains were shown to be attenuated for virulence. Histopathological studies on lung tissues of infected mice show a higher inflammatory response in tissues infected with the mutant strain in comparison to an isogenic wild-type strain suggesting a role for urease during primary infection by C. neoformans. Finally, extracellular phospholipase activity has been linked to virulence in C. albicans  and phospholipase activity has also been identified in C. neoformans. Strains with higher phospholipase activity on average showed increased capsule size and higher virulence. A phospholipase B gene, PLB1, has been identified in C. neoformans, and virulence studies of plb1 mutants revealed that phospholipase B activity is required for virulence in C. neoformans.

O-glycosylation, the addition of carbohydrates to serine or threonine residues of respective target proteins, is a very important modification of eukaryotic proteins and has been studied extensively in the yeasts Saccharomyces cerevisiae and Candida albicans, but has also been identified in other fungal species like Apergillus, Fusarium or Neurospora. While O-glycosylation has also been identified in higher eukaryotes the structure of O-glycosyl chains varies considerably in these organisms as the initial sugar residue added to proteins is usually N-acetylgalactosamine. Additional residues found in O-glycosylation of mammals include galactose, fucose, N-acetylglucosamine and sialic acid.

The initial step of O-glycosylation occurs at the lumenal side of the endoplasmatic reticulum (ER) membrane and is mediated by the highly conserved Pmt-family of protein O-mannosyltransferases. These membrane-bound proteins catalyse the addition of the first mannose residue from dolichol phosphate mannose (Dol-P-Man) to serine or threonine residues of the target proteins, a process that is thought to occur cotranslationally at the secretory pore complex. Genes encoding members of the Pmt-protein family have been identified in several fungal species and mammals by genome sequence analysis, however, at present Pmt encoding genes were not found in plants.  In S. cerevisiae and C. albicans genes for 7 and 5 Pmts were identified in the genome respectively. The Pmts could be grouped into three major subfamilies, Pmt1, Pmt2 and Pmt4, based on protein similarities (Fig. 3). Although the Pmt proteins of a single organism can vary considerably in length (C. albicans: 725-877 aa) they all share a similar structure. The proteins show a 7 transmembrane domain structure with the amino-terminal region facing the ER lumen and the carboxy-terminal region projecting into the cytoplasm. A marked loop between helices 5 and 6, which faces the ER lumen, was found to be indispensable for enzymatic activity of these proteins. While direct interactions of different Pmt proteins were found and seem to be necessary for full activity of the enzymes it is still unclear whether the Pmt-proteins act as single proteins, as homo-dimers, or as hetero-dimers.

Only few direct targets of a singular Pmt protein have been identified at present, however, various proteins that are O-glycosylated have been identified in S. cerevisiae and C. albicans. In contrast to mammalian cells only few cytoplasmic proteins seem to be affected, but most proteins identified are either secreted proteins or proteins located in the cell-membrane or cell-wall. Proteins found include chitinases, proteases, proteins involved in glucan synthesis, heat-shock proteins, and cell-surface antigens found to be important in virulence in C. albicans. In addition, various receptors have been identified, whose function seem to be affected by protein O-glycosylation.

The N-glycosylation of proteins plays a vital role in correct folding of secreted proteins by linking glycoproteins to the glycoprotein-folding machinery and thus affects proper function and activity of these proteins. Furthermore, a glycoprotein quality control has been identified in the secretory pathway. This glycan-based disposal system removes misfolded, transport-deficient glycoproteins from the early secretory system and directs them towards degradation. In addition, glycosylation ensures proper sorting and transport of glycoproteins through the secretory pathway (ER and Golgi). While data elucidating the role of O-glycosylation are still scarce similar functions have been proposed for O-glycosylation in analogy to N-glycosylation.

Aim of Research and Research Programm

Actual virulence of the basidiomycete C. neoformans is mainly determined by “extracellular” factors including a polysaccharide capsule, extracellular enzymes, cell-wall components, and cell-surface antigens (see above). Interestingly, O-glycosylation seems to have a huge impact on secretion and function of many extracellular and cell-wall components. Any major mis-regulation of O-glycosylation in C. neoformans thus should have dramatic consequences on virulence of the organism. Therefore, our major goal with this project is first, to identify and characterize members of the highly conserved PMT-gene family in C. neoformans, and second, to determine their influence on various virulence factors in vitro as well as infectivity of pmt-deletion mutants in vivo.

In contrast to S. cerevisiae and C. albicans, but similar to what was found by sequence database analysis for other fungal species, only three PMT-genes could be identified in both the serotype A and serotype D sequence database of C. neoformans doing a blast search with S. cerevisiae Pmt1p as a bait. The Pmt proteins Pmt1, Pmt2, and Pmt4 each represent a member of the three major subfamilies of Pmts identified in other organisms (Fig. 3). Since serotype A and serotype D gene sequences usually deviate around 5-10% in base-pairs, a comparison of the corresponding genomic regions usually allows to determine the exon/intron structure of a specific open reading frame (ORF). Yet, to confirm the gene structure of the PMT-genes in C. neoformans we first need to generate cDNA of these genes, including 5’ and 3’ RACE, in order to determine the exact exon/intron structure and transcriptional start points of the PMT-genes as well as poly-adenylation sites of their mRNAs.

In addition, many virulence factors including capsule material and melanin are produced to varying amounts under different growth conditions (see above). Therefore, Northern analyses using RNA isolated from cells grown under such different conditions may give us a first idea which of the PMT-genes may be important for the function of a specific virulence factor.

Phenotypic and in vitro analysis of pmt-mutants in C. neoformans

In vivo virulence studies of C. neoformans

In addition, because of the simplicity of these new virulence models they may be useful for screening large numbers of of C. neoformans mutants for strains that are able to survive the killing by A. castellanii or C. elegans or are attenuated for virulence.

Publications

Loftus BJ, Fung E, Roncaglia P, Rowley D, Amedeo P, Bruno D, Vamathevan J, Miranda M, Anderson IJ, Fraser JA, Allen JE, Bosdet IE, Brent MR, Chiu R, Doering TL, Donlin MJ, D'Souza CA, Fox DS, Grinberg V, Fu J, Fukushima M, Haas BJ, Huang JC, Janbon G, Jones SJ, Koo HL, Krzywinski MI, Kwon-Chung JK, Lengeler KB, Maiti R, Marra MA, Marra RE, Mathewson CA, Mitchell TG, Pertea M, Riggs FR, Salzberg SL, Schein JE, Shvartsbeyn A, Shin H, Shumway M, Specht CA, Suh BB, Tenney A, Utterback TR, Wickes BL, Wortman JR, Wye NH, Kronstad JW, Lodge JK, Heitman J, Davis RW, Fraser CM, Hyman RW. The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans Science. 2005 Feb 25;307(5713):1321-4. Epub 2005 Jan 13

Fraser JA, Diezmann S, Subaran RL, Allen A, Lengeler KB, Dietrich FS, Heitman J. Convergent evolution of chromosomal sex-determining regions in the animal and fungal kingdoms PLoS Biol. 2004 Dec;2(12):e384. Epub 2004 Nov 9

Barreto de Oliveira MT, Boekhout T, Theelen B, Hagen F, Baroni FA, Lazera MS, Lengeler KB, Heitman J, Rivera IN, Paula CR. Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil J Clin Microbiol. 2004 Mar;42(3):1356-9

Wang P, Nichols CB, Lengeler KB, Cardenas ME, Cox GM, Perfect JR, Heitman J. Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans Eukaryot Cell. 2002 Apr;1(2):257-72

Lengeler KB, Fox DS, Fraser JA, Allen A, Forrester K, Dietrich FS, Heitman J. Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes Eukaryot Cell. 2002 Oct;1(5):704-18

Schein JE, Tangen KL, Chiu R, Shin H, Lengeler KB, MacDonald WK, Bosdet I, Heitman J, Jones SJ, Marra MA, Kronstad JW. Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans Genome Res. 2002 Sep;12(9):1445-53.

Lengeler KB, Cox GM, Heitman J. Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus Infect Immun. 2001 Jan;69(1):115-22

Lengeler KB, Wang P, Cox GM, Perfect JR, Heitman J. Identification of the MATa mating-type locus of Cryptococcus neoformans reveals a serotype A MATa strain thought to have been extinct Proc Natl Acad Sci U S A. 2000 Dec 19;97(26):14455-60

Lengeler KB, Davidson RC, D'souza C, Harashima T, Shen WC, Wang P, Pan X, Waugh M, Heitman J. Signal transduction cascades regulating fungal development and virulence Microbiol Mol Biol Rev. 2000 Dec;64(4):746-85. Review

Sia RA, Lengeler KB, Heitman J. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic Fungal Genet Biol. 2000 Apr;29(3):153-63