Klaus

Research interest

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 on Home). 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.

Characterization and deletion of the PMT-genes in C. neoformans

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. 1). 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.

Figure 1: Phylogenetic analysis of Pmt proteins derived from DNA sequences obtained from C. neoformans genome sequence databases. Pmt proteins of C. albicans (Ca), S. cerevisiae (Sc) and C. neoformans (Cn) serotype D were aligned using the ClustalX algorythm. The alignment was transformed into a phylogenetic tree using the TREEVIEW programm. The bar represents an evolutionary distance of 0.1 nucleotide substitutions per site. C. neoformans Pmt proteins are highlighted.

In addition, many virulence factors including capsule material and melanin are produced to varying amounts under different growth conditions (see on Home). 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.

Simultaneously, we will delete each of the three PMT-genes in C. neoformans. O-glycosylation is a vital process in most eukaryotic organisms. Therefore, we will begin by deleting a single allele of each PMT-gene in diploid serotype D strains of C. neoformans. These strains can proceed through the sexual life-cycle of the organism resulting in haploid progeny. Analysis of these progenies would enable us to determine whether any of the three PMT-genes is essential in C. neoformans. In addition, we could generate deletion mutants in each mating-type background that would enable us to determine the effects of pmt-gene deletions in the sexual development of C. neoformans. Furthermore, we would be able to generate strains carrying various combinations of the pmt-mutations by simple genetic crosses. In a second step we will also delete the PMT-genes in the serotype A background for the following reasons. First, it was found that several genes deleted in the various serotypic backgrounds cause differences in their phenotypes indicative of the genetic divergence of the various varieties of C. neoformans. Therefore, the phenotypes of pmt-deletions found in serotype D may vary from the ones found in serotype A. This is of special interest since the serotype A strains are commonly more virulent than the serotype D strains. Finally, because serotype A strains are more virulent, virulence studies are preferentially done in this genetic background (see below).

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

With respect to virulence of C. neoformans it will further be of interest whether the deletion of any PMT-gene will have an effect on the various virulence factors identified for this organism. Capsule formation can easily be tested for in vitro by growing the pmt-deletion mutants and wild-type reference strains under capsule inducing conditions (low glucose/low iron) and stain the cells using india ink (see on Home). In addition, several proteins have been identified that are closely associated with capsule formation (Caps). Introducing tagged alleles of these proteins into C. neoformans by standard molecular techniques we should be able to determine by Western analysis whether these proteins are glycosylated and whether the deletion of the PMT-genes has an effect on glycosylation of these proteins. Similar to capsule analysis, melanin production can be analysed using a simple colorimetric plate assay. While wild-type strains produce a characteristic brownish pigment when provided with phenol based compounds such as DOPA, mutants defective in melanin biosynthesis stay white (see on Home). Finally, using enzymatic assays we should be able to determine whether defects in O-glycosylation have any effect on the secretion and/or function of easy to test extracellular enzymes including lipases or proteases. The analysis of the various virulence factors should provide us with vital information whether O-glycosylation has an effect on virulence of C. neoformans.

In vivo virulence studies of C. neoformans

To complete the data provided by the analysis of specific virulence factors we would like to test the pmt-mutant strains for infectivity and virulence in a virulence model based on living host cells. Since we are not able to do mouse or rabbit experiments in our institute we would like to establish virulence assays in our laboratory that are based on the interaction of C. neoformans with the lower eukaryotes Caenorhabditis elegans or Acanthamoeba castellanii. These organisms are killed by wild-type C. neoformans strains when used as feed for the lower eukaryotes while mutants known to be reduced for virulence have no influence of survival of these organisms. It has recently been shown that the infection and interaction of these organisms with C. neoformans are comparable to the interaction of C. neoformans with macrophages and neutrophils in the human host or in a mouse model. However, both model systems are much less cost effective and seem to be easy to do experiments. In both models C. elegans or A. castellanii are grown on wild-type or mutant C. neoformans strains and killing-rates are determined by screening mobility for C. elegans or using viability stains for A. castellanii. Killing of A. castellanii or C. elegans is more dramatic using serotype A strains of C. neoformans in contrast to serotype D strains thus corroborating the necessity to generate pmt-deletions in the serotype A background.

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

Schubert D, Lengeler KB, Kothe E.
Identification of mating-type dependent genes by non-radioactive, arbitrarily primed PCR in Schizophyllum commune
J Basic Microbiol. 2000;40(1):65-70

Lengeler KB, Kothe E.
Mated: a putative peptide transporter of Schizophyllum commune expressed in dikaryons
Curr Genet. 1999 Sep;36(3):159-64

Lengeler KB, Kothe E.
Identification and characterization of brt1, a gene down-regulated during B-regulated development in Schizophyllum commune
Curr Genet. 1999 Jun;35(5):551-6

Wendland J, Vaillancourt LJ, Hegner J, Lengeler KB, Laddison KJ, Specht CA, Raper CA, Kothe E.
The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes
EMBO J. 1995 Nov 1;14(21):5271-8

Lengeler K, Kothe E.
Molecular characterization of ura1, a mutant allele for orotidine-5'- monophosphate decarboxylase in Schizophyllum commune
FEMS Microbiol Lett. 1994 Jun 1;119(1-2):243-7