The contribution of the White Collar complex to Cryptococcus neoformans virulence is independent of its light-sensing capabilities
Graphical abstract
Introduction
The effects of light on fungi impact aspects of their biology as diverse as sporulation, circadian clock function, primary metabolism, and the biosynthesis of secondary metabolites such as toxins or pigments (Fuller et al., 2016, Tisch and Schmoll, 2010). Those responses are controlled by a small number of photosensory proteins (Fischer et al., 2016, Idnurm et al., 2010, Rodriguez-Romero et al., 2010). Among them the blue light receptor White Collar-1 (WC-1), first characterized in the model ascomycete Neurospora crassa, appears to have originated early and then been well-conserved during the evolution of the fungi.
In N. crassa the light-regulated processes are controlled by WC-1 and WC-2, which form a GATA-type zinc finger transcription factor complex. WC-1 contains a number of domains: for DNA binding, two PAS domains (named after the Per, Arnt and Sim proteins, in which they were first observed) involved in protein-protein interactions, two putative transcriptional activation domains, a nuclear localization signal, and a LOV domain which is a specialized type of PAS domain involved in environmental sensing of light, oxygen and voltage (Froehlich et al., 2002, He et al., 2002, Wang et al., 2015). The chromophore flavin adenine dinucleotide (FAD), accommodated in the LOV domain, is essential for the light sensing activity of WC-1. A motif of NCRFLQ amino acids that contributed to the α’A helix is a highly conserved region among LOV domains, as the cysteine (C) residue is essential for the covalent attachment of the flavin chromophore to the sensor protein in the presence of light (Cheng et al., 2003). WC-1 interacts with a partner protein WC-2, another GATA-type transcription factor, to form the White Collar complex (WCC) that activates the expression of light-responsive genes upon light stimulation (Collett et al., 2002, Froehlich et al., 2002, Liu et al., 2003).
The availability of fungal genome sequences has allowed the identification of photoreceptors, especially wc-1 and wc-2 orthologs, in varied fungal species. Mutation of these white collar genes in most cases abolishes the effects of blue light on the species. However, analysis of such mutants has also revealed phenotypes in the absence of light, such as changes in sporulation or secondary metabolite production (Fuller et al., 2016, Fuller et al., 2015), leading to questions about how this complex functions above and beyond its perception of light.
One property that may be unrelated to light status is virulence in pathogenic species. In the human pathogenic fungi Cryptococcus neoformans and C. deneoformans [their previous names are C. neoformans var. grubii/serotype A and C. neoformans var. neoformans/serotype D, respectively (Hagen et al., 2015)], light inhibits mating and induces a protective response against ultraviolet light. Mutation of the orthologs of N. crassa wc-1 and wc-2 impairs these effects (Idnurm and Heitman, 2005, Lu et al., 2005, Verma and Idnurm, 2013). An additional phenotype associated with mutating the BWC1 or BWC2 genes in C. neoformans and the related species C. deuterogattii is a reduction in virulence in animal models of disease (Idnurm and Heitman, 2005, Zhu et al., 2013). Cryptococcosis refers to a set of diseases caused by the C. neoformans species complex, currently made up of seven species that are most problematic when growing in the lungs and central nervous systems of people (Heitman et al., 2011). While a number of factors and genes have been identified that control virulence, mutation of the WCC does not impact on any of these that are known to date (Alspaugh, 2015, Brown et al., 2014).
The role of light or the WCC in fungal virulence is more widespread than the Cryptococcus species. For instance, mutation of the WC homologs also alters the pathogenicity in plant pathogens. In the maize leaf pathogen Cercospora zeae-maydis, the WC-1 ortholog is required for tropism toward the host stomata and lesion formation (Kim et al., 2011a). In the rice blast pathogen Pyricularia (Magnaporthe) oryzae, light represses asexual spore releases and a dark-phase immediately after pathogen–host contact plays a critical role in successful disease development, with spore release and the light-dependent repression accomplished by the photoreceptor MGWC-1 (Kim et al., 2011b, Lee et al., 2006). A T-DNA insertion within the WC-2 ortholog also causes a decrease in pathogenicity of P. oryzae (Jeon et al., 2007). Mutation of wc1 in Fusarium oxysporum, which is normally a plant pathogen, causes a decrease in virulence in a mouse model of disease (Ruiz-Roldán et al., 2008). In the gray mold pathogen Botrytis cinerea, loss of BcWCL1 results in attenuated virulence in the host plant when light is present, and this is due to BcWCL1 being required for coping with excessive reactive oxygen species generated by the host’s oxidative burst and photooxidative stress (Canessa et al., 2013). A light-responsive transcription factor, BcLTF1, downstream of BcWCL1 is responsible for anti-oxidative stress activities and is thus required for advanced host infection by B. cinerea (Schumacher et al., 2014). In addition, the influence of the circadian clock, which is controlled by light, on the disease outcome in the Botrytis cinerea-Arabidopsis thaliana interaction is dependent on the effects of the clock and light from the fungal pathogen, rather than changes in plant defense systems (Hevia et al., 2015). In addition to the genetic contributions of the WCC to virulence, pre-exposing fungi to light can impact subsequent virulence outcomes (Campbell and Berliner, 1973, Oliveira et al., 2018).
It is not yet clear in the pathogenic fungi how light or the light-sensing proteins contribute to virulence. This study used C. neoformans as the model organism to test if light sensing is responsible for the function of the conserved White Collar complex in virulence.
Section snippets
Generation of plasmids
The LITMUS 28i vector (New England Biolabs, Ispwich, MA) was double digested with the restriction enzymes KpnI and BamHI. The BWC1 open reading frame and adjacent 5′ and 3′ regulatory regions were amplified from the genomic DNA of strain KN99α with the primer pair PK003/PK004, and then cloned into linearized LITMUS 28i using T4 DNA ligase, yielding the LITMUS 28i-BWC1 vector. Primer sequences are given in Table 1. A plasmid without errors in BWC1 was identified by sequencing the inserts. The
The flavin-binding site in the LOV domain of Bwc1 is required for full light responses in C. neoformans
Plasmid vectors were created with three versions of the BWC1 gene: a wild type copy, and those that cause a cysteine to alanine or serine substitution in the flavin-binding part of the encoded protein. These substitutes were based on previous work on the Neurospora crassa WC-1 and oat phototropin proteins (Cheng et al., 2003, Salomon et al., 2000), with the rationale that the new isoforms would retain their other functions apart from a loss in the ability to transmit the light signal. These
Discussion
The LOV domains are essential for the light sensing capabilities of photosensory proteins in plants, fungi and bacteria (Briggs and Spudich, 2005), and LOV-domain containing proteins are implicated as virulence factors in pathogenic fungi and bacteria. In fungi, the most common and best-characterized LOV domain protein is White Collar 1 (Fischer et al., 2016). This gene, and a second gene wc-2, were first identified and cloned based on mutant phenotypes in the ascomycete N. crassa (Ballario et
Acknowledgments
We thank Barbara Howlett for suggestions and edits on the manuscript. We are grateful for support from the Chinese Scholarship Council to P.Z. and National Institutes of Health NIAID (grant AI094364) and Australian Research Council (grant FT130100146) to A.I.
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