Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided in determining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

Chitosan-Deficient Cryptococcus as Whole-Cell Vaccines.

Creating a safe and effective vaccine against infection by the fungal pathogen Cryptococcus neoformans is an appealing option that complements the discovery of new small molecule antifungals. Recent animal studies have yielded promising results for a variety of vaccines that include live-attenuated and heat-killed whole-cell vaccines, as well as subunit vaccines formulated around recombinant proteins. Some of the recombinantly engineered cryptococcal mutants in the chitosan biosynthesis pathway are avirulent and very effective at conferring protective immunity. Mice vaccinated with these avirulent chitosan-deficient strains are protected from a lethal pulmonary infection with C. neoformans strain KN99. Heat-killed derivatives of the vaccination strains are likewise effective in a murine model of infection. The efficacy of these whole-cell vaccines, however, is dependent on a number of factors, including the inoculation dose, route of vaccination, frequency of vaccination, and the specific mouse strain used in the study. Here, we present detailed methods for identifying and optimizing various factors influencing vaccine potency and efficacy in various inbred mouse strains using a chitosan-deficient cda1Δcda2Δcda3Δ strain as a whole-cell vaccine candidate. This chapter describes the protocols for immunizing three different laboratory mouse strains with vaccination regimens that use intranasal, orotracheal, and subcutaneous vaccination routes after the animals were sedated using two different types of anesthesia.

Cell wall composition in Cryptococcus neoformans is media dependent and alters host response, inducing protective immunity.

Introduction: Cryptococcus neoformans is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence. Objective: The purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response. Methods: Yeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, C. neoformans was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection. Results: We observed that the growth of wild-type C. neoformans KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells. Conclusion: These findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the C. neoformans cell wall, as well as their impact on fungal virulence and the host response.

Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin.

The fungal pathogen Cryptococcus neoformans causes up to 278 000 infections each year globally, resulting in up to 180,000 deaths annually, mostly impacting immunocompromised people. Therapeutic options for C. neoformans infections are very limited. Caspofungin, a member of the echinocandin class of antifungals, is generally well tolerated but clinically ineffective against C. neoformans. We sought to identify biological processes that can be targeted to render the cell more susceptible to echinocandins by screening the available libraries of gene deletion mutants made in the KN99α background for caspofungin sensitivity. We adapted a Candida albicans fungal biofilm assay for the growth characteristics of C. neoformans and systematically screened 4,030 individual gene deletion mutants in triplicate plate assays. We identified 25 strains that showed caspofungin sensitivity. We followed up with a dose dependence assay, and 17 of the 25 were confirmed sensitive, 5 of which were also sensitive in an agar plate assay. We made new deletion mutant strains for four of these genes: CFT1, encoding an iron transporter; ERG4, encoding a sterol desaturase; MYO1, encoding a myosin heavy chain; and YSP2, encoding a sterol transporter. All were more sensitive to membrane stress and showed significantly increased sensitivity to caspofungin at higher temperatures. Surprisingly, none showed any obvious cell wall defects such as would be expected for caspofungin-sensitive strains. Our microscopy analyses suggested that loss of membrane integrity contributed to the caspofungin sensitivity, either by allowing more caspofungin to enter or remain in the cell or by altering the location or orientation of the enzyme target to render it more susceptible to inhibition. IMPORTANCE The intrinsic resistance of Cryptococcus neoformans to the cell wall inhibitor caspofungin limits the available therapies for treating cryptococcal infections. We screened a collection of more than 4,000 gene deletion strains for altered caspofungin sensitivity to identify biological processes that could be targeted to render the cell more susceptible to caspofungin. We identified multiple genes with an effect on caspofungin susceptibility and found that they were associated with altered membrane permeability rather than the expected cell wall defects. This suggests that targeting these genes or other genes affecting membrane permeability is a viable path for developing novel therapies for treating this global fungal pathogen.

Cryptococcus neoformans Cda1 and Cda2 coordinate deacetylation of chitin during infection to control fungal virulence.

Chitosan, a deacetylated form of chitin, is required for the virulence of Cryptococcus neoformans. There are three chitin deacetylase genes (CDA) that are essential for chitosan production, and deletion of all three genes results in the absence of chitosan, loss of virulence, and induction of a protective host response when used as a vaccine. Cda1 plays a major role in deacetylating chitin during pulmonary infection of CBA/J mice. Inoculation with the cda1Δ strain did not lead to a lethal infection. However, the infection was not cleared. The persistence of the fungus in the host suggests that chitin is still being deacetylated by Cda2 and/or Cda3. To test this hypothesis, we subjected strains deleted of two CDA genes to fungal virulence in CBA/J, C57BL/6 and BALB/c and found that cda1Δcda2Δ was avirulent in all mouse lines, as evidenced by its complete clearance. Consistent with the major role of Cda1 in CBA/J, we found that cda2Δcda3Δ was as virulent as its wild-type progenitor KN99. On the other hand, cda1Δcda3Δ displayed virulence comparable to that of cda1Δ. The virulence of each mutant correlates with the amount of chitosan produced when grown under host-mimicking culture conditions. In addition, the avirulence of cda1Δcda2Δ was followed by the induction of a protective immune response in C57BL/6 and CBA/J mice, when a live or heat-killed form of the mutant was used as a vaccine respectively. Taken together, these data imply that, in C. neoformans, coordinated activity of both Cda1 and Cda2 is essential for mediating fungal virulence.

Cryptococcus neoformans Chitin Synthase 3 Plays a Critical Role in Dampening Host Inflammatory Responses.

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human-pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan-deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan-deficient C. neoformans strain lacking three chitin deacetylases (cda1Δcda2Δcda3Δ) was found to confer protective immunity to a subsequent challenge with a virulent wild-type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase-mediated formation of chitosan. Mice inoculated with the chs3Δ strain at a dose previously shown to induce protection with the cda1Δcda2Δcda3Δ strain die within 36 h after installation of the organism. Mortality was not dependent on viable fungi, as mice inoculated with a heat-killed preparation of the chs3Δ strain died at the same rate as mice inoculated with a live chs3Δ strain, suggesting that the rapid onset of death was host mediated, likely caused by an overexuberant immune response. Histology, cytokine profiling, and flow cytometry indicate a massive neutrophil influx in the mice inoculated with the chs3Δ strain. Mice depleted of neutrophils survived chs3Δ inoculation, indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcus-induced host inflammatory responses.IMPORTANCECryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in ∼200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the host's immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different from the cell walls of many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study, we investigated the host responses to a chs3Δ strain, a chitosan-deficient strain, and found that mice inoculated with the chs3Δ strain all died within 36 h and that death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

Chitosan Biosynthesis and Virulence in the Human Fungal Pathogen Cryptococcus gattii.

Cryptococcus gattii R265 is a hypervirulent fungal strain responsible for the recent outbreak of cryptococcosis in Vancouver Island of British Columbia in Canada. It differs significantly from Cryptococcus neoformans in its natural environment, its preferred site in the mammalian host, and its pathogenesis. Our previous studies of C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains a two- to threefold larger amount of chitosan than that of C. neoformans The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDAs) have, however, been modified. To deduce their roles, single and double CDA deletion strains and a triple CDA deletion strain were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in strain R265, Cda3 is critical for virulence. Deletion of either CDA3 alone or in combination with another CDA (cda1Δ3Δ or cda2Δ3Δ) or both (cda1Δ2Δ3Δ) rendered the fungus avirulent and cleared from the infected host. Moreover, the cda1Δ2Δ3Δ strain of R265 induced a protective response to a subsequent infection with R265. These studies begin to illuminate the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.IMPORTANCE The fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offer new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

Novel Synthetic Polyamines Have Potent Antimalarial Activities in vitro and in vivo by Decreasing Intracellular Spermidine and Spermine Concentrations.

Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.

Cryptococcus neoformans Cda1 and Its Chitin Deacetylase Activity Are Required for Fungal Pathogenesis.

Chitin is an essential component of the cell wall of Cryptococcus neoformans conferring structural rigidity and integrity under diverse environmental conditions. Chitin deacetylase genes encode the enyzmes (chitin deacetylases [Cdas]) that deacetylate chitin, converting it to chitosan. The functional role of chitosan in the fungal cell wall is not well defined, but it is an important virulence determinant of C. neoformans Mutant strains deficient in chitosan are completely avirulent in a mouse pulmonary infection model. C. neoformans carries genes that encode three Cdas (Cda1, Cda2, and Cda3) that appear to be functionally redundant in cells grown under vegetative conditions. Here we report that C. neoformans Cda1 is the principal Cda responsible for fungal pathogenesis. Point mutations were introduced in the active site of Cda1 to generate strains in which the enzyme activity of Cda1 was abolished without perturbing either its stability or localization. When used to infect CBA/J mice, Cda1 mutant strains produced less chitosan and were attenuated for virulence. We further demonstrate that C. neoformans Cda genes are transcribed differently during a murine infection from what has been measured in vitroIMPORTANCECryptococcus neoformans is unique among fungal pathogens that cause disease in a mammalian host, as it secretes a polysaccharide capsule that hinders recognition by the host to facilitate its survival and proliferation. Even though it causes serious infections in immunocompromised hosts, reports of infection in hosts that are immunocompetent are on the rise. The cell wall of a fungal pathogen, its synthesis, composition, and pathways of remodelling are attractive therapeutic targets for the development of fungicides. Chitosan, a polysaccharide in the cell wall of C. neoformans is one such target, as it is critical for pathogenesis and absent in the host. The results we present shed light on the importance of one of the chitin deacetylases that synthesize chitosan during infection and further implicates chitosan as being a critical factor for the pathogenesis of C. neoformans.

A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome.

C. neoformans is an encapsulated fungal pathogen with defined asexual and sexual life cycles. Due to the availability of genetic and molecular tools for its manipulation, it has become a model organism for studies of fungal pathogens, even though it lacks a reliable system for maintaining DNA fragments as extrachromosomal plasmids. To compensate for this deficiency, we identified a genomic gene-free intergenic region where heterologous DNA could be inserted by homologous recombination without adverse effects on the phenotype of the recipient strain. Since such a site in the C. neoformans genome at a different location has been named previously as "safe haven", we named this locus second safe haven site (SH2). Insertion of DNA into this site in the genome of the KN99 congenic strain pair caused minimal change in the growth of the engineered strain under a variety of in vitro and in vivo conditions. We exploited this 'safe' locus to create a genetically stable highly fluorescent strain expressing mCherry protein (KN99mCH); this strain closely resembled its wild-type parent (KN99α) in growth under a variety of in vitro stress conditions and in the expression of virulence traits. The efficiency of phagocytosis and the proliferation of KN99mCH inside human monocyte-derived macrophages were comparable to those of KN99α, and the engineered strain showed the expected organ dissemination after inoculation, although there was a slight reduction in virulence. The mCherry fluorescence allowed us to measure specific association of cryptococci with leukocytes in the lungs and mediastinal lymph nodes of infected animals and, for the first-time, to assess their live/dead status in vivo. These results highlight the utility of KN99mCH for elucidation of host-pathogen interactions in vivo. Finally, we generated drug-resistant KN99 strains of both mating types that are marked at the SH2 locus with a specific drug resistant gene cassette; these strains will facilitate the generation of mutant strains by mating.

Induction of Protective Immunity to Cryptococcal Infection in Mice by a Heat-Killed, Chitosan-Deficient Strain of Cryptococcus neoformans.

UNLABELLED: Cryptococcus neoformans is a major opportunistic fungal pathogen that causes fatal meningoencephalitis in immunocompromised individuals and is responsible for a large proportion of AIDS-related deaths. The fungal cell wall is an essential organelle which undergoes constant modification during various stages of growth and is critical for fungal pathogenesis. One critical component of the fungal cell wall is chitin, which in C. neoformans is predominantly deacetylated to chitosan. We previously reported that three chitin deacetylase (CDA) genes have to be deleted to generate a chitosan-deficient C. neoformans strain. This cda1Δ2Δ3Δ strain was avirulent in mice, as it was rapidly cleared from the lungs of infected mice. Here, we report that clearance of the cda1Δ2Δ3Δ strain was associated with sharply spiked concentrations of proinflammatory molecules that are known to be critical mediators of the orchestration of a protective Th1-type adaptive immune response. This was followed by the selective enrichment of the Th1-type T cell population in the cda1Δ2Δ3Δ strain-infected mouse lung. Importantly, this response resulted in the development of robust protective immunity to a subsequent lethal challenge with a virulent wild-type C. neoformans strain. Moreover, protective immunity was also induced in mice vaccinated with heat-killed cda1Δ2Δ3Δ cells and was effective in multiple mouse strains. The results presented here provide a strong framework to develop the cda1Δ2Δ3Δ strain as a potential vaccine candidate for C. neoformans infection. IMPORTANCE: The most commonly used anticryptococcal therapies include amphotericin B, 5-fluorocytosine, and fluconazole alone or in combination. Major drawbacks of these treatment options are their limited efficacy, poor availability in limited resource areas, and potential toxicity. The development of antifungal vaccines and immune-based therapeutic interventions is promising and an attractive alternative to chemotherapeutics. Currently, there are no fungal vaccines in clinical use. This is the first report of a C. neoformans deletion strain with an avirulent phenotype in mice exhibiting protective immunity when used as a vaccine after heat inactivation, although other strains that overexpress fungal or murine proteins have recently been shown to induce a protective response. The data presented here demonstrate the potential for developing the avirulent cda1Δ2Δ3Δ strain into a vaccine-based therapy to treat C. neoformans infection.

Intracellular Action of a Secreted Peptide Required for Fungal Virulence.

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence.

Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division.

UNLABELLED: The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (Δprmt1) by deletion of the PRMT1 gene. Δprmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the Δprmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, Δprmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the Δprmt1 strain relative to the complemented Δprmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1's primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite. IMPORTANCE: Apicomplexan parasites include several important pathogens, including Toxoplasma gondii, a major cause of opportunistic infections and congenital birth defects. These parasites divide using a unique form of cell division called endodyogeny that is different from those of most eukaryotes. PRMT1 is a conserved arginine methyltransferase that was thought to regulate gene expression of T. gondii by modifying histone methylation. Using genetic techniques, we show that disruption of PRMT1 affects the parasite's ability to perform accurate cell division. Our studies reveal an unexpected role for arginine methylation in centrosome biology and regulation of parasite replication.

Cross talk between the cell wall integrity and cyclic AMP/protein kinase A pathways in Cryptococcus neoformans.

UNLABELLED: Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis. IMPORTANCE: Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

Cryptococcus at work: gene expression during human infection.

Meningitis is a frequent manifestation of infection due to Cryptococcus neoformans and a major cause of increased morbidity in patients with AIDS. Numerous in vitro gene expression and genetic studies of the fungus have predicted a myriad of genes, pathways, and biological processes that may be critical for pathogenesis, and many studies using animal models have supported the role of these processes during infection. However, the relevance of these hypotheses based on in vitro and animal models has often been questioned. A recent study by Chen et al. [Y. Chen, D. L. Toffaletti, J. L. Tenor, A. P. Litvintseva, C. Fang, T. G. Mitchell, T. R. McDonald, K. Nielsen, D. R. Boulware, T. Bicanic, and J. R. Perfect, mBio 5(1):e01087-13, 2014] represents an important step in understanding the cryptococcal response during human infection.

Sulphiredoxin plays peroxiredoxin-dependent and -independent roles via the HOG signalling pathway in Cryptococcus neoformans and contributes to fungal virulence.

Mechanisms of oxidative stress resistance are crucial virulence factors for survival and proliferation of fungal pathogens within the human host. In this study we have identified and functionally characterized the role of sulphiredoxin, Srx1, in oxidative stress resistance of Cryptococcus neoformans causing fungal meningoencephalitis and regulation of peroxiredoxins, Tsa1 and Tsa3, and thioredoxins, Trx1 and Trx2. The C. neoformans HOG (High Osmolarity Glycerol response) pathway was essential for the transcriptional regulation of SRX1 under peroxide stress conditions. A gene deletion study revealed that Srx1 was required for cells to counteract peroxide stress, but not other oxidative damaging agents. HOG1 was found to be essential for the induction of adaptive response to peroxide stress with concurrent repression of ergosterol biosynthesis in an SRX1-independent manner. Consistent with this, phosphorylation of C. neoformans Hog1 was modulated by both low and high doses of exogenous hydrogen peroxide treatment. Immunoblot analysis using the C. neoformans Tsa1 specific antibody revealed that both Srx1 and Trx1 were essential for recycling of oxidized Tsa1. In addition to its role in peroxide sensing and response C. neoformans Srx1 was also found to be required for a peroxiredoxin-independent function in promoting fungicide-dependent cell swelling and growth arrest. Finally we showed the importance of C. neoformans Srx1 in fungal pathogenesis by demonstrating its requirement for full virulence using a mouse infection model.

Global transcriptome profile of Cryptococcus neoformans during exposure to hydrogen peroxide induced oxidative stress.

The ability of the opportunistic fungal pathogen Cryptococcus neoformans to resist oxidative stress is one of its most important virulence related traits. To cope with the deleterious effect of cellular damage caused by the oxidative burst inside the macrophages, C. neoformans has developed multilayered redundant molecular responses to neutralize the stress, to repair the damage and to eventually grow inside the hostile environment of the phagosome. We used microarray analysis of cells treated with hydrogen peroxide (H(2)O(2)) at multiple time points in a nutrient defined medium to identify a transcriptional signature associated with oxidative stress. We discovered that the composition of the medium in which fungal cells were grown and treated had a profound effect on their capacity to degrade exogenous H(2)O(2). We determined the kinetics of H(2)O(2) breakdown by growing yeast cells under different conditions and accordingly selected an appropriate media composition and range of time points for isolating RNA for hybridization. Microarray analysis revealed a robust transient transcriptional response and the intensity of the global response was consistent with the kinetics of H(2)O(2) breakdown by treated cells. Gene ontology analysis of differentially expressed genes related to oxidation-reduction, metabolic process and protein catabolic processes identified potential roles of mitochondrial function and protein ubiquitination in oxidative stress resistance. Interestingly, the metabolic pathway adaptation of C. neoformans to H(2)O(2) treatment was remarkably distinct from the response of other fungal organisms to oxidative stress. We also identified the induction of an antifungal drug resistance response upon the treatment of C. neoformans with H(2)O(2). These results highlight the complexity of the oxidative stress response and offer possible new avenues for improving our understanding of mechanisms of oxidative stress resistance in C. neoformans.

Response of adipose tissue to early infection with Trypanosoma cruzi (Brazil strain).

Brown adipose tissue (BAT) and white adipose tissue (WAT) and adipocytes are targets of Trypanosoma cruzi infection. Adipose tissue obtained from CD-1 mice 15 days after infection, an early stage of infection revealed a high parasite load. There was a significant increase in macrophages in infected adipose tissue and a reduction in lipid accumulation, adipocyte size, and fat mass and increased expression of lipolytic enzymes. Infection increased levels of Toll-like receptor (TLR) 4 and TLR9 and in the expression of components of the mitogen-activated protein kinase pathway. Protein and messenger RNA (mRNA) levels of peroxisome proliferator-activated receptor γ were increased in WAT, whereas protein and mRNA levels of adiponectin were significantly reduced in BAT and WAT. The mRNA levels of cytokines, chemokines, and their receptors were increased. Nuclear Factor Kappa B levels were increased in BAT, whereas Iκκ-γ levels increased in WAT. Adipose tissue is an early target of T. cruzi infection.

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1-5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.