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Background/Purpose In 2004, a heritable occurrence of spina bifida was reported in sheep on a farm in the United States. We maintained and characterized the spina bifida phenotype in this flock to assess its potential as an alternative surgical model. Methods A breeding strategy was developed in which the sheep were crossed to maintain or increase the occurrence of spina bifida. Measurements and observations were recorded regarding lesion size, birthweight, ambulatory capacity, or urological function, and necropsies were performed on spina bifida afflicted lambs in conjunction with magnetic resonance imaging to determine the character of the spina bifida defects and assess the presence of Chiari-like malformations or hydrocephalus. Results The defects were observed to be more prevalent in ram lambs, and the rate of spina bifida per litter could be increased through backcrossing or by selection of a productive ewe breed. The lambs displayed a range of ambulatory and urological deficits which could be used to evaluate new fetal repair methodologies. Finally, affected lambs were shown to demonstrate severe Chiari malformations and hydrocephalus. Conclusions We have determined that use of these sheep as a natural source for spina bifida fetuses is feasible and could supplement the deficits of current sheep models for myelomeningocele repair. Level of evidence Level IV.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a deadly infection for which new antifungal therapies are needed. Here, we demonstrate the efficacy of a Gwt1 inhibitor, APX2041, and its prodrug, APX2104, against A. fumigatus. The wild-type, azole-resistant, and echinocandin-resistant A. fumigatus strains were equally susceptible to APX2041 in vitro. APX2104 treatment in vivo significantly prolonged survival of neutropenic mice challenged with the wild-type and azole-resistant strains, revealing APX2104 as a potentially promising therapeutic against IA.

PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies., Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.

We previously observed a substantial burden of cryptococcal meningitis in Vietnam atypically arising in HIV-uninfected individuals. This disease was associated with a single genotype ofCryptococcus neoformans(Sequence Type (ST)5), which was significantly less common in HIV-infected individuals. Aiming to compare the phenotypic characteristics of ST5 and non-ST5 C. neoformans we selected 30 representative Vietnamese isolates, compared theirin vitropathogenic potential andin vivovirulence. ST5 and non-ST5 organisms exhibited comparable characteristics with respect toin vitrovirulence markers including melanin production, replication at 37°C, and growth in cerebrospinal fluid. However, the ST5 isolates had significantly increased variability in cellular and capsular sizing compared with non-ST5 organisms (pp

Appendices for publication:Shwab EK, Juvvadi PR, Waitt G, Shaheen S, Allen J, IV, Soderblom EJ, Bobay BG, Asfaw YG, Moseley MA, Steinbach WJ. 2020. The protein kinase A-dependent phosphoproteome of the human pathogen Aspergillus fumigatus reveals diverse virulenceassociated kinase targets. mBio 11:e02880-20.

The procedures necessary to perform testing in a veterinary diagnostic laboratory have inherent associated risks to personnel in regard to exposure to infectious agents. In research institutions animals can be experimentally infected, acquire naturally occurring infections and can also be exposed to other hazards such as toxic chemicals or radiologic entities. A critical component of the use of animals in a research environment is the collaboration between the responsible researcher and the veterinary diagnostic laboratory with the institutional health and safety professionals to ensure that the proper engineering controls, personal protective equipment, laboratory procedures and training are in place for personnel working with the animals or their specimens. Unlike the typical researcher, the veterinary diagnostic laboratory generally has to be equipped to safely process and work with a wide range of potential hazards where the communication of pertinent information from the researcher to the diagnostic laboratory regarding the identity of the potential hazard is paramount. Diagnostic laboratory design, safety equipment, personal protective equipment, laboratory procedures, occupational health program and personnel training must be sufficient to address hazards based on a risk assessment performed in conjunction with safety professionals. This article will summarize safety considerations with the various areas of concern in the operation of a diagnostic laboratory for research animal specimens.

Background Aspergillus fumigatus is the leading cause of invasive aspergillosis (IA), a lethal infection among immunocompromised patients. Guideline-recommended antifungal therapy against IA is a triazole antifungal, with other secondary options including an echinocandin and amphotericin B. Concerns about drug-host toxicity and antifungal resistance have been globally reported, so new, safe, and effective therapeutics are imperative. Methods In vitro, CLSI standards were upheld as we tested APX2041, voriconazole, caspofungin, and amphotericin B against various A. fumigatus strains. In vivo we assessed toxicity and efficacy of APX2104 in immunocompromised mice respectively. Neutropenia was induced with 150 mg/kg of cyclophosphamide on days -2/+3 and 250 mg/kg of cortisone acetate on days -1/+6. Immunocompromised mice were infected in an inhalation chamber via 12 mL of aerosolized spores of A. fumigatus CEA10 at a concentration of 1x109 spores/mL (Day 0). Treatment started day +1 and ended day +7. Results In vitro, APX2041, the active-form of APX2104, has over a 16-fold lower minimum effective concentration (MEC) when compared to voriconazole, caspofungin, and amphotericin B against various A. fumigatus strains, including echinocandin- and azole-resistant strains. In vivo, given preliminary pharmacokinetic data, APX2104 was tested in non-infected immunocompromised mice at 60 mg/kg and 78 mg/kg once per day (QD). Deaths due to toxicity were seen only at a dose of 78 mg/kg, so 60 mg/kg was set as a safe dose for our in vivo efficacy studies. In IA-challenged neutropenic mice, treatment with either posaconazole (20 mg/kg BID) or APX2104 (60 mg/kg QD) equally prolonged survival in 14 of 15 (93%) mice 14 days post-infection (p= 0.985). Untreatment control yielded a survival of 3 of 15 (20%) 14 days post-infection (p= < 0.001). Consistent with our survival studies, H&E and GMS histological samples also demonstrated that APX2104 treatment decreased fungal burden within the lungs of neutropenic mice when compared to the untreated group. Conclusion Future studies will test the efficacy of APX2104 and posaconazole against azole antifungal resistant strains in vivo, as our preliminary findings suggest that APX2104 is a plausible solution to cure IA disease and combat antifungal resistance. Disclosures All Authors: No reported disclosures

Calcium signaling through calcineurin and its major transcription factor, CrzA, is integral to hyphal growth, stress response, and virulence of the pathogenic fungus Aspergillus fumigatus, the leading etiology of invasive aspergillosis. Dephosphorylation of CrzA by calcineurin activates the transcription factor, but the specific phosphorylation sites and their roles in the activation/inactivation mechanism are unknown. Mass spectroscopic analysis identified twenty phosphorylation sites, the majority of which were specific to filamentous fungi and distributed throughout the CrzA protein, with particular concentration in a serine-rich region N-terminal to the conserved DNA-binding domain (DBD). Site-directed mutagenesis of phosphorylated residues revealed that CrzA activity during calcium stimulation can only be suppressed by a high degree of phosphorylation in multiple regions of the protein. Our findings further suggest that this regulation is not solely accomplished through control of CrzA nuclear import. Additionally, we demonstrate the importance of the CrzA phosphorylation state in regulating growth, conidiation, calcium and cell-wall stress tolerance, and virulence. Finally, we identify two previously undescribed nuclear localization sequences in the DBD. These findings provide novel insight into the phosphoregulation of CrzA which may be exploited to selectively target A. fumigatus.

Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans . However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin ( myoB ) is dispensable for growth, the class V myosin ( myoE ) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the Δ myoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The Δ myoE strain showed fungal growth in the murine lung, while the Δ myoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies.

BACKGROUND: Glycogen storage disease type Ia (GSD Ia) in dogs closely resembles human GSD Ia. Untreated patients with GSD Ia develop complications associated with glucose-6-phosphatase (G6Pase) deficiency. Survival of human patients on intensive nutritional management has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatocellular adenomas (HCA), and a high risk for hepatocellular carcinoma (HCC). Affected dogs fail to thrive with dietary therapy alone. Treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia in affected dogs. However, long-term complications have not been described to date. METHODS: Five GSD Ia affected dogs treated with AAV-G6Pase were evaluated. Dogs were euthanized due to reaching humane endpoints related to liver and/or kidney involvement, at 4 to 8 years of life. Necropsies were performed and tissues were analyzed. RESULTS: Four dogs had liver tumors consistent with HCA and HCC. Three dogs developed renal failure, but all dogs exhibited progressive kidney disease histologically. Urolithiasis was detected in two dogs; uroliths were composed of calcium oxalate and calcium phosphate. One affected and one carrier dog had polycystic ovarian disease. Bone mineral density was not significantly affected. CONCLUSIONS: Here we show that the canine GSD Ia model demonstrates similar long-term complications as GSD Ia patients in spite of gene replacement therapy. Further development of gene therapy is needed to develop a more effective treatment to prevent long-term complications of GSD Ia.

Heat shock protein 90 (Hsp90) is an essential chaperone involved in the fungal stress response that can be harnessed as a novel antifungal target for the treatment of invasive aspergillosis. We previously showed that genetic repression of Hsp90 reduced Aspergillus fumigatus virulence and potentiated the effect of the echinocandin caspofungin. In this study, we sought to identify sites of posttranslational modifications (phosphorylation or acetylation) that are important for Hsp90 function in A. fumigatus . Phosphopeptide enrichment and tandem mass spectrometry revealed phosphorylation of three residues in Hsp90 (S49, S288, and T681), but their mutation did not compromise Hsp90 function. Acetylation of lysine residues of Hsp90 was recovered after treatment with deacetylase inhibitors, and acetylation-mimetic mutations (K27A and K271A) resulted in reduced virulence in a murine model of invasive aspergillosis, supporting their role in Hsp90 function. A single deletion of lysine K27 or an acetylation-mimetic mutation (K27A) resulted in increased susceptibility to voriconazole and caspofungin. This effect was attenuated following a deacetylation-mimetic mutation (K27R), suggesting that this site is crucial and should be deacetylated for proper Hsp90 function in antifungal resistance pathways. In contrast to previous reports in Candida albicans , the lysine deacetylase inhibitor trichostatin A (TSA) was active alone against A. fumigatus and potentiated the effect of caspofungin against both the wild type and an echinocandin-resistant strain. Our results indicate that the Hsp90 K27 residue is required for azole and echinocandin resistance in A. fumigatus and that deacetylase inhibition may represent an adjunctive anti- Aspergillus strategy.

Septins are a conserved family of GTPases that form hetero-oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspB (mt) -T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A. fumigatus hyphal septation.

Ras is a highly conserved GTPase protein that is essential for proper polarized morphogenesis offilamentous fungi. Localization of Ras proteins to the plasma membrane and endomembranes through posttranslational addition of farnesyl and palmitoyl residues is an important mechanism through which cells provide specificity to Ras signal output. Although the Aspergillus fumigatusRasA protein is known to be a major regulator of growth and development, the membrane distribution of RasA during polarized morphogenesis and the role of properly localized Ras signaling in virulence of a pathogenic mold remain unknown. Here we demonstrate that Aspergillus fumigatus RasA localizes primarily to the plasma membrane of actively growing hyphae. We show that treatment with the palmitoylation inhibitor 2-bromopalmitate disrupts normal RasA plasma membrane association and decreases hyphal growth. Targeted mutations of the highly conserved RasA palmitoylation motif also mislocalized RasA from the plasma membrane and led to severe hyphal abnormalities, cell wall structural changes, and reduced virulence in murine invasive aspergillosis. Finally, we provide evidence that proper RasA localization is independent of the Ras palmitoyltransferase homolog, encoded by erfB, but requires the palmitoyltransferase complex subunit, encoded by erfD. Our results demonstrate that plasma membrane-associated RasA is critical for polarized morphogenesis, cell wall stability, and virulence in A. fumigatus. T heRasGTPasesaremembrane-associatedbinaryswitchesthat controltheactivityofamultitudeofsignaltransductionpathways.SpecificityofRassignaloutputoccursinpartviasubcellular localization of the Ras protein to various membranes, as well as through compartmentalization to specific regions with the membrane (23, 37). Differential localization of Ras proteins is accomplished through posttranslational modification of the hypervariable domain, the region in which Ras homologs are most divergent. The hypervariable domains of nascent Ras proteins contain an extreme C-terminal “CAAX” motif that serves as a recognition sequence for cytoplasmic farnesyltransferase (5). The farnesyltransferase enzyme modifies the CAAX motif by addition of a farnesyl lipid moiety to the cysteine residue. Farnesyl modification has been shown to be necessary and sufficient for direction of the Ras protein to the endoplasmic reticulum (ER) and Golgi membranes(5).Oncelocalizedtotheendomembranesystem,Ras

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca 2+ signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed. In this study, we investigate the role of CbpA, the Aspergillus fumigatus member of the calcipressin family, and report that deletion of the cbpA gene resulted in reduced hyphal growth and limited attenuated virulence. Interestingly, under high-calcium-level conditions, the Δ cbpA strain displayed improved Ca 2+ tolerance compared to the wild-type strain and revealed increased expression of vcxA , chsA , and cnaA , which encode the vacuolar Ca 2+ /H + exchanger VcxA, chitin synthase A, and the calcineurin catalytic subunit CnaA, respectively. The increased transcript levels of these three genes were reversed in the presence of the calcineurin inhibitor FK506, indicating a calcineurin-dependent mechanism. Overexpression of cbpA resulted in decreased transcription of vcxA , chsA , and cnaA , associated with wild-type sensitivity to Ca 2+ . Taken together, our study highlights the importance of CbpA in the regulation of hyphal growth and calcium adaptation of A. fumigatus and provides evidence that CbpA may serve as a feedback inhibitor in some aspects of calcineurin functions.

Glycogen storage disease type Ia (GSD-Ia) in dogs closely resembles human GSD-Ia where untreated patients develop complications associated with glucose-6-phosphatase (G6Pase) deficiency (hypoglycemia, hyperlipidemia, growth retardation, and early death). For the past 3 decades survival of human patients that are placed under intensive nutritional management with uncooked corn starch has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatic adenomas, and a high risk for hepatocellular carcinoma. Affected dogs fail to thrive with dietary therapy alone, but treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia. In this study, 7 GSD-Ia affected dogs treated with AAV-G6Pase were followed up to 8 years of life to describe long-term complications. All required readministration of AAV vector(s) pseudotyped as a new serotype to avoid anti-AAV antibodies, due to decreased ability to maintain normoglycemia during fasting. Two dogs developed chronic renal disease as denoted by polyuria, polydipsia, and azotemia at 6 and 8 years of age. Calcium oxalate and phosphate urolithiasis was detected in 2 dogs; 1 dog had evidence of polycystic ovarian disease. Four of the 7 dogs were euthanized due to reaching humane endpoints related to liver and/or kidney disease, at 3 to 8 years of life, and 1 dog succumbed to acute hypoglycemia. Necropsy revealed several focal hepatic lesions in all deceased dogs; 2 dogs had lesions confirmed histologically as hepatocellular carcinoma, the largest tumor reached 8 cm in diameter (Fig. 1). One dog demonstrated lesions consistent with hepatocellular adenoma and in the other 2, there was hepatocellular hyperplasia. There was no significant difference in amount of vector DNA in hepatic tumors compared with normal tissue (n=4 dogs), suggesting that insertional mutagenesis by the AAV vector was not the mechanism for tumor formation (Fig. 2). View Large Image | Download PowerPoint Slide View Large Image | Download PowerPoint SlideGlomerular sclerosis, fibrosis of Bowman's capsules and focal interstitial nephritis were found in the 2 dogs with clinical renal disease. Two dogs, both female, remain in good health with no evidence of liver masses appreciable on ultrasound examination; they are 4 and 5 years of age. Hepatocellular carcinoma, kidney disease, polycystic ovaries and urolithiasis are common findings among GSD-Ia patients. Here we show that the canine GSD-Ia model demonstrates similar long-term complications in spite of AAV-mediated gene replacement therapy. Further development of gene therapy is needed to prevent long-term complications of GSD-Ia.

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A ( cnaA ) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The Δ cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the Δ cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the Δ cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the Δ cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

Background Respiratory exposure to crystalline silica in man is associated with autoimmunity that includes a prominent humoral component. We hypothesize that autoimmune B cell recruitment and regulation are altered in silica-exposed lungs in genetically susceptible individuals. Methods Wildtype and autoAb transgenic (Tg+) mice of B6 and lupus-prone NZB, MRL, and BXSB backgrounds were exposed to silica dust or vehicle by oropharyngeal aspiration. After 1–3 months, lung pathology and lymphocytic infiltrates were scored. Lupus and vasculitis autoAb levels were measured in bronchoalveolar lavage fluid (BALF), serum, and supernatants from cultured lung and spleen cells. Results All silica-exposed strains showed extensive lung pathology and focal B and T cell infiltrates consistent with ectopic lymphoid structures (ELS). In wildtype mice, % lung area containing ELS after silica exposure varied by strain (BXSB>MRL>B6>NZB, p=0.03). Significant increases in autoAb production with silica exposure were observed in BALF, lung cell supernatants, and serum, and varied by genetic background. Among Tg+ mice, Tg+ Ig levels in BALF were higher in silica- vs vehicle-exposed mice in B6 and BXSB strains (p Conclusions Respiratory exposure to silica leads to pulmonary B and T cell accumulation with ELS formation and enhanced autoAb production. This occurs in a strain-dependent manner, highlighting the impact of genetic influences on silica-induced autoimmunity. Evidence of local autoAb secretion implicates dysregulated control of autoreactive B cells at the environment/lung interface, possibly through the intermediary of silica-induced ELS.

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans . Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans . This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D −/− mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D −/− mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D −/− mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans -driven pulmonary IL-5 and eosinophil infiltration.

Invasive aspergillosis is a deadly infection for which new antifungal therapies are needed. Heat shock protein 90 (Hsp90) is an essential chaperone in Aspergillus fumigatus representing an attractive antifungal target. Using a thiamine-repressible promoter (pthiA), we showed that genetic repression of Hsp90 significantly reduced virulence in a murine model of invasive aspergillosis. Moreover, substituting the A. fumigatus hsp90 promoter with 2 artificial promoters (potef, pthiA) and the Candida albicans hsp90 promoter resulted in hypersensitivity to caspofungin and abolition of the paradoxical effect (resistance at high caspofungin concentrations). By inducing truncations in the hsp90 promoter, we identified a 100-base pair proximal sequence that triggers a significant increase of hsp90 expression (≥1.5-fold) and is essential for the paradoxical effect. Preventing this increase of hsp90 expression was sufficient to abolish the paradoxical effect and therefore optimize the antifungal activity of caspofungin.

The Aspergillus fumigatus Δ pmrA (Golgi apparatus Ca 2+ /Mn 2+ P-type ATPase) strain has osmotically suppressible basal growth defects and cationic tolerance associated with increased expression of calcineurin pathway genes. Despite increased β-glucan and chitin content, it is hypersensitive to cell wall inhibitors but remains virulent, suggesting a role for PmrA in cation homeostasis and cell wall integrity.

Invasive pulmonary aspergillosis (IPA) is a devastating disease characterized by extensive invasion of lung tissue in immunocompromised patients by hyphae of saprophytic molds in the genus Aspergillus (26, 48, 61). As the number of immunocompromised patients continues to rise with advances in medical technology, there has been a concomitant increase in the number of patients with IPA (29). Despite expanding treatment options for IPA, current therapy still has a dismal 40 to 50% success rate (19, 28). Thus, the development of new antifungal therapies to augment existing treatment strategies is urgently needed. One potential antifungal drug target in Aspergillus species is the calcineurin pathway (55). The importance of the calcineurin signaling pathway in fungal physiology is vividly demonstrated by gene expression analyses in Saccharomyces cerevisiae that identified 163 genes regulated by the calcineurin/Crz1 signaling pathway. These regulated genes were involved in ion/small-molecule transport, signal transduction, cell wall maintenance, and vesicular transport, indicating the important role that this pathway plays in fungal physiology (66). Unlike the case for many new candidate antifungal drug targets, drugs that target the calcineurin pathway already exist. The calcineurin inhibitors FK506 and cyclosporine, which are used routinely to inhibit human calcineurin and have revolutionized modern organ transplantation, exhibit antifungal activity in vitro, indicating their potential as therapeutic agents for IPA (3, 20, 54, 56, 57). In addition, our group and another independent research group have shown that deletion of the gene encoding the calcineurin A catalytic subunit in Aspergillus fumigatus resulted in a strain with significant defects in hyphal growth. Furthermore, these ΔcnaA strains are no longer capable of causing IPA in distinct experimental murine models (14, 53). The importance of the calcineurin pathway in fungal physiology and the dramatic phenotypes associated with pharmacologic inhibition and genetic deletions of this pathway make it an attractive antifungal drug target. However, the critical role of calcineurin in mammalian immune system responses brings into question the feasibility of directly targeting fungal calcineurin pathways in vivo with existing calcineurin inhibitors (8, 27, 34, 36). The impact of calcineurin inhibition on the host, via immunosuppression, may be stronger than its antifungal activity, with the net result being exacerbation of the disease. Thus, further studies are needed to identify fungus-specific components of the calcineurin signaling pathway that are not present in mammals. In this study, we characterize a calcineurin effector protein, CrzA, which is not present in mammals but is integrally linked with calcineurin-mediated signal transduction in many fungi (12, 21, 23, 43, 50). In S. cerevisiae, calcineurin-dependent transcription is regulated in part by the zinc finger transcription factor Crz1 (30, 50). Calcineurin regulates Crz1 activity via dephosporylation of Crz1, which results in nuclear localization of cytoplasmic Crz1 and requires the karyopherin Nmd5 (39, 51). S. cerevisiae Δcrz1 mutants display many phenotypes, including growth and viability defects, that are similar to those of S. cerevisiae calcineurin mutants (30, 50). Additional downstream effectors of calcineurin have also been identified. In S. cerevisiae, Hph1 and Hph2 have been shown to be novel calcineurin-regulated response components and Crz1-independent downstream effectors of calcineurin (18). Additional downstream effectors of calcineurin are also likely to exist in the opportunistic human fungal pathogen Candida albicans. C. albicans crz1 mutant strains display a pathobiologic phenotype different from that of C. albicans calcineurin mutants (23, 37, 43). For example, C. albicans Δcrz1/Δcrz1 mutant strains, unlike C. albicans calcineurin mutant strains, are not sensitive to serum and do not display a decrease in virulence in murine models of candidiasis. These results suggest the presence of additional calcineurin downstream effectors in this pathogenic yeast. However, in the pathogenic mold A. fumigatus downstream calcineurin effectors have not been identified or functionally characterized. In this study, we identified the A. fumigatus Crz1 homolog, CrzA, and tested the hypothesis that CrzA is a primary downstream effector of the calcineurin pathway in A. fumigatus. Our results suggest that CrzA is an important downstream effector for calcineurin in A. fumigatus but that additional calcineurin downstream effectors also exist in this mold. We observed that CrzA is critical for in vivo fungal growth, development, and pathogenicity and thus is an excellent antifungal drug target for treatment of invasive aspergillosis due to its role in pathogenesis and lack of a conserved mammalian homolog.

The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis., Author Summary Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into tangible clinical benefit has been difficult due to the fact that fungal pathogens and their hosts have similar physiology. The calcineurin pathway is an important signaling cascade in all eukaryotes, and calcineurin inhibitors are powerful immunosuppressants that have revolutionized medicine. Through both genetic and pharmacologic inhibition, we have established that calcineurin is vital for invasive fungal disease. Although the currently available calcineurin inhibitors are active in vitro against the major invasive fungal pathogens, they are also immunosuppressive in the host, limiting therapeutic effectiveness. Here we defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi that is responsible for virulence in the opportunistic human pathogen, Aspergillus fumigatus. This phosphorylation occurs on a cluster of four serine residues located in a unique serine-proline rich domain of calcineurin that is absent in humans. This finding of a new fungal-specific mechanism controlling hyphal growth and virulence represents a new potential target for antifungal drug therapy.