Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.
G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein-coupled receptors (GPCRs) and negatively controlled by regulator of G protein signaling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein α subunits (Gpa1, Gpa2, and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1-cAMP signaling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell-cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1Q284L enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1-cAMP-virulence and pheromone-mating signaling cascades and hypothesize it may serve as a molecular interface between these two central signaling conduits.
G protein-coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors, and are primary targets for drug development. A variety of detection systems have been reported to study ligand-GPCR interactions. Using Saccharomyces cerevisiae to express foreign proteins has long been appreciated for its low cost, simplicity, and conserved cellular pathways. The yeast pheromone responsive pathway has been utilized to assess a range of different GPCRs. We have identified a pheromone-like receptor, Cpr2, that is located outside of the MAT locus in the human fungal pathogen Cryptococcus neoformans. To characterize its function and potential ligands, we expressed CPR2 in a yeast heterologous expression system. To optimize for CPR2 expression in this system, pheromone receptor Ste3, regulator of G protein signaling (RGS) Sst2, and the cyclin-dependent kinase inhibitor Far1 were mutated. The lacZ gene was fused with the promoter of the FUS1 gene that is activated by the yeast pheromone signal and then introduced into yeast cells. Expression of CPR2 in this yeast heterologous expression system revealed that Cpr2 could activate the pheromone responsive pathway without additional of potential ligands, suggesting it is a naturally-occurring, constitutively active receptor. Mutation of a single amino acid, Leu222, was sufficient to reverse the constitutive activity of Cpr2. In this report, we summarize methods used for assessing the constitutive activity of Cpr2 and its mutants, which could be beneficial for other GPCR studies.
Although routinely done, there has been no evaluation of the utility of performing routine cerebrospinal fluid (CSF) examination in patients with active coccidioidomycosis and high complement fixation (IgG) antibody titers or other risk factors for disseminated infection. In our review 100% of patients diagnosed with coccidioidal meningitis had at least one sign or symptom consistent with infection of the central nervous system, headache was present in 100% of those with meningitis, while no patients without signs/symptoms of CNS infection were found to have coccidioidal meningitis, irrespective of antibody titers or other risk factors. Thus routine lumbar puncture may be unnecessary for patients with coccidioidomycosis who lack suggestive clinical symptoms.
Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.
Cryptococcus neoformans is the most common cause of fungal meningitis, with high mortality and morbidity. The reason for the frequent occurrence of Cryptococcus infection in the central nervous system (CNS) is poorly understood. The facts that human and animal brains contain abundant inositol and that Cryptococcus has a sophisticated system for the acquisition of inositol from the environment suggests that host inositol utilization may contribute to the development of cryptococcal meningitis. In this study, we found that inositol plays an important role in Cryptococcus traversal across the blood-brain barrier (BBB) both in an in vitro human BBB model and in in vivo animal models. The capacity of inositol to stimulate BBB crossing was dependent upon fungal inositol transporters, indicated by a 70% reduction in transmigration efficiency in mutant strains lacking two major inositol transporters, Itr1a and Itr3c. Upregulation of genes involved in the inositol catabolic pathway was evident in a microarray analysis following inositol treatment. In addition, inositol increased the production of hyaluronic acid in Cryptococcus cells, which is a ligand known to binding host CD44 receptor for their invasion. These studies suggest an inositol-dependent Cryptococcus traversal of the BBB, and support our hypothesis that utilization of host-derived inositol by Cryptococcus contributes to CNS infection.
Cryptococcus neoformans is an AIDS-associated human fungal pathogen that annually causes over 1 million cases of meningitis world-wide, and more than 600,000 attributable deaths. Cryptococcus often causes lung and brain infection and is the leading cause of fungal meningitis in immunosuppressed patients. Why Cryptococcus frequently infects the central nervous system to cause fatal meningitis is an unanswered critical question. Our previous studies revealed a sophisticated inositol acquisition system in Cryptococcus that plays a central role in utilizing environmental inositol to complete its sexual cycle. Here we further demonstrate that inositol acquisition is also important for fungal infection in the brain, where abundant inositol is available. We found that inositol promotes the traversal of Cryptococcus across the blood-brain barrier (BBB), and such stimulation is fungal inositol transporter dependent. We also identified the effects of host inositol on fungal cellular functions that contribute to the stimulation of fungal penetration of the BBB. We propose that inositol utilization is a novel virulence factor for CNS cryptococcosis. Our work lays an important foundation for understanding how fungi respond to available host inositol and indicates the impact of host inositol acquisition on the development of cryptococcal meningitis.
Cryptococcal induced visual loss is a devastating complication in survivors of cryptococcal meningitis (CM). Early detection is paramount in prevention and treatment. Subclinical optic nerve dysfunction in CM has not hitherto been investigated by electrophysiological means. We undertook a prospective study on 90 HIV sero-positive patients with culture confirmed CM. Seventy-four patients underwent visual evoked potential (VEP) testing and 47 patients underwent Humphrey's visual field (HVF) testing. Decreased best corrected visual acuity (BCVA) was detected in 46.5% of patients. VEP was abnormal in 51/74 (68.9%) right eyes and 50/74 (67.6%) left eyes. VEP P100 latency was the main abnormality with mean latency values of 118.9 (±16.5) ms and 119.8 (±15.7) ms for the right and left eyes respectively, mildly prolonged when compared to our laboratory references of 104 (±10) ms (p<0.001). Subclinical VEP abnormality was detected in 56.5% of normal eyes and constituted mostly latency abnormality. VEP amplitude was also significantly reduced in this cohort but minimally so in the visually unimpaired. HVF was abnormal in 36/47 (76.6%) right eyes and 32/45 (71.1%) left eyes. The predominant field defect was peripheral constriction with an enlarged blind spot suggesting the greater impact by raised intracranial pressure over that of optic neuritis. Whether this was due to papilloedema or a compartment syndrome is open to further investigation. Subclinical HVF abnormalities were minimal and therefore a poor screening test for early optic nerve dysfunction. However, early optic nerve dysfunction can be detected by testing of VEP P100 latency, which may precede the onset of visual loss in CM.
Background and Purpose
Successful outcomes from bacterial meningitis require rapid antibiotic treatment; however, unnecessary treatment of viral meningitis may lead to increased toxicities and expense. Thus, improved diagnostics are required to maximize treatment and minimize side effects and cost. Thirteen clinical decision rules have been reported to identify bacterial from viral meningitis. However, few rules have been tested and compared in a single study, while several rules are yet to be tested by independent researchers or in pediatric populations. Thus, simultaneous test and comparison of these rules are required to enable clinicians to select an optimal diagnostic rule for bacterial meningitis in settings and populations similar to ours.
A retrospective cross-sectional study was conducted at the Infectious Department of Pediatric Hospital Number 1, Ho Chi Minh City, Vietnam. The performance of the clinical rules was evaluated by area under a receiver operating characteristic curve (ROC-AUC) using the method of DeLong and McNemar test for specificity comparison.
Our study included 129 patients, of whom 80 had bacterial meningitis and 49 had presumed viral meningitis. Spanos's rule had the highest AUC at 0.938 but was not significantly greater than other rules. No rule provided 100% sensitivity with a specificity higher than 50%. Based on our calculation of theoretical sensitivity and specificity, we suggest that a perfect rule requires at least four independent variables that posses both sensitivity and specificity higher than 85–90%.
No clinical decision rules provided an acceptable specificity (>50%) with 100% sensitivity when applying our data set in children. More studies in Vietnam and developing countries are required to develop and/or validate clinical rules and more very good biomarkers are required to develop such a perfect rule.
The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown.
Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway.
Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the powerful competitive ability of plant cell wall degrading fungi in nature.
The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.
Many diseases are contracted from the environment, rather than from sick people. It is unclear why those species are able to cause disease, since the selective pressures in the environment are presumed to be very different from those found within the host. Cryptococcus neoformans is a fungus that causes life-threatening lung and central nervous system disease in approximately one million people each year. The fungus is inhaled from environmental sources. One hypothesis to account for C. neoformans virulence is that amoeba are predators for this fungus, and surviving strains are pre-selected to be virulent in the human host. On the other hand, experiments have found that amoeba eat C. neoformans. A pseudohyphal cell type can survive, and while protecting against amoeba these cells are unable to cause disease in mouse models. We predicted that the pseudohyphal morphology reflected a change in function of a pathway of genes, and found that all pseudohyphal isolates contain mutations within genes for this pathway. The pseudohyphal trait is unstable, with reversion to normal yeast growth by counter-acting mutations. These mutations can occur during the course of mammalian infection. Our results show that mutation events account for a microevolution system currently described as phenotypic switching, and that mutations, at least under experimental conditions, can regulate pathogen adaptation and influence its host range.
Casein kinases regulate a wide range of cellular functions in eukaryotes, including phosphorylation of proteins that are substrates for degradation via the ubiquitin-proteasome system (UPS). Our previous study demonstrated that Fbp1, a component of the SCFFBP1 E3 ligase complex, was essential for Cryptococcus virulence. Because the Saccharomyces cerevisiae homolog of Fbp1, Grr1, requires casein kinase I (Yck1 and Yck2) to phosphorylate its substrates, we investigated the function of casein kinase I in Cryptococcus neoformans. In this report, we identified a C. neoformans casein kinase I protein homolog, Cck1. Similar to Fbp1, the expression of Cck1 is negatively regulated by glucose and during mating. cck1 null mutants showed significant virulence attenuation in a murine systemic infection model, but Cck1 was dispensable for the development of classical virulence factors (capsule, melanin, and growth at 37°C). cck1 mutants were hypersensitive to SDS treatment, indicating that Cck1 is required for cell integrity. The functional overlap between Cck1 and Fbp1 suggests that Cck1 may be required for the phosphorylation of Fbp1 substrates. Interestingly, the cck1 mutant also showed increased sensitivity to osmotic stress and oxidative stress, suggesting that Cck1 regulates both cell integrity and the cellular stress response. Our results show that Cck1 regulates the phosphorylation of both Mpk1 and Hog1 mitogen-activated protein kinases (MAPKs), demonstrating that Cck1 regulates cell integrity via the Mpk1 pathway and regulates cell adaptation to stresses via the Hog1 pathway. Overall, our study revealed that Cck1 plays important roles in regulating multiple signaling pathways and is required for fungal pathogenicity.
The titan cell is a recently described morphological form of the pathogenic fungus Cryptococcus neoformans. Occurring during the earliest stages of lung infection, titan cells are 5 to 10 times larger than the normal yeast-like cells, thereby resisting engulfment by lung phagocytes and favoring the persistence of infection. These enlarged cells exhibit an altered capsule structure, a thickened cell wall, increased ploidy, and resistance to nitrosative and oxidative stresses. We demonstrate that two G-protein-coupled receptors are important for induction of the titan cell phenotype: the Ste3a pheromone receptor (in mating type a cells) and the Gpr5 protein. Both receptors control titan cell formation through elements of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. This conserved signaling pathway, in turn, mediates its effect on titan cells through the PKA-regulated Rim101 transcription factor. Additional downstream effectors required for titan cell formation include the G1 cyclin Pcl103, the Rho104 GTPase, and two GTPase-activating proteins, Gap1 and Cnc1560. These observations support developing models in which the PKA signaling pathway coordinately regulates many virulence-associated phenotypes in diverse human pathogens.
Fungal meningitis is a serious disease caused by a fungal infection of the central nervous system (CNS) mostly in individuals with immune system deficiencies. Fungal meningitis is often fatal without proper treatment, and the mortality rate remains unacceptably high even with antifungal drug interventions. Currently, cryptococcal meningitis is the most common fungal meningitis in HIV-1/AIDS, and its disease mechanism has been extensively studied. The key steps for fungi to infect brain and cause meningitis after establishment of local infection are the dissemination of fungal cells to the bloodstream and invasion through the blood brain barrier to reach the CNS. In this review, we use cryptococcal CNS infection as an example to describe the current molecular understanding of fungal meningitis, including the establishment of the infection, dissemination, and brain invasion. Host and microbial factors that contribute to these infection steps are also discussed.
Cryptococcus neoformans; blood-brain barrier; central nervous system; fungus; infection; meningitis
In the rice blast fungus Magnaporthe oryzae, the PMK1 mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of M. oryzae and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them, PIC1 and PIC5, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of PIC1 had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of PIC5 led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The pic5 mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both PIC1 and PIC5 are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that PIC5 is a novel virulence factor involved in appressorium differentiation and pathogenesis in M. oryzae.
G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily.
Cryptococcus neoformans is the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Although fbp1 mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, as fbp1 mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating between fbp1 mutants, even though normal dikaryotic hyphae were observed during mating. In vitro assays of stress responses revealed that fbp1 mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in both Saccharomyces cerevisiae and C. neoformans via its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence in C. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.
The ubiquitin-proteasome system is one of the major protein turnover mechanisms that plays important roles in the regulation of a variety of cellular functions. It is composed of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 ubiquitin ligases that transfer ubiquitin to the substrates that are subjected to degradation in the 26S proteasome. The Skp1, Cullin, F-box protein (SCF) E3 ligases are the largest E3 gene family, in which the F-box protein is the key component to determine substrate specificity. Although the SCF E3 ligase and its F-box proteins have been extensively studied in the model yeast Saccharomyces cerevisiae, only limited studies have been reported on the role of F-box proteins in other fungi. Recently, a number of studies revealed that F-box proteins are required for fungal pathogenicity. In this communication, we review the current understanding of F-box proteins in pathogenic fungi.
Cryptococcus neoformans; E3 ligase; F-box; Fungi; Virulence
Cryptococcus neoformans is an AIDS-associated human fungal pathogen and the most common cause of fungal meningitis, with a mortality rate over 40% in AIDS patients. Significant advances have been achieved in understanding its disease mechanisms. Yet the underlying mechanism of a high frequency of cryptococcal meningitis remains unclear. The existence of high inositol concentrations in brain and our earlier discovery of a large inositol transporter (ITR) gene family in C. neoformans led us to investigate the potential role of inositol in Cryptococcus-host interactions. In this study, we focus on functional analyses of two major ITR genes to understand their role in virulence of C. neoformans. Our results show that ITR1A and ITR3C are the only two ITR genes among 10 candidates that can complement the growth defect of a Saccharomyces cerevisiae strain lacking inositol transporters. Both S. cerevisiae strains heterologously expressing ITR1A or ITR3C showed high inositol uptake activity, an indication that they are major inositol transporters. Significantly, itr1a itr3c double mutants showed significant virulence attenuation in murine infection models. Mutating both ITR1A and ITR3C in an ino1 mutant background activates the expression of several remaining ITR candidates and does not show more severe virulence attenuation, suggesting that both inositol uptake and biosynthetic pathways are important for inositol acquisition. Overall, our study provides evidence that host inositol and fungal inositol transporters are important for Cryptococcus pathogenicity.
Predicting future species invasions presents significant challenges to researchers and government agencies. Simply considering the vast number of potential species that could invade an area can be insurmountable. One method, recently suggested, which can analyse large datasets of invasive species simultaneously is that of a self organising map (SOM), a form of artificial neural network which can rank species by establishment likelihood. We used this method to analyse the worldwide distribution of 486 fungal pathogens and then validated the method by creating a virtual world of invasive species in which to test the SOM. This novel validation method allowed us to test SOM's ability to rank those species that can establish above those that can't. Overall, we found the SOM highly effective, having on average, a 96–98% success rate (depending on the virtual world parameters). We also found that regions with fewer species present (i.e. 1–10 species) were more difficult for the SOM to generate an accurately ranked list, with success rates varying from 100% correct down to 0% correct. However, we were able to combine the numbers of species present in a region with clustering patterns in the SOM, to further refine confidence in lists generated from these sparsely populated regions. We then used the results from the virtual world to determine confidences for lists generated from the fungal pathogen dataset. Specifically, for lists generated for Australia and its states and territories, the reliability scores were between 84–98%. We conclude that a SOM analysis is a reliable method for analysing a large dataset of potential invasive species and could be used by biosecurity agencies around the world resulting in a better overall assessment of invasion risk.
Despite the great morbidity and mortality that childhood bacterial meningitis (BM) is experiencing in Africa, diagnosis of BM in resource-limited contexts is still a challenge. Several algorithms and clinical predictors have been proposed to help physicians in decision-making but a lot of these markers used variables that are calculable only in well-equipped laboratories. Predictors or algorithm based on parameters that can be easily performed in basic laboratories can help significantly in BM diagnosis, even in resource-limited settings, rural hospitals or health centers.
This retrospective study examined 145 cerebral-spinal fluid (CSF) specimens from children from 2 months to 14 years. CSF specimens were divided into two groups, according to the presence or not of a clinical diagnosis of BM. For each specimen, CSF aspect, CSF white blood cells (WBC) count, CSF glucose and protein concentration were analyzed and statistical analysis were performed. CSF WBC count ≥10/µl is no more a valuable predictor of BM. CSF protein concentration ≥50 mg/dl has a better sensitivity for BM diagnosis and when used with CSF glucose concentration ≤40 mg/dl, can help to diagnose correctly almost all the BM cases. An algorithm including CSF protein concentration, glucose concentration and WBC count has been proposed to rule out BM and to correctly diagnose it.
In resource-limited health centers, the availability of a combination of easy-to-obtain parameters can significantly help physicians in BM diagnosis. The prompt identification of a BM case can be rapid treated or transferred to adequate structures and can modify the outcome in the patient.
The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.
We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca2+ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.
This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.
Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.
G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks. This review highlights recent research advances in fungal GPCRs, including classification, extracellular sensing, and G protein-signaling regulation. The involvement of GPCRs in pheromone and nutrient sensing has been studied extensively over the past decade. Following recent advances in fungal genome sequencing projects, a panoply of GPCR candidates has been revealed and some have been documented to play key roles sensing diverse extracellular signals, such as pheromones, sugars, amino acids, nitrogen sources, and even photons. Identification and deorphanization of additional putative GPCRs may require the development of new research tools. Here, we compare research on GPCRs in fungi with information derived from mammalian systems to provide a useful road map on how to better understand ligand–GPCR–G protein interactions in general. We also emphasize the utility of yeast as a discovery tool for systemic studies of GPCRs from other organisms.
G protein-coupled receptor; G protein; extracellular sensing; fungus
Signaling by extracellular adenosine 5′-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a “danger signal”, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this “danger signal”.
Methods and Principal Findings
Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells.
Conclusions and Significance
These data suggest that bacteria may sense extracellular dATP/ATP as a signal of “danger” and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an “inter-domain” warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells.