Investigation of the basis of virulence in serotype A strains of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from apparently immunocompetent individuals

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.     

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme,for Capsule Formation,and for Virulence

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.     

S. pombe sck2+, a second homologue of S. cerevisiae SCH9 in fission yeast, encodes a putative protein kinase closely related to PKA in function

The Schizosaccharomyces pombe sck2 gene, originally identified as SPAC22E12.14c in the genome-sequencing project, encodes a putative protein kinase highly similar to Saccharomyces cerevisiae Sch9p and S. pombe Sck1p, both of which can suppress loss of cAMP-dependent protein kinase (PKA) if over-produced. Over-expression of sck2 suppressed typical phenotypes of PKA-defective cells, including ectopic mating, slow growth and short cell morphology. Wild-type cells over-expressing sck2 behaved like the PKA-hyperactive mutant. Disruption of sck2 caused no obvious phenotype, but it intensified de-repression for sexual development when combined with the disruption of sck1. The pka1 sck1 sck2 triple disruptant could grow but only very slowly. Whereas disruption of sck1 enhanced the inefficiency of Δpka1 spores in germination, disruption of sck2 did not. These results suggest that the molecular function of Sck2p largely overlaps with that of Sck1p, but also that they differ somewhat either quantitatively or qualitatively. Received: 30 December 1997 / 20 January 1998     

Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.     

Chronological aging is associated with biophysical and chemical changes in the capsule of Cryptococcus neoformans

Does the age of a microbial cell affect its virulence factors? To our knowledge, this question has not been addressed previously, but the answer is of great relevance for chronic infections where microbial cells persist and age in hosts. Cryptococcus neoformans is an encapsulated human-pathogenic fungus notorious for causing chronic infections where cells of variable age persist in tissue. The major virulence factor for C. neoformans is a polysaccharide (PS) capsule. To understand how chronological age could impact the cryptococcal capsule properties, we compared the elastic properties, permeabilities, zeta potentials, and glycosidic compositions of capsules from young and old cells and found significant differences in all parameters measured. Changes in capsular properties were paralleled by changes in PS molecular mass and density, as well as modified antigenic density and antiphagocytic properties. Remarkably, chronological aging under stationary-phase growth conditions was associated with the expression of α-1,3-glucans in the capsule, indicating a new structural capsular component. Our results establish that cryptococcal capsules are highly dynamic structures that change dramatically with chronological aging under prolonged stationary-phase growth conditions. Changes associated with cellular aging in chronic infections could contribute to the remarkable capacity of this fungus to persist in tissues by generating phenotypically and antigenically different capsules.     

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to T-2 toxin

Summary T-2 toxin, a trichothecene mycotoxin, inhibits the growth of Saccharomyces cerevisiae. We have isolated nine spontaneous S. cerevisiae mutants resistant to this toxin. The mutants were distinguished from the wild type according to their degree of resistance to T-2 toxin on media with dextrose or glycerol as the carbon source. Generation time, mutation stability and level of cross-resistance to roridin A, another trichothecene, were determined for each mutant. The T-2 toxin resistant mutants were further characterized by subsequent tests involving cross-resistance and collateral sensitivtiy to chlorampenicol, neomycin, paromomycin, ethidium bromide and thiolutin. Mutants have been placed into three subgroups and the mechanism of T-2 toxin resistance in each group has been postulated. Mutant HK1 is the first S. cerevisiae isolate resistant to roridin A. One particular isolate, mutant HK1 l , carries a single recessive nuclear mutation. This mutation was termed ttt (for T-2 toxin resistant).     

The novel virulence-related gene stp of Streptococcus suis serotype 9 strain contributes to a significant reduction in mouse mortality

Streptococcus suis is an important swine pathogen responsible for a diverse range of diseases. S. suis serotype 2 (SS2) and S. suis serotype 9 (SS9) are the prevalent serotypes in diseased Chinese pigs. Little is known about SS9 virulence factors. Two strains, GZ0565 and SH040917, were isolated from a diseased pig and a healthy pig, respectively. Suppression subtractive hybridization (SSH) was used to identify SS9 virulence genes associated with pathogenicity. We identified 30 gene fragments unique to GZ0565, including stp which encodes a serine/threonine protein phosphatase known to affect the virulence and morphology of bacteria. To investigate the role of stp in pathogenesis of SS9, an isogenic stp mutant (Δstp) and a complementation strain (CΔstp) were constructed. The results demonstrated that the stp affected the expression of a few genes involving in adhesion and virulence for bacteria. The Δstp exhibited a significant decrease in HEp-2 cell adherence, compared with the wild type, and a reduced survival ratio in whole blood. The Δstp was attenuated in a CD1 murine model of infection and its LD50 values was seven-fold higher than the wild type. Our data suggest that stp is involved in the pathogenesis of SS9.     

Expression of both M protein and hyaluronic acid capsule by group A streptococcal strains results in a high virulence for chicken embryos

The human pathogenic microorganismStreptococcus pyogenes can resist against phagocytic attack of human granulocytes. Streptococcal M protein and hyaluronic acid were identified as virulence factors involved in this protection. So far, no experiments have been reported which describe the contribution of both components together in one system. We used the chicken embryo as an in vivo phagocytosis model to investigate the role of both components on the virulence of streptococci. For this, isogeneic mutants of group A streptococcal strains (GAS) which lack hyaluronic acid capsule (cap⁻) or M protein (M⁻) expression were used for infection and their virulence was compared with laboratory strains which had lost their ability to produce one or both virulence factors after long-time laboratory passages on blood agar. The experiments revealed that strains producing both M protein and hyaluronic capsule were higly, virulent. Only 1–10 colonyforming units were enough to cause a 50% lethality of 12-day-old chicken embryos. Those strains lacking one of these components showed a significant decrease in virulence. Finally, strains which failed to express either hyaluronic acid or M protein showed an additional tenfold decrease in virulence. This indicates a partial contribution of both M protein and hyaluronic acid to the virulence of GAS in the chicken embryo.     

Cryptococcus neoformans mating and virulence are regulated by the G-protein α subunit GPA1 and cAMP

This study explores signal transduction pathways that function during mating and infection in the opportunistic, human fungal pathogen Cryptococcus neoformans. The gene encoding a G-protein α subunit homolog, GPA1, was disrupted by homologous recombination. The gpa1 mutant strain was viable but exhibited a defect in mating in response to nitrogen starvation. Additionally, the gpa1 mutant strain failed to induce two well-established virulence factors—melanin synthesis, in response to glucose starvation; and capsule production, in response to iron limitation. As a consequence, virulence of the gpa1 mutant strain was significantly attenuated in an animal model of cryptococcal meningitis. Reintroduction of the wild-type GPA1 gene complemented the gpa1 mutant phenotypes and restored mating, melanin and capsule production, and virulence. Similarly, exogenous cAMP also suppressed the gpa1 mutant phenotypes, restoring mating and production of melanin and capsule. These observations support a model in which GPA1 has a role in sensing diverse environmental signals required for mating and virulence by regulating cAMP metabolism in C. neoformans.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Extrachromosomal genetics in the yeast Kluyveromyces lactis. Isolation and characterization of antimycin-resistant mutants

Summary Antimycin-resistant (A^R) mutants of the yeast Kluyveromyces lactis, obtained either spontaneously or after manganese treatment, were isolated and genetically characterized. Most of the mutants obtained after manganese mutagenesis and two spontaneous mutants, tolerated high antimycin concentrations (more than 10 /gmg/ml) and were extrachromosomal. One mutant which grew only in low antimycin (1 /gmg/ml) showed a Mendelian type of inheritance. The extrachromosomal mutants could be assigned to at least two genetic loci (A _I ^R and A _II ^R ). Mutants representative of these two groups showed increased resistance to the antibiotic when the respiration of whole cells or mitochondria was studied. Extrachromosomal mutants of Saccharomyces cerevisiae resistant to antimycin were also induced with manganese, isolated and characterized. Comparative studies of the antimycin-resistant mutants of K. lactis and S. cerevisiae permitted the following observations: a) K. lactis is more resistant to antimycin, funiculosin, mucidin and diuron than S. cerevisiae, as are the A^R mutants; b) K. lactis shows correlated sensitivity to funiculosin differing in this aspect from S. cerevisiae; c) the antimycin-resistant mutants of K. lactis belonging to group 11 (A _II ^R ) were also resistant to diuron, tolerating concentrations of more than 200 /gmg/ml; d) all extrachromosomal antimycin-resistant-mutants of S. cerevisiae and some of the A^R mutants of K. lactis were more sensitive to mucidin than the wild type.Abbreviations diuron or DCMU 3-(3,4-Dichlorophenyl)-1,1dimethylurea - HQNO 2-n Heptyl-4-hydroxyquinoline N-oxide     

Roles of putative His-to-Asp signaling modules HPT-1 and RRG-2, on viability and sensitivity to osmotic and oxidative stresses in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1; hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated. hpt-1 mutation partially recovered the osmotic sensitivity of os-2 mutants, implying the involvement of yeast Skn7-like RRG-2 in osmoregulation. However, the rrg-2 disruption did not change the osmotic sensitivity of the wild-type strain and the os-2 mutant, suggesting that rrg-2 did not participate in the osmoregulation. Both rrg-2 and os-2 single mutation slightly increased sensitivity to t-butyl hydroperoxide, and rrg-2 and hpt-1 mutations increased the os-2 mutant’s sensitivity. Although OS-1 is considered as a positive regulator of OS-2 MAP kinase, our results suggested that HPT-1 negatively regulated downstream MAP kinase cascade, and that OS-2 and RRG-2 probably participate independently in the oxidative stress response in N. crassa.     

Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in<Emphasis Type="Italic"> Gibberella zeae</Emphasis>

Gibberella zeae (anamorph, Fusarium graminearum) is an important pathogen of cereal crops found in many regions of the world. In this study, we have characterized two auxotrophic strains, designated S4B1279 and S4B3008, which were discovered from a collection of insertional mutants of G. zeae generated by restriction enzyme-mediated integration (REMI). Both mutant strains exhibited pleiotropic phenotypic changes that include reduction of mycelial growth and virulence and abolished sexual reproduction. Molecular analysis of the REMI mutants has shown that the auxotrophy of S4B1279 is due to a mutation of the ARG2 gene encoding an acetylglutamate synthase, and the auxotrophy of S4B3008 is due to a mutation of the ADE5 gene encoding a phosphoribosylamine-glycine ligase. Subsequent gene disruption and complementation studies have confirmed the functions for ARG2 and ADE5, respectively, in G. zeae. Our study has demonstrated the feasibility of using the REMI technique in studying G. zeae virulence mechanisms, in addition to providing two new selectable markers allowing genetic transformation of the fungus.     

Hypothetical gene C18orf42 encodes a novel protein kinase A‐binding protein

The substrate specificity of cAMP‐dependent protein kinase A (PKA) is controlled by its interaction with the A‐kinase anchoring protein (AKAP) family. Individual AKAP members are localized to particular intracellular sites and tether PKA specifically to the subcellular compartments where target substrates exist. Here, we report that the human hypothetical gene C18orf42 encodes a novel PKA‐binding protein that potentially regulates PKA–AKAP interactions. C18orf42 is expressed preferentially in neural tissues. Functional motif searching predicted that C18orf42 may encode a short protein that contains a putative PKA‐binding motif. To confirm this possibility, we applied the CRISPR/Cas9 genome‐editing system to incorporate the FLAG tag into the C‐terminus of the endogenous C18orf42 protein in the mouse neural cell line Neuro2a. Immunoprecipitation and immunoblotting using anti‐FLAG antibody showed translation of the endogenous C18orf42 protein and the physical interaction of the C18orf42 protein with PKA subunits. Immunoprecipitation and pull‐down assays showed that C18orf42 binds specifically to the type II regulatory subunits of PKA. Unlike the expression of many AKAPs, that of C18orf42 could block the AKAP‐mediated subcellular localization of PKA. These findings suggest that C18orf42 may be a novel PKA signaling gene that serves as an endogenous disruptor peptide for PKA–AKAP interactions.     

Role of Ferric Reductases in Iron Acquisition and Virulence in the Fungal Pathogen Cryptococcus neoformans

Iron acquisition is critical for the ability of the pathogenic yeast Cryptococcus neoformans to cause disease in vertebrate hosts. In particular, iron overload exacerbates cryptococcal disease in an animal model, defects in iron acquisition attenuate virulence, and iron availability influences the expression of major virulence factors. C. neoformans acquires iron by multiple mechanisms, including a ferroxidase-permease high-affinity system, siderophore uptake, and utilization of both heme and transferrin. In this study, we examined the expression of eight candidate ferric reductase genes and their contributions to iron acquisition as well as to ferric and cupric reductase activities. We found that loss of the FRE4 gene resulted in a defect in production of the virulence factor melanin and increased susceptibility to azole antifungal drugs. In addition, the FRE2 gene was important for growth on the iron sources heme and transferrin, which are relevant for proliferation in the host. Fre2 may participate with the ferroxidase Cfo1 of the high-affinity uptake system for growth on heme, because a mutant lacking both genes showed a more pronounced growth defect than the fre2 single mutant. A role for Fre2 in iron acquisition is consistent with the attenuation of virulence observed for the fre2 mutant. This mutant also was defective in accumulation in the brains of infected mice, a phenotype previously observed for mutants with defects in high-affinity iron uptake (e.g., the cfo1 mutant). Overall, this study provides a more detailed view of the iron acquisition components required for C. neoformans to cause cryptococcosis.     

spoT-mediated stringent response influences environmental and nutritional stress tolerance,biofilm formation and antimicrobial resistance in Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen with significant potential for virulence and multidrug resistance. Treatment failure often occurs because the pathogen may couple virulence and drug resistance with the stringent response. This study assessed the role of the spoT gene in environmental and nutritional stress tolerance, exopolysaccharide capsule production and biofilm formation. spoT mutants were constructed using the lambda red recombinase technique, and mutant and wild-type (WT) strains were exposed to limiting concentrations of carbon (glucose), phosphate and aminoacid, and environmental stresses of ethanol, salt and heat. Cell viability, capsule production and cell length were assessed as well as the ability to grow biofilm under antibiotic pressure using gentamicin and ceftazidime. spoT mutants were more susceptible to stresses versus WT; the reverse was true for survival during biofilm susceptibility assay (p < 0.05), especially when carbon and phosphate were present. spoT mutants were elongated and lacked a capsule versus WT and non-starved strains. The inability to produce capsule in mutants before and after starvation was likely a general effect of spoT mutation. These data suggest that the spoT-mediated stringent response is important for K. pneumoniae in conditions of nutrient limitation, environmental stress and antimicrobial pressure.     

Characterization of temperature-sensitive Akabane virus mutants and their roles in attenuation

Summary Akabane virus (AKAV) of the genus Orthobunyavirus in the family Bunyaviridae is an important animal pathogen; however, studies on AKAV biology are scarce. Therefore, we generated temperature-sensitive (ts) mutants of AKAV in order to study its pathogenesis. The ts AKAV mutants were generated by incubating the virulent OBE-1 strain with the chemical mutagen 5-fluorouracil. Each ts mutant was inoculated intracerebrally into mice to assess its virulence, and the genomic sequences of the attenuated mutants were also determined. Three of the twelve ts mutants studied showed a mortality rate of less than 10%. Although no mutation was detected in the S RNA segment of these three mutants, amino acid substitutions were observed in both the M and L RNA segments. Three of the mutants and the wild-type virus demonstrated a similar pattern of immunoreactivity in an ELISA with anti-Gc monoclonal antibodies. On the other hand, using a minireplicon system, the level of L protein activity of each ts mutant decreased as the temperature increased. These results suggest that the L RNA segment could be involved in the virulence of AKAV, which increases our understanding of how the viral gene products contribute to pathogenesis.