[Show abstract][Hide abstract] ABSTRACT: The Candida albicans RHR2 gene, which specifies a glycerol biosynthetic enzyme, is required for biofilm formation in vitro and in vivo. Prior studies indicate that RHR2 is ultimately required for expression of adhesin genes, such as ALS1. In fact, RHR2 is unnecessary for biofilm formation when ALS1 is overexpressed from an RHR2-independent promoter. Here, we describe two additional biological processes that depend upon RHR2: invasion into an abiotic substrate and pathogenicity in an abdominal infection model. We report here that abiotic substrate invasion occurs concomitantly with biofilm formation, and a screen of transcription factor mutants indicates that biofilm and hyphal formation ability correlates with invasion ability. However, analysis presented here of the rhr2Δ/Δ mutant separates biofilm formation and invasion. We found that an rhr2Δ/Δ mutant forms a biofilm upon overexpression of the adhesin gene ALS1 or the transcription factor genes BRG1 or UME6. However, the biofilm-forming strains do not invade the substrate. These results indicate that RHR2 has an adhesin-independent role in substrate invasion, and mathematical modeling argues that RHR2 is required to generate turgor. Previous studies have shown that abdominal infection by C. albicans has two aspects: infection of abdominal organs and persistence in abscesses. We report here that an rhr2Δ/Δ mutant is defective in both of these infection phenotypes. We find here that overexpression of ALS1 in the mutant restores infection of organs, but does not improve persistence in abscesses. Therefore, RHR2 has an adhesin-independent role in abdominal infection, just as it does in substrate invasion. This report suggests that RHR2, through glycerol synthesis, coordinates adherence with host- or substrate-interaction activities that enable proliferation of the C. albicans population.
[Show abstract][Hide abstract] ABSTRACT: Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the
septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful
analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic
fungus Candida albicans has shown that septins also have vital roles in cell wall regulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing
phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors
has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins,
which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles
have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway
as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of Candida glabrata infections is poorly understood. We studied pathogenesis of intra-abdominal candidiasis (IAC) in mice that were infected intra-peritoneally with C. glabrata and sterile stool. C. glabrata BG2 (5×10(8) CFU) caused 100% mortality. Sublethal inocula of BG2 (1×10(8) or 1×10(7) CFU) caused peritonitis that progressed to abscesses. Three clinical C. glabrata strains (5×10(8) CFU) caused 80-100% mortality, compared to 29% for a fourth strain (#346). Following sublethal inocula (1×10(7) CFU), intra-abscess burdens of virulent strain #356 were ∼ 1-log higher than those of #346. C. glabrata Δplb1-2 (disruption of phospholipase B genes) killed mice as well as BG2. Following sublethal inocula, however, Δplb1-2 was associated with more rapid abscess resolution and lower intra-abscess burdens; these findings were reversed by PLB1-2 re-insertion. Δplb1-2 was also more susceptible than BG2 to killing by human neutrophils in vitro. BG2 and Δplb1-2 were indistinguishable during hematogenously disseminated candidiasis. C. albicans SC5314 was more virulent than C. glabrata BG2 during IAC, causing 100% mortality following challenge with 5×10(7) CFU. In contrast, sublethal inocula (1×10(7) CFU) of BG2 caused less neutrophil infiltration and higher burdens in peritoneal fluid than SC5314, and abscesses that persisted longer and contained higher burdens. In conclusion, a mouse model of C. glabrata IAC mimics disease in humans and distinguishes the relative virulence of clinical and gene disruption strains. C. glabrata differed from C. albicans during IAC by causing attenuated mortality and eliciting dampened neutrophil responses, but resulting in more persistent peritonitis and abscesses.
Infection and immunity 05/2014; 82(7). DOI:10.1128/IAI.00062-14 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
The pathogenesis of intra-abdominal candidiasis is poorly understood.
Mice were intraperitoneally infected with Candida albicans (1 × 10(6) colony-forming units) and sterile stool. nanoString assays were used to quantitate messenger RNA for 145 C. albicans genes within the peritoneal cavity at 48 hours.
Within 6 hours after infection, mice developed peritonitis, characterized by high yeast burdens, neutrophil influx, and a pH of 7.9 within peritoneal fluid. Organ invasion by hyphae and early abscess formation were evident 6 and 24 hours after infection, respectively; abscesses resolved by day 14. nanoString assays revealed adhesion and responses to alkaline pH, osmolarity, and stress as biologic processes activated in the peritoneal cavity. Disruption of the highly-expressed gene RIM101, which encodes an alkaline-regulated transcription factor, did not impact cellular morphology but reduced both C. albicans burden during early peritonitis and C. albicans persistence within abscesses. RIM101 influenced expression of 49 genes during intra-abdominal candidiasis, including previously unidentified Rim101 targets. Overexpression of the RIM101-dependent gene SAP5, which encodes a secreted protease, restored the ability of a rim101 mutant to persist within abscesses.
A mouse model of intra-abdominal candidiasis is valuable for studying pathogenesis and C. albicans gene expression. RIM101 contributes to persistence within intra-abdominal abscesses, at least in part through activation of SAP5.
The Journal of Infectious Diseases 09/2013; 208(9). DOI:10.1093/infdis/jit335 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of
doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml.
Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135
GD, IS5, and wild-type/other mutants, respectively (P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8
μg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins
aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.
[Show abstract][Hide abstract] ABSTRACT: We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) clinical isolates, and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to ST258 clonal group, and harbored blaKPC-2, blaSHV-12, and blaTEM-1. By time-kills, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted bactericidal effects (range: 0.39-2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12) and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R2 = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than low expression (100% (8/8) vs 0% (0/4); p=0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (p≤0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC-Kp, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC-Kp isolates may be a practical tool for identifying effective combination regimens.
[Show abstract][Hide abstract] ABSTRACT: Candida albicans IRS4 encodes a protein that regulates phosphatidylinositol- (4, 5)-bisphosphate, which was shown to contribute to hematogenously disseminated candidiasis (DC) after several days in the standard mouse model. Our objective was to more accurately define temporal contributions of IRS4 to pathogenesis. During competition assays in vitro, an irs4 null mutant (Δirs4) exhibited wild-type fitness. In DC experiments, mice were infected intravenously with Δirs or CAI-12 (1×10(5) CFU), or a mixture of the strains (0.5×10(5) CFU each). In single-strain infections, qPCR revealed reduced Δirs4 burdens within kidneys at days 1, 4 and 7, but not 6 hours. In competitive infections, Δirs4 was outcompeted by CAI-12 in each mouse at ≥6 hours (competitive indices, p≤0.0001). At 4 and 7 days, Δirs4 burdens during competitive infections were significantly lower than single-strain infections (p=0.01 and <0.001, respectively), suggesting increased susceptibility to inflammatory responses. Phagocytic infiltration of kidneys in response to CAI-12 or competitive infections was significantly greater than Δirs4 at days 1 and 4 (p<0.001), and Δirs4 was more susceptible to phagocytosis and killing by human polymorphonuclear cells (p=0.01 and 0.006, respectively) and mouse macrophages in vitro (p=0.04 and 0.01, respectively). Therefore, IRS4 contributes to tissue invasion at early stages of DC, and mediates resistance to phagocytosis as DC progresses. Microarray revealed remarkably similar gene expression by Δirs4 and reference strain CAI-12 within blood, suggesting that IRS4 is not significantly involved in the hematogenous stage of disease. A competitive DC model detects attenuated virulence that is not evident with the standard model.
Infection and immunity 02/2013; 81(5). DOI:10.1128/IAI.00743-12 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Caspofungin exerts candidacidal activity by inhibiting cell wall (1, 3)-β-D-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced C. albicans cell death.Methods. Apoptosis (programmed cell death) and necrosis were studied after exposing C. albicans SC5314 to caspofungin at 0.06, 0.125 and 0.5 μg/mL (0.5x, 1x and 4x MIC, respectively) for three hours.Results. Caspofungin at 0.125 and 0.5 μg/mL reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin-V and propidium iodine staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20-25% and 5-7% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively (p=NS). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 μg/mL (48%) than 0.06 μg/mL (26%; p=0.003 and 0.003, respectively). The induction of apoptosis at concentrations ≤ MIC was corroborated by DHR-123 and DHE staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and TUNEL and DAPI staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/mL of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining.Conclusions. Caspofungin exerts activity against C. albicans by directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target this process may augment the benefits of antifungal agents.
[Show abstract][Hide abstract] ABSTRACT: The ability to identify patients at particularly low risk for invasive aspergillosis (IA) would facilitate more efficient targeting of antifungal prophylaxis. We measured baseline serum immunoglobulin responses against 6 purified recombinant Aspergillus fumigatus proteins before hematopoietic stem cell transplantation (HSCT) or chemotherapy in 73 subjects, including 19 patients who subsequently developed proven or probable IA and 54 uninfected controls. We also assessed responses at the time of IA diagnosis and 4 weeks later (acute and convalescent sera, respectively). Baseline IgG responses against enolase, Ahp1, Hsp90, Crf1, and Cdc37 were significantly higher in the patients with IA compared with controls (P < .05). Cutoff concentrations identified by reveiver-operating characteristic curve analysis were 67%-84% sensitive and 52%-67% specific. In a population with a 15% likelihood of developing IA, positive and negative predictive values would be 22%-26% and 92%-95%, respectively. Positive IgG responses against Hsp90, Pep2, Crf1, and Cdc37 were specifically associated with early-onset IA (<40 days) rather than late-onset IA (P ≤ .009). Increased IgG concentrations against Hsp90, Pep2, and Crf1 in convalescent sera versus baseline sera were more likely in the patients with IA who survived (P ≤ .01). IgG responses in acute sera were not correlated with outcomes, and IgM and IgA responses did not differ in baseline, acute, or convalescent sera between the patients and controls. In conclusion, baseline IgG responses against Aspergillus proteins may be useful screening tests for patients at low risk for IA. Our data suggest that some patients with IA have significant colonization or ongoing Aspergillus infections before immunosuppression. As such, IA may reflect unique predispositions to infection and/or progression from endogenous sources.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 07/2012; 18(12). DOI:10.1016/j.bbmt.2012.07.013 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Echinocandins are frontline agents against invasive candidiasis (IC), but predictors for echinocandin therapeutic failure have not been well defined. Mutations in Candida FKS genes, which encode the enzyme targeted by echinocandins, result in elevated MICs and have been linked to therapeutic failures. In this study, echinocandin MICs by broth microdilution and FKS1 and FKS2 mutations among C. glabrata isolates recovered from patients with IC at our center were correlated retrospectively with echinocandin therapeutic responses. Thirty-five patients with candidemia and 4 with intra-abdominal abscesses were included, 92% (36/39) of whom received caspofungin. Twenty-six percent (10) and 74% (29) failed and responded to echinocandin therapy, respectively. Caspofungin, anidulafungin, and micafungin MICs ranged from 0.5 to 8, 0.03 to 1, and 0.015 to 0.5 μg/ml, respectively. FKS mutations were detected in 18% (7/39) of C. glabrata isolates (FKS1, n = 2; FKS2, n = 5). Median caspofungin and anidulafungin MICs were higher for patients who failed therapy (P = 0.04 and 0.006, respectively). By receiver operating characteristic (ROC) analyses, MIC cutoffs that best predicted failure were >0.5 (caspofungin), >0.06 (anidulafungin), and >0.03 μg/ml (micafungin), for which sensitivity/specificity were 60%/86%, 50%/97%, and 40%/90%, respectively. Sensitivity/specificity of an FKS mutation in predicting failure were 60%/97%. By univariate analysis, recent gastrointestinal surgery, prior echinocandin exposure, anidulafungin MIC of >0.06 μg/ml, caspofungin MIC of >0.5 μg/ml, and an FKS mutation were significantly associated with failure. The presence of an FKS mutation was the only independent risk factor by multivariate analysis (P = 0.002). In conclusion, detection of C. glabrata FKS mutations was superior to MICs in predicting echinocandin therapeutic responses among patients with IC.
[Show abstract][Hide abstract] ABSTRACT: We previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintaining Candida albicans cell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response of C. albicans to caspofungin. First, we studied caspofungin-hypersusceptible C. albicans irs4 and inp51 mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5'-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes, irs4 and inp51 mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants. irs4 and inp51 were linked most closely to the gin4 mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-type C. albicans to caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those of irs4, inp51, and gin4 mutants. Taken together, our data suggest that the C. albicans Irs4-Inp51 5'-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.
[Show abstract][Hide abstract] ABSTRACT: The sensitivity of blood cultures for diagnosing invasive candidiasis (IC) is poor.
We performed a validated Candida real-time polymerase chain reaction (PCR) and the Fungitell 1,3-β-D-glucan (BDG) assay on blood samples collected from prospectively identified patients with IC (n = 55) and hospitalized controls (n = 73). Patients with IC had candidemia (n = 17), deep-seated candidiasis (n = 33), or both (n = 5). Controls had mucosal candidiasis (n = 5), Candida colonization (n = 48), or no known Candida colonization (n = 20).
PCR using plasma or sera was more sensitive than whole blood for diagnosing IC (P = .008). Plasma or sera PCR was more sensitive than BDG in diagnosing IC (80% vs 56%; P = .03), with comparable specificity (70% vs 73%; P = .31). The tests were similar in diagnosing candidemia (59% vs 68%; P = .77), but PCR was more sensitive for deep-seated candidiasis (89% vs 53%; P = .004). PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% vs 17%; P = .0005 and .003, respectively). PCR and culture identified the same Candida species in 82% of patients. The sensitivity of blood cultures combined with PCR or BDG among patients with IC was 98% and 79%, respectively.
Candida PCR and, to a lesser extent, BDG testing significantly enhanced the ability of blood cultures to diagnose IC.
[Show abstract][Hide abstract] ABSTRACT: Background: C. albicans Irs4 regulates phosphoinositides, cell wall integrity, and invasion, but not colonization of mouse kidneys. Attenuated virulence of irs4 mutants within kidneys may reflect intrinsic cell defects and/or increased susceptibility to host defenses.
Methods: We used qPCR to quantitate wild-type (WT) C. albicans and irs4 cells during competitive mixed infections in vitro and within mouse kidneys, and assessed mouse PMN and CK responses.
Results: Using qPCR, we demonstrated that WT and irs4 strains had similar growth rates in liquid media. We infected ICR mice via the lateral tail vein with 1x105 CFU/mL of WT alone, irs4 alone, or mixed WT+irs4 (5x104 each). There were no differences in kidney burdens at 6 hours between groups. At 1 and 4 days, burdens of irs4 were 1.5-2 log lower than wild-type by qPCR when strains were inoculated alone. During mixed infections, differences at both time points were >3-log (p<0.001 vs. strains alone). Mutants were not impaired in hyphal formation in liquid media, but on histopathology of mouse kidneys, they exhibited fewer hyphae and were less tissue penetration. PMN infiltration of kidneys of mice infected with irs4 was reduced at 6 hours, but similar to WT and mixed infection kidneys at 1 and 4 days despite lower burdens. Likewise, Th1, Th2 and Th17 responses for mice infected with irs4, mixed or WT strains were similar. irs4 mutant was significantly more susceptible than WT to killing by human PMN in vitro (p=0.001).
Conclusion: Virulence of C. albicans irs4 within mouse kidneys was more attenuated during mixed infection than when inoculated alone, which was not explained by altered PMN or CK responses. Therefore, attenuated virulence reflected factors intrinsic to mutant cells, rather than altered susceptibility to host defenses. Since irs4 grows in liquid media and colonizes kidneys comparably to WT C. albicans, the renal parenchyma must present a unique environment in which WT is more competitive. We hypothesize that impaired cell wall integrity responses of the irs4 mutant exact a fitness cost under conditions of cell wall stress within kidneys.
Infectious Diseases Society of America 2011 Annual Meeting; 10/2011
[Show abstract][Hide abstract] ABSTRACT: Paradoxical growth of Candida in vitro at echinocandin concentrations exceeding the MIC is well described, but the clinical relevance is unknown. We assessed echinocandin
paradoxical effects against Candida bloodstream isolates (BSI) in the presence or absence of human serum and investigated regulatory mechanisms. As determined
by broth microdilution, a paradoxical effect was evident for 60% (18/30), 23% (7/30), and 13% (4/30) of Candida albicans BSI exposed to caspofungin, anidulafungin, and micafungin, respectively, at achievable human serum concentrations (≤8 μg/ml).
A paradoxical effect was not evident among 34 C. glabrata BSI and was observed only for caspofungin against C. parapsilosis (4%, 1/23). As determined in time-kill studies, a caspofungin paradoxical effect was demonstrated by C. albicans (2/3), C. glabrata (1/3), and C. parapsilosis (1/3), including BSI that were determined to be negative by microdilution. In 50% human serum, a paradoxical effect was eliminated
at caspofungin concentrations up to 64 μg/ml for 100% (8/8) of the C. albicans BSI. A caspofungin paradoxical effect was also eliminated by chitin synthase inhibitor nikkomycin Z and at achievable concentrations
of calcineurin pathway inhibitors, tacrolimus and cyclosporine. Moreover, these agents were synergistic with caspofungin against
100, 100, and 88% (7/8) of C. albicans, respectively, and exerted their own paradoxical effects. Finally, paradoxical growth was eliminated in C. albicans irs4- and inp51-null mutants, which lack phosphatidylinositol-(4,5)-bisphosphate 5′-phosphatase. Our findings suggest that the paradoxical
effect is unlikely to be important in vivo but remains an important tool to study cell wall stress responses. We implicate the Irs4-Inp51 phosphatidylinositol-(4,5)-bisphosphate
5′-phosphatase as a novel regulator of paradoxical growth.
[Show abstract][Hide abstract] ABSTRACT: In this study, we measured time-kills and post-antifungal effects (PAFEs) for micafungin against Candida albicans (n=4), Candida glabrata (n=3), Candida parapsilosis (n=3) and Candida krusei (n=2) isolates and further characterised the PAFEs. Minimum inhibitory concentrations (MICs) were 0.5-1.0 mg/L against C. parapsilosis and 0.008-0.125 mg/L against the other species. Micafungin caused kills >1 log at 1 x MIC, 4 x MIC (range 1.19-3.10 log) and 16 x MIC (2.27-3.68 log), achieving fungicidal levels (> or = 3 log) against nine isolates. One-hour drug exposure during PAFE experiments resulted in kills of 0.73-2.88 log and 1.72-3.55 log at 4 x and 16 x MIC, respectively, achieving fungicidal levels against five isolates. Isolates of each species collected 8 h after a 1-h exposure to micafungin (4 x and 16 x MIC) were hypersusceptible to sodium dodecyl sulphate (SDS) and Calcofluor White. Cells tested during the PAFE period demonstrated cell wall disturbances as evident on electron micrographs as well as significant reductions in adherence to epithelial cells. Phagocytosis by J774 macrophages was significantly enhanced for three PAFE isolates tested. Micafungin is fungicidal and exerts PAFEs that kill diverse Candida spp., disturb cell walls of viable organisms, reduce adherence and enhance susceptibility to phagocytosis.
International journal of antimicrobial agents 11/2009; 35(1):80-4. DOI:10.1016/j.ijantimicag.2009.09.003 · 4.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anidulafungin targets the cell walls of Candida species by inhibiting β-1,3-glucan synthase, thereby killing isolates and exerting prolonged postantifungal effects (PAFEs).
We performed time-kill and PAFE experiments on Candida albicans (n = 4), C. glabrata (n = 3), C. parapsilosis (n = 3), and C. krusei (n = 2) isolates and characterized the PAFEs in greater detail. MICs were 0.008 to 0.125 μg/ml against C. albicans, C. glabrata, and C. krusei and 1.0 to 2.0 μg/ml against C. parapsilosis. During time-kill experiments, anidulafungin caused significant kills at 16× MIC (range, log 2.68 to 3.89) and 4× MIC (log
1.87 to 3.19), achieving fungicidal levels (≥log 3) against nine isolates. A 1-hour drug exposure during PAFE experiments
resulted in kills ranging from log 1.55 to 3.47 and log 1.18 to 2.89 (16× and 4× MIC, respectively), achieving fungicidal
levels against four isolates. Regrowth of all 12 isolates was inhibited for ≥12 h after drug washout. Isolates of each species
collected 8 h after a 1-hour exposure to anidulafungin (16× and 4× MIC) were hypersusceptible to sodium dodecyl sulfate (0.01
to 0.04%) and calcofluor white (40 μg/ml). Moreover, PAFEs were associated with major cell wall disturbances, as evident in
electron micrographs of viable cells, and significant reductions in adherence to buccal epithelial cells (P ≤ 0.01). Finally, three of four PAFE isolates tested were hypersusceptible to killing by J774 macrophages (P ≤ 0.007). Our data suggest that the efficacy of anidulafungin in the treatment of candidiasis might stem from both direct
fungicidal activity and indirect PAFEs that lessen the ability of Candida cells to establish invasive disease and to persist within infected hosts.
[Show abstract][Hide abstract] ABSTRACT: We previously showed that Candida albicans orf19.4590, which we have renamed RFX2, expresses a protein that is reactive with antibodies in persons with candidiasis. In this study, we demonstrate that C. albicans RFX2 shares some functional redundancy with Saccharomyces cerevisiae RFX1. Complementation of an S. cerevisiae rfx1 mutant with C. albicans RFX2 partially restored UV susceptibility and the repression of DNA damage response genes. DNA damage- and UV-induced genes RAD6 and DDR48 were derepressed in a C. albicans rfx2 null mutant strain under basal conditions, and the mutant was significantly more resistant to UV irradiation, heat shock,
and ethanol than wild-type strain SC5314. The rfx2 mutant was hyperfilamentous on solid media and constitutively expressed hypha-specific genes HWP1, ALS3, HYR1, ECE1, and CEK1. The mutant also demonstrated increased invasion of solid agar and significantly increased adherence to human buccal epithelial
cells. During hematogenously disseminated candidiasis, mice infected with the mutant had a significantly delayed time to death
compared to the wild type. During oropharyngeal candidiasis, mice infected with the mutant had significantly lower tissue
burdens in the oral cavity and esophagus at 7 days and they were less likely to develop disseminated infections because of
mucosal translocation. The data demonstrate that C. albicans Rfx2p regulates DNA damage responses, morphogenesis, and virulence.
[Show abstract][Hide abstract] ABSTRACT: Investigators have long used antibody-based screening strategies to identify Candida albicans immunogenic proteins and the genes that encode them during infections. With the recent availability of the C. albicans genome sequence and the development of genomic and proteomic technologies, it is now possible to efficiently conduct large-scale screening in standard research labs. C. albicans proteins and genes identified with a variety of screening methods have been implicated as important determinants of candidal virulence and exploited as vaccine and therapeutic targets. In this chapter, we describe methods used in our lab, in which sera recovered from patients with candidiasis are used to screen a C. albicans genomic DNA expression library. Immunoreactive colonies are detected by reaction with anti-human immunoglobulin, and the corresponding open reading frames are identified using the genome sequence database. The methods are also suitable for use with cDNA expression libraries, and they are complementary to proteomic screening strategies described elsewhere in this volume.
[Show abstract][Hide abstract] ABSTRACT: Animal models are powerful tools to study the pathogenesis of diverse types of candidiasis. Murine models are particularly attractive because of cost, ease of handling, technical feasibility, and experience with their use. In this chapter, we describe methods for two of the most popular murine models of disease caused by Candida albicans. In an intravenously disseminated candidiasis (DC) model, immunocompetent mice are infected by lateral tail vein injections of a C. albicans suspension. Endpoints include mortality, tissue burdens of infection (most importantly in the kidneys, although spleens and livers are sometimes also assessed), and histopathology of infected organs. In a model of oral/esophageal candidiasis, mice are immunosuppressed with cortisone acetate and inoculated in the oral cavities using swabs saturated with a C. albicans suspension. Since mice do not die from oral candidiasis in this model, endpoints are tissue burden of infection and histopathology. The DC and oral/esophageal models are most commonly used for studies of C. albicans virulence, in which the disease-causing ability of a mutant strain is compared with an isogenic parent strain. Nevertheless, the basic techniques we describe are also applicable to models adapted to investigate other aspects of pathogenesis, such as spatiotemporal patterns of gene expression, specific aspects of host immune response and assessment of antifungal agents, immunomodulatory strategies, and vaccines.
[Show abstract][Hide abstract] ABSTRACT: We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P(2) levels and the cell integrity pathway, and that PI(4,5)P(2) homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.