Mucosal Infection with Unmasked Candida albicans Cells Impacts Disease Progression in a Host Niche-Specific Manner.

Shielding the immunogenic cell wall epitope ß(1, 3)-glucan under an outer layer of mannosylated glycoproteins is an essential virulence factor deployed by Candida albicans during systemic infection. Accordingly, mutants with increased ß(1, 3)-glucan exposure (unmasking) display increased immunostimulatory capabilities in vitro and attenuated virulence during systemic infection in mice. However, little work has been done to assess the impact of increased unmasking during the two most common manifestations of candidiasis, namely, oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC). We have shown previously that the expression of a single hyperactive allele of the MAP3K STE11ΔN467 induces unmasking via the Cek1 MAPK pathway, attenuates fungal burden, and prolongs survival during systemic infection in mice. Here, we expand on these findings and show that infection with an unmasked STE11ΔN467 mutant also impacts disease progression during OPC and VVC murine infection models. Male mice sublingually infected with the STE11ΔN467 mutant showed a significant reduction in tongue fungal burden at 2 days postinfection and a modest reduction at 5 days postinfection. However, we find that selection for STE11ΔN467 suppressor mutants that no longer display increased unmasking occurs within the oral cavity and is likely responsible for the restoration of fungal burden trends to wild-type levels later in the infection. In the VVC infection model, no attenuation in fungal burden was observed. However, polymorphonuclear cell recruitment and interleukin-1ß (IL-1ß) levels within the vaginal lumen, markers of immunopathogenesis, were increased in mice infected with unmasked STE11ΔN467 cells. Thus, our data suggest a niche-specific impact for unmasking on disease progression.

Disruption to de novo uridine biosynthesis alters ß-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of ß-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing ß-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of ß-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly ß-1,3-glucan. However, ß-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase ß-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

Rapid Hypothesis Testing in Candida albicans Clinical Isolates Using a Cloning-Free, Modular, and Recyclable System for CRISPR-Cas9 Mediated Mutant and Revertant Construction.

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 104 homozygous deletion mutants per µg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δals3, Δ/Δcph1 Δ/Δefg1, Δ/Δece1) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1ß response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1-flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1- and CaHygB-flipper cassettes were simultaneously integrated at loci with high efficiency (104 transformants per µg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.

Effect of conventional transcranial direct current stimulation devices and electrode sizes on motor cortical excitability of the quadriceps muscle.

BACKGROUND: There is a growing concern among the scientific community that the effects of transcranial direct current stimulation (tDCS) are highly variable across studies. The use of different tDCS devices and electrode sizes may contribute to this variability; however, this issue has not been verified experimentally. OBJECTIVE: To evaluate the effects of tDCS device and electrode size on quadriceps motor cortical excitability. METHODS: The effect of tDCS device and electrode size on quadriceps motor cortical excitability was quantified across a range of TMS intensities using a novel evoked torque approach that has been previously shown to be highly reliable. In experiment 1, anodal tDCS-induced excitability changes were measured in twenty individuals using two devices (Empi and Soterix) on two separate days. In experiment 2, anodal tDCS-induced excitability changes were measured in thirty individuals divided into three groups based on the electrode size. A novel Bayesian approach was used in addition to the classical hypothesis testing during data analyses. RESULTS: There were no significant main or interaction effects, indicating that cortical excitability did not differ between different tDCS devices or electrode sizes. The lack of pre-post time effect in both experiments indicated that cortical excitability was minimally affected by anodal tDCS. Bayesian analyses indicated that the null model was more favored than the main or the interaction effects model. CONCLUSIONS: Motor cortical excitability was not altered by anodal tDCS and did not differ by devices or electrode sizes used in the study. Future studies should examine if behavioral outcomes are different based on tDCS device or electrode size.

Evaluation of motor cortical excitability using evoked torque responses: A new tool with high reliability.

BACKGROUND: Motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) are typically recorded via surface electromyography (EMG). However, another suitable alternative may be recording torque output associated with MEPs, especially when studying multiheaded muscles (e.g. quadriceps) for which EMG may not be ideal. METHODS: We recorded the motor evoked torque elicited by TMS along with conventional EMG-based MEPs (MEPEMG) over a range of TMS intensities (100-140 % of active motor threshold [AMT]) from twenty healthy young adults on two different days. MEPs were normalized using different normalization procedures (raw, normalized to maximum voluntary isometric contraction [MVIC], and peak MEP). Additionally, motor evoked torque was normalized to TMS-evoked peripheral resting twitch torque. Intraclass correlation coefficients (ICCs) were determined for each of these variables to compute reliability. RESULTS: Motor evoked torque showed good to excellent reliability (ICC: 0.65-0.90) at TMS intensities ≥ 110 % AMT, except when normalized by peak MEP. The reliability of raw MEPEMG and MVIC normalized MEPEMG was fair to excellent only at ≥ 130 % AMT (ICC: 0.42-0.82) and at ≥ 120 % AMT (ICC: 0.41-0.83), respectively. The reliability of both MEPEMG and motor evoked torque generally increased with increasing TMS intensities, with motor evoked torque normalized to the resting twitch torque yielding the best ICC scores. COMPARISON WITH EXISTING METHODS: When compared with conventional MEPEMG, motor evoked torque offers superior and reliable estimates of corticospinal excitability, particularly when normalized to resting twitch torque. CONCLUSIONS: TMS-induced motor evoked torque can reliably be used to measure corticospinal excitability in the quadriceps muscles.