Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

Butts, Arielle; DeJarnette, Christian; Peters, Tracy L.; Parker, Josie E.; Kerns, Morgan E.; Eberle, Karen E.; Kelly, S. L.; Palmer, Glen E.

doi:10.1128/msphere.00379-17

Cited by 11 publications

(23 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Strains and growth conditions. Biofilm-forming C. albicans prototypical isolate SC5314 and C. albicans CAI4ϩpKE4-GFP␥ (single IRO1-URA3 locus restored) were used in this study and are as described previously (68,69). Both organisms were maintained as frozen stocks at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Willems

Stultz

Coltrane

et al. 2019

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Receipt of parenteral nutrition (PN) remains an independent risk factor for developing catheter-related bloodstream infections (CR-BSI) caused by fungi, including by the polymorphic fungus Candida albicans, which is notoriously adept at forming drug-resistant biofilm structures. Among a variety of macronutrients, PN solutions contain lipid emulsions to supply daily essential fats and are often delivered via central venous catheters (CVCs). Therefore, using an in vitro biofilm model system, we sought to determine whether various clinical lipid emulsions differentially impacted biofilm growth in C. albicans. We observed that the lipid emulsions Intralipid and Omegaven both stimulated C. albicans biofilm formation during growth in minimal medium or a macronutrient PN solution. Conversely, Smoflipid inhibited C. albicans biofilm formation by approximately 50%. Follow-up studies revealed that while Smoflipid did not impair C. albicans growth, it did significantly inhibit hypha formation and hyphal elongation. Moreover, growth inhibition could be recapitulated in Intralipid when supplemented with capric acid—a fatty acid present in Smoflipid but absent in Intralipid. Capric acid was also found to dose dependently inhibit C. albicans biofilm formation in PN solutions. This is the first study to directly compare different clinical lipid emulsions for their capacity to affect C. albicans biofilm growth. Results derived from this study necessitate further research regarding different lipid emulsions and rates of fungus-associated CR-BSIs.

show abstract

Section: Methodsmentioning

confidence: 99%

Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Willems

Stultz

Coltrane

et al. 2019

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…To facilitate replacement of endogenous transcriptional promoters in C. albicans, a previously described plasmid constructed in pGEMHIS1 with the P TEF1 promoter sequence (565 bp) was utilized (28).…”

Section: Methodsmentioning

confidence: 99%

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion

Tournu

Butts

Palmer

2019

mSphere

Self Cite

View full text Add to dashboard Cite

A recent study demonstrated that the insertion of poly-adenosine (poly-A) tracts into an open reading frame can suppress expression of the encoded protein in both prokaryotic and eukaryotic species. Furthermore, the degree of suppression is proportional to the length of the poly-A insertion, which can therefore provide a reliable and predictable means to titrate a specific protein’s expression. The goal of this study was to determine if this methodology can be applied to modulate the expression of proteins in the prevalent human fungal pathogen, Candida albicans. Insertion of increasing numbers of AAA codons encoding lysine at the N terminus of the C. albicans lanosterol demethylase (Erg11p) progressively diminished expression without significantly reducing the levels of mRNA. This suggests that Erg11p expression was attenuated at the posttranscriptional level. A direct correlation between the number of AAA codons inserted and C. albicans susceptibility to the Erg11p inhibitor fluconazole was also noted, indicating a progressive loss of Erg11p activity. Finally, we constructed a series of C. albicans strains with 3 to 12 AAA codons inserted at the 5′ end of the ARO1 gene, which encodes a pentafunctional enzyme catalyzing five sequential steps of the aromatic amino acid biosynthetic pathway. Increasing numbers of AAA codons progressively reduced the growth rate of C. albicans in standard laboratory medium, indicating a progressive loss of ARO biosynthetic activity. These data unequivocally demonstrate the potential utility of the poly-A insertion method to examine the phenotypic consequences of titrating target protein function in C. albicans. IMPORTANCE Investigating a protein’s functional importance at the whole-organism level usually involves altering its expression level or its specific activity and observing the consequences with respect to physiology or phenotype. Several approaches designed to partially or completely abolish the function of a gene, including its deletion from the genome and the use of systems that facilitate conditional expression, have been widely applied. However, each has significant limitations that are especially problematic in pathogenic microbes when it is desirable to determine if a particular gene is required for infection in an animal model. In this study, we sought to determine if an alternative approach—the insertion of poly-A repeats within the coding sequence of the gene—is sufficient to modulate its function in the prevalent human fungal pathogen C. albicans. Our results confirm that this approach enables us to predictably and gradually titrate the expression level of a protein and thus to investigate the phenotypic consequences of various levels of gene/protein function.

show abstract

“…All oligonucleotides used in this study are listed in Table S1 in the supplemental material. Plasmids pKE1 (34), pKE1Nluc (35), pKE4 (16), pKE4CDR1 (16), pAR8 (36), pAR8-ERG11 (36), pKE1:GFP-YPT72 (25), and pDUP3 (37) were described previously.…”

Section: Methodsmentioning

confidence: 99%

Loss of C-5 Sterol Desaturase Activity in Candida albicans : Azole Resistance or Merely Trailing Growth?

Luna-Tapia

Butts

Palmer

2019

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Increased expression of drug efflux pumps and changes in the target enzyme Erg11p are known to contribute to azole resistance in Candida albicans, one of the most prevalent fungal pathogens. Mutations that inactivate ERG3, which encodes sterol Δ5,6-desaturase, also confer in vitro azole resistance. However, it is unclear whether the loss of Erg3p activity is sufficient to confer resistance within the mammalian host, and relatively few erg3 mutants have been reported among azole-resistant clinical isolates. Trailing growth (residual growth in the presence of the azoles) is a phenotype observed with many C. albicans isolates and, in its extreme form, can be mistaken for resistance. The purpose of this study was to determine whether the growth of Erg3p-deficient C. albicans mutants in the presence of the azoles possesses the characteristics of azole resistance or of an exaggerated form of trailing growth. Our results demonstrate that, similar to trailing isolates, the capacity of an erg3Δ/Δ mutant to endure the consequences of azole exposure is at least partly dependent on both temperature and pH. This contrasts with true azole resistance that results from enhanced drug efflux and/or changes in the target enzyme. The erg3Δ/Δ mutant and trailing isolates also appear to sustain significant membrane damage upon azole treatment, further distinguishing them from resistant isolates. However, the insensitivity of the erg3Δ/Δ mutant to azoles is unaffected by the calcineurin inhibitor cyclosporin A, distinguishing it from trailing isolates. In conclusion, the erg3 mutant phenotype is qualitatively and quantitatively distinct from both azole resistance and trailing growth.

show abstract

Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

Cited by 11 publications

References 39 publications

Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Titrating Gene Function in the Human Fungal Pathogen Candida albicans through Poly-Adenosine Tract Insertion

Loss of C-5 Sterol Desaturase Activity in Candida albicans : Azole Resistance or Merely Trailing Growth?

Contact Info

Product

Resources

About