Aneuploidy enables cross-tolerance to unrelated antifungal drugs in Candida parapsilosis.

Candida parapsilosis is an emerging major human fungal pathogen. Echinocandins are first-line antifungal drugs for the treatment of invasive Candida infections. In clinical isolates, tolerance to echinocandins in Candida species is mostly due to point mutations of FKS genes, which encode the target protein of echinocandins. However, here, we found chromosome 5 trisomy was the major mechanism of adaptation to the echinocandin drug caspofungin, and FKS mutations were rare events. Chromosome 5 trisomy conferred tolerance to echinocandin drugs caspofungin and micafungin and cross-tolerance to 5-flucytosine, another class of antifungal drugs. The inherent instability of aneuploidy caused unstable drug tolerance. Tolerance to echinocandins might be due to increased copy number and expression of CHS7, which encodes chitin synthase. Although copy number of chitinase genes CHT3 and CHT4 was also increased to the trisomic level, the expression was buffered to the disomic level. Tolerance to 5-flucytosine might be due to the decreased expression of FUR1. Therefore, the pleiotropic effect of aneuploidy on antifungal tolerance was due to the simultaneous regulation of genes on the aneuploid chromosome and genes on euploid chromosomes. In summary, aneuploidy provides a rapid and reversible mechanism of drug tolerance and cross-tolerance in C. parapsilosis.

Modulating and optimizing Pluronic F-68 concentrations and feeding for intensified perfusion Chinese hamster ovary cell cultures.

Perfusion culture is often performed with micro-sparger to fulfill the high oxygen demand from the densified cells. Protective additive Pluronic F-68 (PF-68) is widely used to mitigate the adverse effect in cell viability from micro-sparging. In this study, different PF-68 retention ratio in alternating tangential filtration (ATF) columns was found to be crucial for cell performance of different perfusion culture modes. The PF-68 in the perfusion medium was found retained inside the bioreactor when exchanged through ATF hollow fibers with a small pore size (50 kD). The accumulated PF-68 could provide sufficient protection for cells under micro-sparging. On the other hand, with large-pore-size (0.2 µm) hollow fibers, PF-68 could pass through the ATF filtration membranes with little retention, and consequently led to compromised cell growth. To overcome the defect, a PF-68 feeding strategy was designed and successfully verified on promoting cell growth with different Chinese hamster ovary (CHO) cell lines. With PF-68 feeding, enhancements were observed in both viable cell densities (20%-30%) and productivity (~30%). A threshold PF-68 concentration of 5 g/L for high-density cell culture (up to 100 × 106 cells/mL) was also proposed and verified. The additional PF-68 feeding was not observed to affect product qualities. By designing the PF-68 concentration of perfusion medium to or higher than the threshold level, a similar cell growth enhancement was also achieved. This study systematically investigated the protecting role of PF-68 in intensified CHO cell cultures, shedding a light on the optimization of perfusion cultures through the control of protective additives.

Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes.

Antifungal drug tolerance is a response distinct from resistance, in which cells grow slowly above the MIC. Here, we found that the majority (69.2%) of 133 Candida albicans clinical isolates, including standard lab strain SC5314, exhibited temperature-enhanced tolerance at 37°C and 39°C, and were not tolerant at 30°C. Other isolates were either always tolerant (23.3%) or never tolerant (7.5%) at these three temperatures, suggesting that tolerance requires different physiological processes in different isolates. At supra-MIC fluconazole concentrations (8 to 128 µg/mL), tolerant colonies emerged rapidly at a frequency of ~10-3. In liquid passages over a broader range of fluconazole concentrations (0.25 to 128 µg/mL), tolerance emerged rapidly (within one passage) at supra-MICs. In contrast, resistance appeared at sub-MICs after 5 or more passages. Of 155 adaptors that evolved higher tolerance, all carried one of several recurrent aneuploid chromosomes, often including chromosome R, alone or in combination with other chromosomes. Furthermore, loss of these recurrent aneuploidies was associated with a loss of acquired tolerance, indicating that specific aneuploidies confer fluconazole tolerance. Thus, genetic background and physiology and the degree of drug stress (above or below the MIC) influence the evolutionary trajectories and dynamics with which antifungal drug resistance or tolerance emerges. IMPORTANCE Antifungal drug tolerance differs from drug resistance: tolerant cells grow slowly in drug, while resistant cells usually grow well, due to mutations in a few known genes. More than half of Candida albicans clinical isolates have higher tolerance at body temperature than they do at the lower temperatures used for most lab experiments. This implies that different isolates achieve drug tolerance via several cellular processes. When we evolved different strains at a range of high drug concentrations above inhibitory levels, tolerance emerged rapidly and at high frequency (one in 1,000 cells) while resistance appeared only later at very low drug concentrations. An extra copy of all or part of chromosome R was associated with tolerance, while point mutations or different aneuploidies were seen with resistance. Thus, genetic background and physiology, temperature, and drug concentration all influence how drug tolerance or resistance evolves.

Aneuploidy Mediates Rapid Adaptation to a Subinhibitory Amount of Fluconazole in Candida albicans.

Candida albicans is a prevalent, opportunistic, human fungal pathogen. Antifungal drug resistance and tolerance are two distinct mechanisms of adaptation to drugs. Studies of mechanisms of drug resistance are limited to the applications of high doses of drugs. Few studies have investigated the effects of subinhibitory amounts of drugs on the development of drug resistance or tolerance. In this study, we found that growth in a subinhibitory amount of fluconazole (FLC), a widely used antifungal drug, for just a short time was sufficient to induce aneuploidy in C. albicans. Surprisingly, the aneuploids displayed fitness loss in the presence of subinhibitory FLC, but a subpopulation of cells could tolerate up to 128 µg/mL FLC. Particular aneuploidy (ChrR trisomy) caused tolerance, not resistance, to FLC. In the absence of FLC, the aneuploids were unstable. Depending on the karyotype, aneuploids might become completely euploid or maintain particular aneuploidy, and, accordingly, the tolerance would be lost or maintained. Mechanistically, subinhibitory FLC was sufficient to induce the expression of several ERG genes and as well as the drug efflux gene MDR1. Aneuploids had a constitutive high-level expression of genes on and outside the aneuploid chromosomes, including most of the ERG genes as well as the drug efflux genes MDR1 and CDR2. Therefore, aneuploids were prepared for FLC challenges. In summary, aneuploidy provides a rapid and reversible strategy of adaptation when C. albicans is challenged with subinhibitory concentrations of FLC. IMPORTANCE Genome instability is a hallmark of C. albicans. Aneuploidy usually causes fitness loss in the absence of stress but confers better fitness under particular stress conditions. Therefore, aneuploidy is considered to be a double-edged sword. Here, we extend the understanding of aneuploidy. We found that aneuploidy arose under weak stress conditions but that it did not confer better fitness to the stress. Instead, it was less fit than its euploid counterparts. If the stress was withdrawn, aneuploidy spontaneously reverted to euploidy. If the stress became stronger, aneuploidy enabled subpopulation growth in a dose-independent manner of the stress. Therefore, we posit that aneuploidy enables the rapid and reversible development of drug tolerance in C. albicans. Further studies are required to investigate whether this is a general mechanism in human fungal pathogens.

Optimized process operations reduce product retention and column clogging in ATF-based perfusion cell cultures.

Product retention in hollow fibers is a common issue in ATF-based cell culture system. In this study, the effects of four major process factors on product (therapeutic antibody/recombinant protein) retention were investigated using Chinese hamster ovary cell. Hollow fibers made of polysulfone presented a product retention rate from 15% ± 8 to 43% ± 18% higher than those made of polyether sulfone varying with specific processes. Higher harvest flowrate and ATF exchange rate increased product retention by 13% ± 10% and up to 31% ± 13%, respectively. Hollow fibers with larger pore sizes (0.65 µm) appeared to have increased product retention by 38% ± 7% compared with smaller ones (0.2 µm) in this study. Further investigation revealed that the effects of pore size on retention could be correlated to the particle size distribution in the cell culture broth. A hollow fiber with a larger pore size (>0.5 µm) may reduce protein retention when small particles (approximately 0.01-0.2 µm in diameter) are dominant in the culture. However, if majority of the particles are larger than 0.2 µm in diameter, hollow fiber with smaller pore sizes (0.2 µm) could be a solution to reducing product retention. Alternatively, process optimization may modulate particle size distribution towards reduced production retention with selected ATF hollow fibers. This study for the first time highlights the importance of matching proper pore sizes of hollow fibers with the cell culture particles distribution and offers methods to reducing product retention and ATF column clogging in perfusion cell cultures. KEY POINTS: The material of ATF column could impact product retention during perfusion culture. Higher harvest flowrate and ATF exchange rate increased product retention. Matching culture particle size and ATF pore size is critical for retention modulation.

Paclitaxel-mediated human aryl hydrocarbon receptor mRNA translation by an internal ribosomal entry site-dependent mechanism.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is best known in mediating the toxicities of dioxins and dioxin-like compounds. AHR is activated by a variety of endogenous ligands and participating in tumor development. Thus, it will provide a new approach for cancer prevention and treatment to study the translation mechanism of AHR in tumor cells. In this study, we show that the 5'-untranslated region (UTR) of AHR mRNA contains an internal ribosome entry site (IRES). After mapping the entire AHR 5'-UTR, we determined that the full-length 5'-UTR is indispensable for the highest IRES activity. Interestingly, we found that AHR expression is induced in ovarian (A2780), breast (MDA-MB231), hepatic (Bel7402) and colorectal cancer cells (SW620) by chemotherapeutic drug paclitaxel (PTX) through IRES-dependent translation mechanism. Moreover, IRES activity is increased in the PTX-resistant ovarian cancer cells in which AHR protein expression was also enhanced. These results strongly suggest an important role for AHR IRES-dependent translation mechanism in cancer cell response to paclitaxel treatment.