Brown, G. D. | Search bioRxiv

bioRxiv2018

Comparisons of a Novel Air Sampling Filter Material, Wash Buffers and Extraction Methods in the Detection and Quantification of Influenza Virus

Quantification of aerosolized influenza virus is used for determining inhalation exposure. Several bioaerosol samplers and analytical methods have been used; however, the detection and quantification of influenza virus among aerosol samples remains challenging. Therefore, improved viral aerosol measurement methods are needed. This study evaluated influenza virus recovery among three filter types polytetrafluoroethylene, polyvinylchloride and polystyrene. Polytetrafluoroethylene, polyvinylchloride are fabricated filter materials and commonly used in the scientific literature to sample for viral aerosols. A novel, electrospun polystyrene filter material may improve viral aerosol recovery during filter-based air sampling. The filter materials were compared across the following conditions: treated with or without air, filter wash buffer (HBSS or PBS), and viral RNA extraction method (QIAamp Viral RNA Mini Kit or Trizol). Twenty trials were completed in a chamber and samples were analyzed using RT-qPCR. Viral recovery was significantly different (p-value < .0001) by filter type. Polystyrene filter use resulted in recovery of the most viral RNA. Air sampling did not affect the recovery of viral RNA from the filter materials (p-values > 0.05). Viral RNA concentrations were significantly different across extraction methods for all comparisons (p-values < 0.05). Our results demonstrated that the novel polystyrene filter material resulted in the highest concentration of extracted RNA compared to the commonly used polytetrafluoroethylene and polyvinylchloride, which we speculate may be related to the chemical composition of the filter material (e.g., polystyrene is an aromatic hydrocarbon whereas polytetrafluoroethylene and polyvinylchloride contain more polar, and thus potentially reactive, carbon-halogen bonds). Air sampling did not have an effect on viral RNA recovery. Using Hanks Balanced Salt Solution with QIAamp Viral RNA Mini Kit, and Phosphate-buffered saline with the Trizol extraction, resulted in the most viral RNA recovery.

microbiology↗

bioRxiv2017

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis

Fungi undergo changes in cell shape in response to environmental stimuli that drive pathogenesis and niche adaptation, such as the yeast-to-hyphal transition of dimorphic fungi in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify bacterial peptidoglycan as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and E. coli co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established and previously undescribed regulators of Titanisation in vitro and investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-titan transition and establish an essential in vitro model for the future characterization of this important morphotype.\n\nAuthor SummaryChanges in cell shape underlie fungal pathogenesis by allowing immune evasion and dissemination. Aspergillus and Candida albicans hyphae drive tissue penetration. Histoplasma capsulatum and C. albicans yeast growth allows evasion and dissemination. As major virulence determinates, morphogenic transitions are extensively studied in animal models and in vitro. The pathogenic fungus Cryptococcus neoformans is a budding yeast that, in the host lung, switches to an unusual morphotype termed the Titan cell. Titans are large, polyploid, have altered cell wall and capsule, and produce haploid daughters. Their size prevents engulfment by phagocytes, yet they are linked to dissemination and altered immune response. Despite their important influence on disease, replicating the yeast-to-Titan switch in vitro has proved challenging. Here we show that Titans are induced by host-relevant stimuli, including serum and bronchio-alveolar lavage fluid. We identify bacterial peptidoglycan as a relevant inducing compound and predict an in vivo Titan defect for a clinical isolate. Genes regulating in vivo Titanisation also influence in vitro formation. Titanisation is a conserved morphogenic switch across the C. neoformans species complex. Together, we show that Titan cells are a regulated morphotype analogous to the yeast-to-hyphal transition and establish new ways to study Titans outside the host lung.

microbiology↗