Novel Insight into Neutrophil Immune Responses by Dry Mass Determination of Candida albicans Morphotypes

Abstract

The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI) is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.

Dual transcriptome of the immediate neutrophil and Candida albicans interplay

“…Since C. albicans is a polymorphic pathogen and mechanisms applied by neutrophils to discriminate between the C. albicans morphotypes remain incompletely understood [ 39 – 41 ], we analyzed transcriptional changes in neutrophils isolated from healthy donors upon yeast and hypha infection separately. DEGs were determined through comparison of infected and uninfected neutrophils.…”

“…DEGs were determined through comparison of infected and uninfected neutrophils. We have shown earlier that quantification based on dry mass was preferable to determine comparable amounts of C. albicans yeasts and hyphae [ 39 ]. In the present study we infected with similar dry masses, which correlated well with surface area of yeast and hyphal cells resulting in comparable infectious loads and contact sites for neutrophils.…”

“…The transcripts of these adjusted, but unchallenged fungal cells served as reference for DEG identification for each time point throughout the course of the infection. To have a comparable infectious load we adjusted the dry mass of Candida as described previously [ 39 ]. An average of 13.1 million reads was sequenced.…”

“…Cell numbers for Candida yeast were calculated by OD 600 – CFU correlation ( C. albicans : OD 600 1 ≙ 2.9 × 10 7 CFUs/ml). Using an XTT / dry mass correlation, the dry mass of yeast and hyphae per milliliter were adjusted before infection [ 39 ].…”

Programming and memory dynamics of innate leukocytes during tissue homeostasis and inflammation

“…For example, infection with a fungal pathogen Candida albicans could dose dependently induce ROS production in human neutrophils. The ROS production reached a maximum plateau in human neutrophils infected with C. albicans at MOI = 2 and subsequently declined in infected neutrophils at higher MOIs [40].…”