微生物学

Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterization of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.

The filamentous ascomycete Fusarium graminearum is the causal agent of Fusarium head blight, a devastating disease of cereals with a worldwide distribution. Fusarium graminearum infections result in a quantitative yield reduction by impairing the growth of the kernels, and a qualitative reduction by poisoning the remaining kernels with mycotoxins toxic to animals and humans. The colonization of wheat florets by phytopathogenic fungus requires high-efficiency energy generation in the mitochondria (Bönnighausen et al., 2015). Mitochondrial activity in microorganisms can be measured using the oxygen consumption rate (OCR) method. Here we describe a method for the assessment of fungal respiration using an XF24 extracellular flux analyzer. The Seahorse XF Analyzer is a microplate-based respirometer which measures oxygen consumption by changes in the fluorescence of immobilized fluorophores (Gerencser et al., 2009). Multiple mitochondrial parameters can be measured by the application of mitochondrial substrates and inhibitors which are injected automatically during the assays via ports (Divakaruni et al., 2014). The experimental work-flow involves the inoculation with conidia and the application of specific inhibitors of mitochondrial functions. The analysis of fungal respiration represents a valuable tool that complements classical phenotypic screenings.

Bacteria live mostly as biofilms, not as planktonic cell populations. Bacterial cells living as biofilms are known to be in different physiological status. Persister cells are one of such physiological conditions and they are recognized as to be a stochastically produced sub-population of non-growing bacterial cells. The following protocol describes a method to determine the respiratory activity of cells within biofilms.