High Resolution Respirometry in Candida albicans

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 characterisation 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 potential of respiration inhibition as a new approach to combat human fungal pathogens

The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence

Passive UHF RFID Voice Prosthesis Mounted Sensor for Microbial Growth Detection

Capacitive loading due to human tissue can lead to low efficiency for implantable Passive Radio Frequency Identification (RFID) antennas. The presented passive UHF antenna sensor provides read distances above 0.5 meters (within a body phantom) by utilizing a convoluted half-wave dipole design. It is able to detect simulated early to mature Candida albicans biofilm growth when mounted upon a voice prosthesis (up to a 30 μm biofilm thickness). Depending on the propagation frequency of interest, as early 4-hour growth (5 to 10 μm biofilm thickness) equivalent could be detected and before any device failure could occur due to the colonization. This was accomplished by utilising thin layers of polyurethane to decouple the saliva from the presented UHF sensor (biofilm growth is known to increase layer hydrophobicity). This presented sensor has better functionality within the US UHF frequency band as it detects changes above 5 μm. If there is a need for implantation within additional tissues with variable dielectric properties, a shunt capacitance of 2.6 pF could allow the system functionality within the permittivity range of 21 to 58. Allowing for immediate medical intervention before medical prosthesis failur

Alternative Oxidase – Aid or obstacle to combat the rise of fungal pathogens?

Fungal pathogens present a growing threat to both humans and global health security alike. Increasing evidence of antifungal resistance in fungal populations that infect both humans and plant species has increased reliance on combination therapies and shown the need for new antifungal therapeutic targets to be investigated. Here, we review the roles of mitochondria and fungal respiration in pathogenesis and discuss the role of the Alternative Oxidase enzyme (Aox) in both human fungal pathogens and phytopathogens. Increasing evidence exists for Aox within mechanisms that underpin fungal virulence. Aox also plays important roles in adaptability that may prove useful within dual targeted fungal-specific therapeutic approaches. As improved fungal specific mitochondrial and Aox inhibitors are under development we may see this as an emerging target for future approaches to tackling the growing challenge of fungal infection

Elevated Levels of Mislocalised, Constitutive Ras Signalling Can Drive Quiescence by Uncoupling Cell-Cycle Regulation from Metabolic Homeostasis

The small GTPase Ras plays an important role in connecting external and internal signalling cues to cell fate in eukaryotic cells. As such, the loss of RAS regulation, localisation, or expression level can drive changes in cell behaviour and fate. Post-translational modifications and expression levels are crucial to ensure Ras localisation, regulation, function, and cell fate, exemplified by RAS mutations and gene duplications that are common in many cancers. Here, we reveal that excessive production of yeast Ras2, in which the phosphorylation-regulated serine at position 225 is replaced with alanine or glutamate, leads to its mislocalisation and constitutive activation. Rather than inducing cell death, as has been widely reported to be a consequence of constitutive Ras2 signalling in yeast, the overexpression of RAS2S225A or RAS2S225E alleles leads to slow growth, a loss of respiration, reduced stress response, and a state of quiescence. These effects are mediated via cAMP/PKA signalling and transcriptional changes, suggesting that quiescence is promoted by an uncoupling of cell-cycle regulation from metabolic homeostasis. The quiescent cell fate induced by the overexpression of RAS2S225A or RAS2S225E could be rescued by the deletion of CUP9, a suppressor of the dipeptide transporter Ptr2, or the addition of peptone, implying that a loss of metabolic control, or a failure to pass a metabolic checkpoint, is central to this altered cell fate. Our data suggest that the combination of an increased RAS2 copy number and a dominant active mutation that leads to its mislocalisation can result in growth arrest and add weight to the possibility that approaches to retarget RAS signalling could be employed to develop new therapies

CO2 enhances the formation, nutrient scavenging and drug resistance properties of C. albicans biofilms

C. albicans is the predominant human fungal pathogen and frequently colonises medical devices, such as voice prostheses, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is the elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance, the effect of CO2 on C. albicans biofilm growth has not been investigated to date. Here we show that physiologically relevant CO2 elevation enhances each stage of the C. albicans biofilm-forming process: from attachment through maturation to dispersion. The effects of CO2 are mediated via the Ras/cAMP/PKA signalling pathway and the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown under elevated CO2 conditions also exhibit increased azole resistance, increased Sef1-dependent iron scavenging and enhanced glucose uptake to support their rapid growth. These findings suggest that C. albicans has evolved to utilise the CO2 signal to promote biofilm formation within the host. We investigate the possibility of targeting CO2-activated processes and propose 2-deoxyglucose as a drug that may be repurposed to prevent C. albicans biofilm formation on medical airway management implants. We thus characterise the mechanisms by which CO2 promotes C. albicans biofilm formation and suggest new approaches for future preventative strategies

Ras signalling in pathogenic yeasts

The small GTPase Ras acts as a master regulator of growth, stress response and cell death in eukaryotic cells. The control of Ras activity is fundamental, as highlighted by the oncogenic properties of constitutive forms of Ras proteins. Ras also plays a crucial role in the pathogenicity of fungal pathogens where it has been found to regulate a number of adaptions required for virulence. The importance of Ras in fungal disease raises the possibility that it may provide a useful target for the development of new treatments at a time when resistance to available antifungals is increasing. New findings suggest that important regulatory sequences found within fungal Ras proteins that are not conserved may prove useful in the development of new antifungals. Here we review the roles of Ras protein function and signalling in the major human yeast pathogensandand discuss the potential for targeting Ras as a novel approach to anti-fungal therapy

Precision Antifungal Treatment Significantly Extends Voice Prosthesis Lifespan in Patients Following Total Laryngectomy

Indwelling silicone valves called voice prostheses (VPs) are the gold standard for speech rehabilitation in patients with laryngeal cancer following total laryngectomy. Reported VP lifespans amongst these patients are highly variable but when devices fail patients experience loss of voice and an increase risk of chest infection. Early failure of VP is a current clinical concern that is associated with regular hospital visits, reduced quality of life and associated medical cost. Poly-microbial biofilms comprised of both bacterial and fungal microorganisms readily colonize VPs and are linked to loss of device performance and its early failure in addition to providing a reservoir for potential infection. Our detailed analysis of poly-microbial biofilm composition on 159 early failing VPs from 48 total laryngectomy patients confirmed Candida albicans as the predominant fungal species and Staphylococcus aureus as the most common bacterial colonizer within our patient cohort. Using a combination of microbiological analysis, patient data and a high-throughput antifungal test assay mimicking in vivo conditions we established an evidence based precision antifungal treatment approach to VP management. Our approach has allowed us to implement a personalized VP management pathway, which increases device in situ lifespan by an average of 270%. Our study represents a significant step forward in both our understanding of the cause of VP failure and a new effective treatment pathway that offers tangible benefit to patients

Passive Wireless UHF RFID Antenna Label for Sensing Dielectric Properties of Aqueous and Organic Liquids

The in situ wireless sensing of dielectric properties for organic aqueous solutions with a wide range of relative permittivities is presented. The use of a UHF passive label antenna design attached to either clear borosilicate glass bottle or petri plate is proposed and which allows for the unobtrusive, safe monitoring of the liquid solutions. The meandered dipole antenna (with a parasitic loop matching component) frequency is highly reliant on the chosen container as well as on the liquid present within, and adjusts with shifting dielectric properties. Tested solutions of high relative permittivity (such as water) along with low permittivity, lossy liquids (such as xylene) presented distinctive frequency characteristics with read distances of up to 7 meters for each type of container tested. The sensor was also able to detect ‘unknown’ solutions and determine the dielectric properties by utilizing standard curve analysis with an accuracy of ± 0.834 relative permittivity and ± 0.050 S·m-1 conductivity (compared to a standard dielectric measurement system available on the market). With the accuracy known, tuning the design to fit any necessary frequency is possible as a means to detect specific changes in any one liquid system. This sensor is a possible candidate for discreet real-time monitoring of liquid storage containers and an alternative for low-cost bulk liquid dielectric property identification which could be implemented in areas requiring, constant, or remote monitoring as needed

A Passive UHF RFID Dielectric Sensor for Aqueous Electrolytes

The one step modification of a commercial RFID sensing tag is demonstrated using polydimethylsiloxane based thin film chemistry to construct reusable passive RFID sensors for changes in the dielectric properties of electrolyte solutions as a function of concentration. The effects of PDMS film thickness were characterized as a function of RFID sensor code value. The output sensor code of the RFMicron RFM2100-AER wireless flexible moisture sensor (taken between 800-860 MHz) was compared to readings taken when the tag was dry and when the tag had a water deposition on the sensor area. The effect of the direct application of liquid water to the tag was to alter the capacitance presented to the integrated chip which auto-tunes to correct for the reactance. By varying the thickness of the PDMS film between the interdigitated sensor and deposited liquid, the sensitivity of the tag to a high dielectric medium could be controlled. Aqueous salt solutions were tested on a 500 m thickness film. It was found that the sensing platform could be used as a means of measuring the concentration of various salt solutions within the range 0-2M, and in turn could be used as a passive UHF RFID dielectric measuring tool. The measurement capability of the platform was subsequently demonstrated using a reduced frequency range (845-865 MHz)