bIn recent decades, pathogenic fungi have become a serious threat to human health, leading to major efforts aimed at characterizing new agents for improved treatments. Promising in this context are antimicrobial peptides produced by animals and plants as part of innate immune systems. Here, we describe an antifungal defensin, NaD1, with activity against the major human pathogen Candida albicans, characterize the mechanism of killing, and identify protection strategies used by the fungus to survive defensin treatment. The mechanism involves interaction between NaD1 and the fungal cell surface followed by membrane permeabilization, entry into the cytoplasm, hyperproduction of reactive oxygen species, and killing induced by oxidative damage. By screening C. albicans mutant libraries, we identified that the high-osmolarity glycerol (HOG) pathway has a unique role in protection against NaD1, while several other stress-responsive pathways are dispensable. The involvement of the HOG pathway is consistent with induction of oxidative stress by NaD1. The HOG pathway has been reported to have a major role in protection of fungi against osmotic stress, but our data indicate that osmotic stress does not contribute significantly to the adverse effects of NaD1 on C. albicans. Our data, together with previous studies with human beta-defensins and salivary histatin 5, indicate that inhibition of the HOG pathway holds promise as a broad strategy for increasing the activity of antimicrobial peptides against C. albicans.

Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata, has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum. The resistance of agp2⌬ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2⌬ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.

Elevated atmospheric CO 2 (eCO 2) affects soil-plant systems by stimulating plant growth, rhizosphere processes and altering the amount and quality of organic matter inputs. This study examined whether these plant-mediated processes indirectly influence the structure and function of soil microbial communities and soil carbon (C) and nitrogen (N) cycling. Surface soils (0-5 and 5-10 cm) of Calcarosol, Chromosol and Vertosol were sampled after 5 years' exposure to either ambient CO 2 (aCO 2 ; 390 ppm) or eCO 2 (550 ppm) using free-air CO 2 enrichment (SoilFACE). Changes in microbial community structure were not detected using automated ribosomal intergenic spacer analyses (ARISA). However, quantitative PCR of targeted organic C decomposition (cu, cbh), N mineralisation (apr, npr), nitrification (amoB, amoA, norA) and denitrification (nirK, narG, nosZ) genes showed that eCO 2 reduced the abundance of half of the functional genes in the Chromosol and Vertosol and their abundance was tightly coupled with total C and N pools. In the Chromosol, total N and C of soil (<2 mm particles) was reduced by up to 20% and was associated with enhanced microbial activity (soil respiration). Soil C was also reduced in the Vertosol (15%, 5-10 cm); however greater laccase, reduced cellulase and lower microbial activity indicated that organic matter decomposition was currently limited by N. The loss of soil organic N and C under eCO 2 was likely driven by greater N demand. This study highlighted that the indirect effects of eCO 2 on functional capacity of soil microbial communities in dryland agricultural system depended on the soil type.

Water quality is largely influenced by the abundance and diversity of indigenous microbes present within an aquatic environment. Physical, chemical and biological contaminants from anthropogenic activities can accumulate in aquatic systems causing detrimental ecological consequences. Approaches exploiting microbial processes are now being utilized for the detection, and removal or reduction of contaminants. Contaminants can be identified and quantified in situ using microbial whole-cell biosensors, negating the need for water samples to be tested off-site. Similarly, the innate biodegradative processes can be enhanced through manipulation of the composition and/or function of the indigenous microbial communities present within the contaminated environments. Biological contaminants, such as detrimental/pathogenic bacteria, can be specifically targeted and reduced in number using bacteriophages. This mini-review discusses the potential application of whole-cell microbial biosensors for the detection of contaminants, the exploitation of microbial biodegradative processes for environmental restoration and the manipulation of microbial communities using phages.