Bacterial-fungal interactions via redox-active small molecules

PI: Yun Wang, Civil and Environmental Engineering

Students: He Zheng, Mathew Liew, Oscar Herrera

Project overview

Bacteria and fungi are the lungs of our planet. Despite their widespread interactions in nature and the clinical environment, little is known about the molecular mechanisms underlying these interactions and their potential impacts on human and ecosystem health. In particular, secreted redox-active molecules are increasingly recognized to mediate many types of interactions between bacteria and fungi, and thus can influence each organism’s behavior and physiology under such a competitive and poly-microbial encounter . These redox-active molecules, e.g., bacteria-produced phenazines, fungi-produced gliotoxin and anthraquinones, are traditionally viewed as “toxins” to fend off their competitors via catalyzing the production of reactive oxygen species. However, more recent studies indicate it is hardly to be a one-sided story because they are often present at concentrations below their toxic thresholds, and interestingly, some are produced under oxygen-limited conditions. These lead us to hypothesize that secreted redox-active molecules may play diverse even opposite roles in bacterial-fungal interactions depending on a variety of factors, including their chemical structures, concentrations, environmental cues (such as pH, oxygen tension) and the ecological niches their producers and non-producers live in. For example, the same molecules that can trigger killing under one scenario might shift to benefit the development of the mixed species communities as a whole in response to the changes of environmental and physiological conditions. To test our hypothesis, we select to study interactions between two model organisms, Pseudomonas aeruginosa and Aspergillus fumigatus, the ubiquitous opportunistic bacterial and fungal pathogens, respectively, via secreted redox-active “toxins”, in particular, phenazine molecules. By combining physiological, genetic and metabolic profiling strategies, we aim to unravel the mechanisms that contribute to the “friend or foe” dynamic response in the small molecule-mediated mixed species interactions. Approach

Bacterial and fungal strains used in this study are listed in Table 1. To perform co-culture biofilm interaction experiments, P. aeruginosa was inoculated onto preformed lawns of A. fumigatus by spotting 10 µl aliquotesm of late exponential phase cultures onto the surface of the plate-grown fungal culture. The A. fumigatus lawns were prepared by spreading 3 ml of fungal spores (106 per ml) followed by incubation at 25oC for 12 hours. After inoculation with P. aeruginosa, the cocultures were incubated at 25 oC for an additional 8 days.

To record P. aeruginosa-A. fumigatus development, cocultures were imaged using a scanner or a digital microscope, and colony diameter growth was followed for P. aeruginosa and conidia formation was followed for A. fumigatus. Production of phenazine derivatives and gliotoxin was analyzed using an HPLC or LC-MS upon full plate extraction with 0.01% tween water and chloroform. Phenazine accumulation within fungal cells was characterized using an epifluorescence microscope.

Major findings To test our hypothesis that different phenazines may affect bacterial-fungal interactions in different ways, we first followed P. aeruginosa and A. fumigatus biofilm development over time in the co-culture experiments performed on solid medium. We observed that A. fumigatus AF293 could greatly inhibit the colony development of the phenazine-null strain of P. aeruginosa PA14 (Δphz), compared to the control condition without fungi (Figs 1, 2). This inhibition can be differentially rescued upon phenazine production. In particular, the PA14 pyocyanin- overproducing mutant exhibited the most efficient rescue, and followed by the PA14 wild type and the pyocyanin-null strains (Figs 1, 2). The phenazine-dependent effect on PA14 colony biofilm formation was clearly induced by the presence of A. fumigatus AF293, because the colony formation for all these PA14 strains was about the same in the absence of AF293 (Fig 2).

We also found that different PA14 strains differentially affected A. fumigatus AF293 development reflected by asexual spore (conidia) formation. Specifically, the phenazine-null strain (Δphz) showed no effect on AF293 conidia formation compared to the control condition without bacteria (Figs 1, 3). The phenazine-producing PA14 wild type and the pyocyanin-null strains dramatically decreased AF293 conidia formation, in accordance with the competitive aspect to enhance their own development (Figs 1, 3). In contrast, the pyocyanin-overproducing strain significantly increased AF293 conidia formation along with eliciting its own colony development (Figs 1, 3). To further correlate the effects on P. aeruginosa and A. fumigatus biofilm development with specific phenazine(s), we simultaneously analyzed phenazines and possible novel metabolites being released into the co-culture plates using HPLC and LC-MS upon plate extraction with water and chloroform. We showed that pyocyanin was only produced by its over-producing PA14 strain, but not by others (Fig 4). Note that PA14 wild type has the genetic component to synthesize pyocyanin, even though pyocyanin was not produced under our experimental conditions. These results indicated that pyocyanin might play the major role in triggering P. aeruginosa colony development together with A. fumigatus conidia formation. While other phenazines, such as phenazine-1-carboxamide (PCN), can rescue its producer’s colony formation defect by inhibiting AF293 development, complying with antifungal activity. Moreover, the A. fumigatus fungus can modify phenazine-1-carboxylic acid (PCA) into at least three other phenazines (Fig 5). We are currently investigating the biological activities of these novel compounds.

In summary, our findings imply that phenazines have profound and diverse effects on each organism’s behavior and physiology, thus contribute to shaping the ecological structure under such a competitive and mixed-species encounter. The mechanistic nature of our approach will inevitably lead us into areas that are as relevant for medicine as for environmental sciences and engineering.

Outputs Although we initially proposed to study the roles of redox-active macromolecular materials in bacterial and fungal physiology, we decided to switch our focus to small molecules because analyzing and extracting these molecules are more technically manageable. These small molecules by no means less important in nature and the clinical environment, with potential outcomes in human and ecosystem health, biogeochemistry and contaminant dynamics. With the ISEN support, we were able to initiate this research project in a very productive manner. We have presented the work at two national conferences, prepared one manuscript to a highly reputable journal. We are also currently preparing an NIH proposal and another proposal to CF foundation. In addition to training one Ph.D. student, the ISEN support allowed my lab to host two undergraduate students to gain research experience. Among the two, Oscar Herrera is a minority student at Northeastern Illinois University. The project exposed Oscar the first time to a research lab. He enjoyed so much and he will come back as an CBG-REU student this summer.

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Table 1. Bacterial and fungal strains used in this study

Strain or plasmid Properties Reference or source

Strains P. aeruginosa bacteria

PA14 Clinical isolate UCBPP-PA14, wild type strain

(1)

PA14 Δphz PA14 with deletions of operons phzA1-G1 and phzA2-G2

(2)

PA14 bigblue PA14 phzM::TnM

PA14 merodiploid strain that overproduces pyocyanin PA14 pyocyanin-null mutant

(3) (4)

A. fumigatus fungi

AF293 Wild type strain Keller NP, UW-Madison

TDWC5.44 (ΔgliZ) Gliotoxin-null mutant Keller NP, UW-Madison

TJW54.2 (ΔlaeA) Secondary metabolite-null mutant Keller NP, UW-Madison

Figure 1. P. aeruginosa bacteria and A. fumigatus fungi affect each other’s development with phenazine molecules in co-culture interactions.

day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 10

big blue

wild type

phzM::TnM

!phz

control

Figure 2. A. fumigatus inhibits P. aeruginosa colony development, which can be differentially rescued by P. aeruginosa PA14 strains with distinct phenazine production capabilities.

Figure 3. A. fumigatus conidia formation can be promoted or retarded, depending on the release of phenazines by P. aeruginosa in co-culture interactions.

Figure 4. phenazine production patterns indicate that different phenazines may have different even opposite effects on P. aeruginosa-A. fumigatus development in co-culture interactions.

Figure 5. A. fumigatus can convert phenazine-1-carboxylic acid (PCA) into three other phenazines in co-culture interactions.

#2,

#1,