Investigating the over sporulation effect of A. fumigatus induced by P. aeruginosa
Oscar Herrera1, Ethan Zheng2, Yun Wang2
1Department
of Chemistry, Northeastern Illinois University, Chicago, IL; 2Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL.
Abstract
Results
Spores are found mostly everywhere within the air, Aspergillus fumigatus
produces spores that can harm immunocompromized patients. It is essential to
understand the pathway that leads to spore production, since a potential inhibitor
can be developed to aid those affected by this fungi specie. The purpose of this
investigation between co-culture interaction focuses on the induction effects and
identification of key components that trigger such mechanism. We approached
the analysis by conducting a series of co-culture crude extracts as treatments for
AF-WT, and additionally recording the amount of spores produced. Our data
demonstrates the phenomena of induction and morphological changes . Further
research will precede to investigate the components responsible that favor oversporulation for each phenotype.
Discussion
• Induction of AF-WT sporulation by PA14-BB occurs
predominantly in the outer regions of the fungal lawn for D-3
PA14-BB combined-extract treatment.
• The mechanism pathway for the component responsible for
over sporulation remains unclear. A more defined extraction
method at early and late time points needs to be performed to
further constrain the component candidates and induction
conditions.
• Research in understanding the mechanism of reaction for the
induction-component combined with the previous outcome
would give us a better understanding about this cross species
interaction which is important in developing novel therapy
strategies for patients affected by pulmonary aspergillosis.
Figure-2. Bacterial treatment-extracts with
respect to time (h.p.i).
D3 PABB vs AFWT
combined extract
D5 PABB vs AFWT
water extract
D10 PABB vs AFWT
water extract
D5 PABB only
water extract
D5 PABB only
Chloroform extract
Introduction
Negative control
YPD medium
Table-1 treatment extracts and strains.
2.5cm
Last year we conducted research that
supported the theory of bacterial
strain Pseudomonas aeruginosa has
indeed an induction effect of oversporulation on A. fumigatus. This year,
our research focuses on the search for
the mechanism of over-sporulation
effect on AF-WT induced by PA14-BB.
Different extracts from the co-culture
conditions were performed and used
as treatment on fungal culture, aiming
to find a proper condition to observe
the over sporulation of the
phenotypes.
Figure-1. Induction effect of
different bacterial-mutants in
A. fumigatus
Methods
• Preparation of fungal-bacterial co-culture petri-dishes.
• Pre-grow fungus for 36 hrs, at the same time co-culture fungal pre-grow
(12hr) and bacterial growth (24hr).
• Extract the co-cultures with milli-Q water and Chloroform(CHCl₃) at
multiple time points to be used as treatments.
• Treat fungal culture AF-WT with different extracts(2ml) using a reservoirtechnique to deposit the liquid inside the petri-dish.
• Monitor the morphological development of the fungal lawn by taking
pictures in the form of scans and count the number spores produced.
Figure-4. Spore formation after 72 hpi for
D-3 treatment extract and control.
Figure-5. Spore formation after 144 hpi for
D-3 treatment extract and control.
Figure-3. Regions from
which spore samples
were taken from.
• Figure-2 A total of five treatments with the control were scanned over a time period of 144 H.P.I. As time progressed, a
morphological difference is observed among the extracts from various co-culture stages. With only the D-3 PA14-BB extract
treatment displaying a dark blue color after an inhibitory zone, we observed the similar over sporulation effect as previous
co-culture experiment.
• Figure-4 Over-sporulation is observed in the outer region of the fungal lawn similar as our previous experiment (Figure-1)
which indicates that induction did occur (2.49E^5 spores/mm2). In contrast the inner region of the fungal lawn that surrounds
the reservoir of treatment with D-3 PA14-BB combined-extract displayed an inhibitory effect (lower spore production at
1.00E^4 spores/mm2). The control (no addition) shows similar values of spore production for both its inner area at 6.00E^4
spores/mm2 and outer area at 5.50E^4 spores/mm2.
• Figure-5 After 144 H.P.I, the induction effect sets to be at a stable number spores with 2.10E^4 spores/mm2 for the outer
region of D-3 PA14-BB combined-extract. The inner region displayed about 2.50E^4 spores/mm2 , which remains to exhibit an
inhibitory behavior. The control (no addition) shows similar amounts of spores produced since 72 H.P.I. at the inner region
with 6.01E^4 spores/mm2 and the outer region at about 5.51^4 spores/mm2 .
• This was the first trial for the set of combination strains with extracts, the component for induction of over-sporulation
remains unknown.
References
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Acknowledgements
Special thanks to: Ethan Zheng, Dr. Wang, Dr. Fant, Dr. Larkin, Dr.
Williams, the Chicago Botanic Garden REU program, and the National
Science Foundation for their support, mentoring and funding.