Exploiting interkingdom interactions for development of small-molecule inhibitors of candida albicans biofilm formation

Article abstract of DOI:10.1128/AAC.00190-16

A rapid decline in the development of new antimicrobial therapeutics has coincided with the emergence of new and more aggressive multidrug-resistant pathogens. Pathogens are protected from antibiotic activity by their ability to enter an aggregative biofilm state. Therefore, disrupting this process in pathogens is a key strategy for the development of next-generation antimicrobials. Here, we present a suite of compounds, based on the Pseudomonas aeruginosa 2-heptyl-4(1H)-quinolone (HHQ) core quinolone interkingdom signal structure, that exhibit noncytotoxic antibiofilm activity toward the fungal pathogen Candida albicans. In addition to providing new insights into what is a clinically important bacterium-fungus interaction, the capacity to modularize the functionality of the quinolone signals is an important advance in harnessing the therapeutic potential of signaling molecules in general. This provides a platform for the development of potent next-generation small-molecule therapeutics targeting clinically relevant fungal pathogens.

Full text of DOI:10.1128/AAC.00190-16

AAC Accepted Manuscript Posted Online 25 July 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.00190-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Antimicrobial Agents and Chemotherapy: Article
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Exploiting interkingdom interactions for the development of
small molecule inhibitors of Candida albicans biofilm
formation
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F. Jerry Reen1, John P. Phelan1, Lorna Gallagher1, David F. Woods1, Rachel M. Shanahan2,
Rafael Cano2, Eoin Ó Muimhneacháin2, Gerard P. McGlacken2 and Fergal O’Gara1,3#
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BIOMERIT Research Centre, School of Microbiology, University College Cork - National
University of Ireland, Cork, Ireland.
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School of Chemistry and Analytical and Biological Chemistry Research Facility (ABCRF),
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University College Cork - National University of Ireland, Cork, Ireland.
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3 School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University,
Perth, WA 6102, Australia.
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Running Head: Hydroxy Alkylquinolone signals target Candida biofilm.
FJR and JPP contributed equally to this work.
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# To whom correspondence should be addressed. Mailing address: Prof. Fergal O’Gara,
BIOMERIT Research Centre, School of Microbiology, University College Cork, Ireland. Phone
number: + 353-21-4901315; Fax number: + 353-21-4275934; E. mail: f.ogara@ucc.ie.
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Abstract
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A rapid decline in the development of new antimicrobial therapeutics has coincided with the
emergence of new and more aggressive multidrug resistant pathogens. Pathogens are protected
from antibiotic activity by their ability to enter an aggregative biofilm state. Therefore, disrupting
this process in pathogens is a key strategy for the development of next generation antimicrobials.
Here we present a suite of compounds, based on the Pseudomonas aeruginosa 2-heptyl-4(1H)-
quinolone (HHQ) core quinolone interkingdom signal structure, that exhibit non-cytotoxic anti-
biofilm activity towards the fungal pathogen Candida albicans. In addition to providing new
insights into what is a clinically important bacterial-fungal interaction, the capacity to modularize
the functionality of the quinolone signals is an important advance in harnessing the therapeutic
potential of signaling molecules in general. This provides a platform for the development of
potent next generation small molecular therapeutics targeting clinically relevant fungal
pathogens.
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Introduction
With the ever increasing emergence of antibiotic resistant pathogens and the lack of new
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antibiotics coming to market, we are entering a ‘post-antibiotic era’ (1-3). This realization has
underpinned a global initiative to identify new and innovative approaches to infection
management. As such, targeting virulence as a potential strategy for developing new anti-
microbial drugs has been the focus of several research initiatives (4-11). In principle, suppressing
virulence behavior and locking pathogens in a vegetative non-biofilm forming lifestyle renders
them less infective and more susceptible to conventional antibiotics (4, 12). While some success
has been achieved against bacterial pathogens (6, 10, 13-19), less focus has been placed on
fungal infections which nevertheless continue to cause serious complications and mortality in
patients (8, 20-22). Indeed, despite the medical and economic damage caused by fungal biofilms,
there remains an urgent and largely unmet need for the identification of compounds able to
specifically and selectively target and inhibit this mode of growth in clinically relevant fungal
pathogens (23).
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The predominant nosocomial fungal pathogens, which include Candida spp., Aspergillus spp.,
and Fusarium spp., are difficult to diagnose and cause high morbidity and mortality, even
following antifungal therapy (21). Candida albicans causes a variety of complications ranging
from mucosal disease to deep seated mycoses, particularly in immunocompromised individuals
(21, 24). Along with other fungal and yeast pathogens, C. albicans are known to form structured
communities known as biofilms on medical devices either pre- or post-implantation leading to
recurring infections and in some cases death (25, 26). Once established in the biofilm phase, C.
albicans presents a significant clinical problem with current treatment options severely limited
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by the intrinsic tolerance of fungal biofilms to antimycotics (20, 27, 28). Recent combination
therapies incorporating antibacterial and antifungal agents have provided some success (29).
However, as with all antibiotic based strategies, reports of resistance continue to emerge (27),
and biofilms themselves are considered a breeding ground for the emergence of antibiotic
resistant strains, effectively hastening the onset of the perfect storm whereby the rapid decline in
new antibiotic production has been met by an equally rapid increase in multidrug resistant
organisms (1). Thus the need to consider new anti-infective strategies that do not target essential
processes in the target organism. While blocking biofilms in these organisms’ remains a major
clinical challenge (26, 30), exploiting our increased understanding of microbial signaling
networks to control virulence and biofilm behavior is one innovative approach with significant
potential.
Many sites of infection are colonized by communities of mixed fungal and bacterial organisms,
and several layers of communication impact significantly on the dynamics and flux of these
populations (31, 32). For example, C. albicans is known to co-exist with Pseudomonas
aeruginosa in the cystic fibrosis (CF) lung, and interkingdom communication between the two
organisms has previously been reported (16, 33). The Pseudomonas Quinolone Signal (PQS), 2-
heptyl-3-hydroxy-4-quinolone, and its biological precursor 2-heptyl-4-quinolone (HHQ) are
important virulence factors produced by P. aeruginosa. Structurally, PQS and HHQ differ by the
presence of a hydrogen at C3 in HHQ and a hydroxyl group in PQS, giving rise to the increased
interest in modulating this position to assign biological function to the structure of these
molecules (34-37). Previously, we have shown that HHQ, but not PQS, suppresses biofilm
formation of C. albicans (10). In response, C. albicans produces farnesol which has been shown
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to modulate PQS production in P. aeruginosa (33). As both PQS and HHQ promote virulence
and pathogenicity of P. aeruginosa (38, 39), their utility as an anti-Candida treatment falls short
of being a viable anti-fungal treatment. However, the amenability of these small molecules to
chemical modification provides an opportunity to develop compounds with specificity of
function.
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The transcriptional data and microscopic imaging described in this study have implicated
components of the cell wall as key factors in the response of C. albicans to P. aeruginosa
alkylhydroxyquinolone (AHQ) signaling. Furthermore, the biological activity of each class of
analogue in bacterial, fungal and host systems provides a new insight into the possible
interkingdom role of AHQs, particularly in a clinical setting such as the CF lung where all three
systems co-exist. From a translational perspective, lead HHQ analogues were identified with four
key features: (1) potent anti-biofilm activity towards C. albicans, (2) selective non-cytotoxicity
towards mammalian cell lines, (3) non-agonistic and (4) potentially antagonistic to the virulent
pathogen P. aeruginosa. Several analogues retained the significant potency of the parent HHQ
molecule against C. albicans biofilm formation, whilst simultaneously becoming inactive in P.
aeruginosa quorum sensing. This suggests that these molecules have the potential to be further
optimized for use as anti-Candida infectives without the concomitant limitation of P. aeruginosa
virulence augmentation.
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Materials and Methods
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C. albicans stock maintenance and culturing conditions.
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C. albicans strain SC5314 was sub-cultured from 15% (v/v) glycerol stocks at -80oC onto Yeast
Peptone Dextrose (YPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v)
dextrose] and incubated at 30°C overnight.
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P. aeruginosa stock maintenance and culturing conditions.
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P. aeruginosa strains, PAO1 and pqsA- mutant, containing the chromosomally inserted pqsA-
lacZ promoter fusion on plasmid pUC18-mini-Tn7, were maintained on Luria Bertani (LB) agar
plates, supplemented with carbenicillin (200 µg/ml) and X-gal (40 µg/ml), and incubated at 37°C
overnight. Single colonies were inoculated into LB broth (20 ml), supplemented with
carbenicillin (200 µg/ml), and incubated at 37°C, shaking at 180 rpm overnight. For subsequent
experiments, the OD600nm was recorded and a starting OD600nm of 0.02 was inoculated into fresh
LB broth, supplemented with carbenicillin (200 µg/ml) and incubated at 37°C, shaking at 180
rpm.
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Structural modification of HHQ
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The synthesis of HHQ, PQS (40, 41) and other HHQ-based analogues (36, 37) was carried out
via previously described methods. Novel compounds and compounds which required modified
syntheses are described vide infra and in the supporting information (Supplemental Data).
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Thin Layer Chromatographic (TLC) analysis.
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Silica TLC plates, activated by soaking in 5% (w/v) K2HPO4 for 30 min were placed in an oven
at 100oC for 1 hr (42). Analogues (5 µl, 10 mM) were spotted approximately 1 cm from the
bottom. The spots were dried and the plate placed in a mobile phase comprising 95:5
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dichloromethane:methanol. The plate was viewed under UV light when the mobile phase had run
5 cm below the top of the plate.
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Biofilm formation, quantification, and visualization.
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C. albicans biofilm formation was carried out in 96 well plates, as previously described (43).
Seeding densities for all subsequent experiments (n=3) were OD600nm 0.05. Biofilm formation
was measured as previously described using a semi-quantitative tetrazolium salt, 2,3-bis-(2-
methoxy-4-nitro-5-sulfophenyl)-2H tetrazolium-5-carboxanilide inner salt (XTT) reduction assay
(44). Experiments were repeated at least three times, with at least eight technical replicates.
Visualization of biofilm formation was performed on glass coverslips in 6-well plates using
Confocal Scanning Laser Microscopy (CSLM). All images were captured using the Zeiss HBO-
100 microscope illuminating system, processed using the Zen AIM application imaging program
and converted to JPGs using Axiovision 40 Ver. 4.6.3.0. A minimum of three independent
biological repetitions were carried out.
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Viable colony biofilm assay
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C. albicans biofilms, supplemented with analogues and parent compounds, were grown in 6-well
plates and incubated overnight at 37°C. Briefly, C. albicans Yeast Nitrogen Base (YNB) cultures
were measured at OD600nm, diluted to 0.05 in YNB-NP supplemented with analogues, plated onto
6-well plates and incubated for 1 hr at 37oC. Media was removed, wells were washed twice with
sterile PBS and supplemented with fresh YNB-NP with analogues. Plates were incubated
overnight at 37°C after which media was removed and wells washed with sterile PBS. For serial
dilutions, biofilms were cell-scraped into 1 ml PBS, vortexed, and serially diluted into sterile
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PBS. Serial dilutions were plated (100 µl) onto YPD agar and incubating overnight at 37°C.
Colonies were counted and recorded the next day.
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C. albicans growth curves
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Overnight C. albicans cultures grown in YNB were diluted to 0.05 in YNB supplemented with
analogues. Cultures (200 µl) were added to each well of a 100 well plate and grown for a 24 hr
period on a Bioscreen C spectrophotometer (Growth Curves USA).
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RNA isolation and qRT-PCR transcriptional analysis.
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Overnight C. albicans cultures were diluted to 0.05 at OD600nm in either YNB or YNB-NP
(Difco). YNB cultures were supplemented with methanol whereas YNB-NP cultures were
supplemented with either 100 µM HHQ or the methanol volume equivalent. Cultures were
grown at 37ºC with agitation (180 rpm) for 6 hr after which they were centrifuged at 4000 rpm,
supernatants discarded and pellets frozen at -20ºC until processing. RNA was isolated using the
MasterPure Yeast RNA purification kit (Cambio Ltd, Cambridge UK) according to
manufacturer's specifications, and was quantified using a ND-1000 Spectrophotometer
(NanoDrop Technologies, USA). Genomic DNA was enzymatically removed using Turbo DNA-
free DNase (Ambion), and samples were confirmed DNA free by PCR. RNA was converted to
cDNA using random primers and AMV reverse transcriptase (Promega) according to
manufacturer's instructions. qRT-PCR was carried out using the Universal ProbeLibrary (UPL)
system (Roche) according to manufacturer's specifications, and samples were normalized to C.
albicans actin transcript expression (ACT1). A full list of primers and UPL probes used in this
study is detailed in Supplemental Table 1.
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Phenazine extraction.
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P. aeruginosa strains were cultured as described above for 24 hr, with the addition of analogues
(100 µM), and pyocyanin was extracted as described previously (45). Cultures were centrifuged
at 4000 rpm for 10 minutes and the cell free supernatant (5 ml) removed. Chloroform (3 ml) was
added, and mixed by vortex. After centrifugation at 4000 rpm for 5 min, the lower aqueous phase
was transferred to 0.2 M HCl (2 ml). Samples were mixed by vortex and centrifuged at 4000 rpm
for 5 min to separate the phases. An aliquot of the top phase (1 ml) was removed and
spectrophotometrically analyzed at OD570nm. Phenazine production was calculated using the
following formula: OD570nm x 2 x 17.072 and the units expressed in µg/ml.
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Promoter fusion based expression analysis.
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Promoter fusion analyses were performed in a 96-well format, with β-galactosidase activity
measured as described previously (46). Briefly, overnight cultures of wild-type PAO1 pqsA-lacZ
(pLP0996) and mutant strain PAO1 pqsA- pqsA-LacZ were diluted to OD600nm=0.02 in LB.
Analogues at 100 µM final concentration were added, mixed, aliquoted into 96 well plates and
incubated overnight at 37ºC with shaking. The next day, OD600nm values were recorded in a plate
reader. Aliquots of cells (0.02 ml) were permeabilized [100 mM dibasic sodium phosphate
(Na2HPO4), 20 mM KCl, 2 mM MgSO4, 0.8 mg/mL CTAB (hexadecyltrimethylammonium
bromide), 0.4 mg/mL sodium deoxycholate, 5.4 μL/mL beta-mercaptoethanol] and added to
substrate solution [60 mM Na2HPO4, 40 mM NaH2PO4, 1 mg/mL o-nitrophenyl-β-D-Galactoside
(ONPG), 2.7 μL/mL β-mercaptoethanol]. The kinetics of color development was monitored and
the reactions were stopped using 1M NaCO3. OD420nm were recorded as above. Miller units were
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calculated using the following equation; 1000 x [OD420nm/(OD600nm) x 0.02 ml x reaction time
(min)].
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Cytotoxicity Assay.
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Lactate dehydrogenase (LDH) release from a panel of mammalian cells was assayed as a
measure of cytotoxicity using an LDH colorimetric kit (Roche) according to manufacturers’
instructions (36). Briefly, IB3-1 lung epithelial cells, A549 human lung adenocarcinoma
epithelial cells, DU-145 human prostate cancer cells and HeLa cervical cancer cells were seeded
onto 96 well plates and treated with methanol (control) and analogues. Following 16 hr
incubation at 37°C and 5% CO2, supernatants were removed and added to catalyst reaction
mixture in a fresh plate and further incubated at 37°C and 5% CO2 for 30 min to allow for color
development. After this period, the plate was analyzed on an ELISA plate reader at OD490nm
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Cytotoxicity was expressed as a percentage of cells treated with 0.1% (v/v) Triton (100%
cytotoxicity).
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Statistical analysis.
All graphs were compiled using GraphPad Prism (version 5.01) unless otherwise stated. All data
were analyzed using built-in GraphPad Prism (version 5.01) functions as specified. The level of
significance was set at p = 0.05 (*) and post-hoc comparisons between groups were performed
using the Bonferroni multiple-comparison test.
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Results
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Key modifications of the quinolone framework retain anti-biofilm activity towards C.
albicans
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The HHQ molecule has previously been shown to suppress biofilm formation in C. albicans at
concentrations from 10 – 100 µM (2.47 – 24.7 µg/ml) independent of any effects on the growth
of planktonic cells (10). Previous structure function analysis of the activity of the quinolone
framework had implicated the C-3 position as a key component of interspecies anti-biofilm
activity (36). We undertook further modification of the HHQ parent molecule with the aim of
developing viable anti-biofilm compounds to target C. albicans. These compounds were
incorporated into a larger collection of alkylquinolone analogues, systematically modified at
different positions on the molecule and classified on the basis of their substitutions relative to the
parent framework HHQ (Table 1).
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The suite of analogues was first tested to establish their potency as anti-biofilm compounds
against C. albicans using an optimized XTT assay, a commonly used quantitative method to
assess Candida biofilm mass and growth (47). As previously described (10), HHQ significantly
suppressed biofilm formation when compared to untreated and methanol treated cells, whereas
PQS appeared to induce biofilm formation (Figure 1). When all analogues were similarly
screened by XTT assay, several had similar anti-biofilm activity to HHQ [1 and 2 (class I;
modified at C-3)], 3, 4, 6, 7, 9 (class II; modified anthranilate ring), and 12 (class III; modified
alkyl chain) (Figure 2a). These analogues were diverse members of classes I, II, and III
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suggesting that several components of the HHQ framework contribute to anti-biofilm activity of
the parent compound. A number of substitutions led to intermediate anti-biofilm activity,
including 5, 8, 10, 11, (class II; modified anthranilate ring) and 15 (class V; modified
anthranilate ring and alkyl chain length), while some analogues had completely lost the ability to
suppress C. albicans biofilm formation e.g. 13 (class IV; modified C-4), and the class V
compounds 14, 16, and 17 (Figure 2a). While modification of the C-3 position to produce PQS
led to loss of anti-biofilm activity (Figure 1), incorporation of an –N-NH2 moiety (2) at the 3-
position or substitution of C-3 with NH (1), did not affect the ability to suppress C. albicans
biofilm formation (Figure 2a). Addition of Cl at the C6 and C8 positions of the anthranilate ring
(6 and 7) also did not lead to loss of anti-biofilm activity. In contrast, the introduction of
considerable steric bulk with the addition of an n-hexyl alkyl chain at C6 of the anthranilate ring
(5), or elaboration of the aromatic group as with the naphthyl compound (8), resulted in
compounds with significantly less potent anti-biofilm activity relative to HHQ. These data
suggest an exquisite level of specificity for the interaction between HHQ and the C. albicans
biofilm intracellular machinery. Modification of the C2 alkyl chain from n-heptyl (HHQ) to n-
nonyl C9 (12) did not affect anti-biofilm activity, while parallel modification of the anthranilate
ring resulted in a complete loss, as with the Class V compounds 16 or 17. Modifying the C-4
position (C=O to C=S) (13), the quinolone thiol exhibited an increase in XTT activity (P<0.05)
relative to controls (Figure 2a), comparable to the increase observed in the presence of PQS
(Figure 1). Previously, we have shown that HHQ elicits a dose-dependent reduction in C.
albicans biofilm formation (10). In order to determine if this also applied to the analogues that
retain anti-biofilm activity, dose response analysis of selected compounds 1, 2, 4, 6, and 12
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representing classes I, II, and IV, was undertaken. This revealed compound specific responses,
with 10 µM of compounds 2, 4, and 12 being sufficient to elicit a statistically significant
reduction in biofilm formation (Figure 2b). All five compounds reduced biofilm formation when
applied at 50 µM and 100 µM. To further confirm the anti-biofilm activity of the lead
compounds, viable colony counts were performed on selected analogues using the maximum 100
µM compound dose. This confirmed the outputs from the XTT assays; all analogues, along with
HHQ, significantly reduced viable biofilm cells in comparison to the control (Figure S1a).
Importantly, the anti-biofilm activity was found to be independent of planktonic growth, which
was unaffected in the presence of selected compounds (Figure S1b).
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Microscopic staining reveals structural changes in C. albicans biofilms
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The formation of biofilms in bacteria, yeast and fungi is a highly ordered process involving
multicellular behavior and has been defined in several stages (22). Confocal microscopy
combined with intracellular staining was used to assess structural integrity and cellular
morphology of C. albicans incubated on coverslips. Biofilms were individually stained with each
of the dyes and multiple fields of view were visualized to accurately represent the effect of the
analogues. The biofilm observed for methanol and untreated controls displayed all the
characteristics of a typical C. albicans biofilm and were classified as wild-type (Figure 1).
Calcofluor, Concanavalin A and FUN-1 staining revealed a uniform distribution of
chitin/cellulose and cell wall mannosyl/glucosyl residues indicative of viable wild-type
morphology (Figure 1). Those analogues identified by XTT assay as causing impaired biofilm
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formation (1, 2, 3, 4, 6, 7, 9, 12, and 15) exhibited markedly disrupted structures when grown on
coverslips and were classified as atypical morphologies (Figure 3). Cells treated with class I
analogues were found to be largely compromised in their biofilm forming capabilities and were
classified as morphologically atypical. Biofilms produced with both 1 and 2 were significantly
distorted, displaying a spindle-like phenotype. Hyphae were short in length, and predominantly
displayed yeast cell types rather than hyphal structures. Other structure disrupting analogues
were from Class II, III and V, suggesting that specific modifications on the anthranilate ring and
alkyl chain variation do not significantly affect the anti-biofilm activity compared to the parent
compound (Figure 3).
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Some analogues, including those that exhibited intermediate activity in the XTT assay, did not
alter the biofilm structure, with 5, 11, 13, 14 and 16 all placed into the wild-type morphology
group. Biofilms formed in the presence of 13 showed hyper-production of short hyphae, creating
a dense mycelial network (Figure S2). The remaining analogues from Class II; 8 and 10 (Figure
S1) caused significant biofilm disruption with fragmented hyphae, stunted vegetative growth and
considerably large cell debris fields. Cells incubated with the Class V molecule 17 induced a
severely compromised phenotype (Figure S2) where debris fields comprising yeast cells and
blastospores characterized the structural phenotype.
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Enhanced gene transcript expression of HWP1, ECE1, ALS3, IHD1 and the
uncharacterized open reading frame ORF 19.2457 provides a molecular mechanism for
alkyl quinolone activity towards C. albicans
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In addition to providing new insights into the interkingdom relationship between these important
pathogens, there has recently been a strong emphasis placed on ligand receptor interactions and
the need to provide molecular mechanisms for the action of any potential therapeutic compound
(48). We previously implicated TUP1 in the HHQ-mediated suppression of biofilm formation in
C. albicans suggesting a role for the cell-wall in this interaction (10, 16). More recently, several
reports have shown changes in expression of cell-wall associated genes linked to biofilm
formation in this organism (16, 20, 28, 49-51). These included a cohort of eight genes that are
proposed to constitute the core filamentous response network; namely ALS3, ECE1, HGT2,
HWP1, IHD1, RBT1, DCK1, and the gene of unknown function open reading frame orf19.2457
(51). Therefore, transcript expression of a cohort of genes implicated in cell wall biogenesis,
hyphal development, biofilm formation and other related functions that were previously shown to
be upregulated during the morphological transition from yeast to filamentous growth was
investigated (Table S1) (16, 51). The housekeeping gene ACT1 was chosen for normalization
based on previous biofilm studies (52). We observed that several transcripts were hyper-
expressed in a HHQ dependent manner; specifically, HWP1, ECE1, ALS3, IDH1 and the as yet
uncharacterized open reading frame (ORF) 19.2457 (Figure 4). The remaining transcripts
(CPH1, EFB1, ESS1, RBT1, TUP1, BCR1, DCK1, and HGT2) yielded expression patterns
similar to control cells (Figure S3). It was perhaps somewhat surprising that, while treatment of
C. albicans with P. aeruginosa supernatants has previously been shown to downregulate
expression of the RBT1, RBT5 and RBT8 genes (16), expression of RBT1 was unaltered in the
presence of HHQ (Figure S3). Taken together, these data suggest that HHQ induces a specific
subset of cell wall proteins in C. albicans. Further work is needed to identify the upstream
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components of this response, although in silico screening of C. albicans genome sequences has
ruled out the presence of an obvious PQS receptor (unpublished data).
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Lead compounds display reduced cytotoxic activity towards specific mammalian cell lines
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Evaluating the cytotoxicity of synthetic compounds is crucial in the context of developing
targeted and highly optimized molecular therapeutics that are benign to human cellular
physiology and ideal for use in a clinical environment. In previous work, we showed that
analogue 1 was significantly less cytotoxic than HHQ, with an 80% reduction in LDH release
relative to the parent compound (36). Therefore, the suite of analogues was tested for in vitro
cytotoxicity towards IB3-1 airway epithelial cells. Class I analogues exhibited reduced
cytotoxicity to IB3-1 cells with 2 displaying approximately 34% toxicity (Figure 5a). Several
class II analogues (4, 6, and 9) exhibited reduced cytotoxicity relative to IB3-1 cells treated with
HHQ, with 7 not reaching statistical significance. The class III analogue 12 was comparable to
HHQ. Of the analogues that did not retain anti-biofilm activity, 5, 8, 10 and 11 exhibited variable
cytotoxicity to IB3-1 cells whereas 13 exhibited considerably reduced cytotoxicity to IB3-1 cells
(Figure S4). Finally, 16 exhibited very low levels of cytotoxicity, while 17 was reduced relative
to HHQ treated cells. Compound 15 was the most toxic killing approximately 91% of all cells
(Figure 5a).
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In order to achieve a more comprehensive understanding of the selective toxicity of the lead
compounds, several additional cell lines were tested (Figure 5b). LDH release assays were
performed in A549, DU145, and HeLa cell lines in the presence of 100 µM of the lead
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compounds revealed distinct cytotoxicity profiles, with 1 and 9 consistently proving the least
cytotoxic of the compounds tested. Compounds 4 and 6 exhibited reduced cytotoxicity in DU145
cells (although not statistically significant) but were comparable to HHQ in both the A549 and
HeLa cell lines, while compound 2 exhibited increased cytotoxicity relative to HHQ in DU145
cells (Table 2). These data suggest that cell-specific cytotoxicity analysis will need to be
performed prior to the introduction of these compounds in an applied setting.
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HHQ analogues display a spectrum of agonist activity towards P. aeruginosa virulence.
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Taken together, compounds 1, 4, 6, and 9 pass both the first and second criteria described above,
i.e. they retain anti-biofilm activity towards C. albicans while exhibiting reduced selective
cytotoxicity towards specific host cell lines. However, both HHQ and PQS are co-inducers of the
virulence associated LysR-Type Transcriptional Regulator; PqsR (41). The structural moieties
that underpin the interaction between HHQ/PQS and PqsR remain to be fully characterized,
although recent studies have reported diverse classes of PqsR antagonist (53-55), and implicated
the hydrophobic pocket situated within the PqsR protein (56). Therefore, in order to assess
whether the lead compounds could elicit a virulence response from P. aeruginosa, phenazine
production and pqsA promoter activity (57) were monitored in a pqsA mutant where the capacity
to produce native HHQ and PQS had been lost.
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Both HHQ and PQS restored phenazine production in the pqsA- strain (Figure 6A). In contrast,
the majority of analogues did not restore phenazine production in this strain, with the notable
exception of compound 9. Several analogues from different classes did partially restore
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phenazine production in the mutant background, including 10, 12, and 17 (Figure 6 and Figure
S5). None of the analogues interfered with phenazine production in the wild-type PAO1 strain,
suggesting that they are ineffective as PQS antagonists (Figure S5).
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Similarly, while some degree of PqsR agonist activity was observed in the presence of
compounds 6, 9, 10, 12, 13, and 17, only HHQ and PQS significantly induced promoter activity.
All other analogues did not influence promoter activity in this system (Figure 6B and Figure
S5). Somewhat surprisingly, antagonistic activity towards pqsA promoter activity was not
observed, with almost all analogues failing to significantly suppress pqsA promoter activity in
the wild-type strain (Figure S5). The relative ineffectiveness of these analogues as PQS
antagonists may in part be due to hydroxylation of HHQ analogues (H at C3) to PQS analogues
(OH at C3), thus establishing the non-antagonistic behavior explained by a recent report by Lu
and colleagues, where the action of PqsH rendered anti-PQS compounds ineffective through
bioconversion (55).
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Discussion
Current antimicrobial therapies tend to be non-pathogen-specific and there is evidence to suggest
that the availability of relatively non-toxic broad-spectrum therapies has contributed to the
emergence of resistance among both targeted and non-targeted microbes (58, 59). Consequently,
there is an urgent need to innovate new options for the targeted prevention of microbial infection
while avoiding the inevitable emergence of resistance that is the hallmark of broad spectrum
antibiotic therapies (59, 60). Increasingly, industry, academia and regulatory bodies have become
interested in single-pathogen therapies to treat highly resistant or totally resistant bacterial
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pathogens, rightly viewed as an area of high unmet need (61-63). Exploiting interkingdom
communication networks, and the mode of action of the chemical messages or signals employed
therein, offers us a powerful platform from which to deliver on this.
Previously we had shown that the HHQ interkingdom signal molecule from P. aeruginosa could
suppress biofilm formation in C. albicans at concentrations ranging from 10 - 100 µM (2.47 –
24.7 µg/ml) (10). This suppression occurred independent of any growth limitation in planktonic
cells, and morphogenesis on spider media was also found to be unaffected (10). The design and
subsequent analysis of a suite of analogues based on the core HHQ quinolone framework has led
to the identification of several lead compounds that retain anti-biofilm activity towards C.
albicans but exhibit significantly reduced cytotoxicity towards IB3-1 epithelial cells when
compared with the parent HHQ molecule (data not shown). The selective cytotoxicity of the lead
compounds together with the dose dependent anti-biofilm effects will be key considerations in
determining the cell line specific therapeutic index of lead analogues as part of the ongoing
development of these compounds. Furthermore, unlike HHQ, these lead compounds are now
inactive towards the P. aeruginosa PqsR quorum sensing system, a critical requirement for their
potential future development as anti-biofilm therapeutics. In addition, the ability to generate
hydrochloride salts of the compounds ((36) and data not shown) suggests that solubility of future
therapeutics based on these scaffolds will not be a bottleneck. Several strategies have been
proposed for the implementation of anti-biofilm compounds as clinical therapeutics to target C.
albicans biofilm infections (64). As the HHQ analogues possess anti-biofilm, but not anti-
Candida activity, they would disrupt the formation of biofilms but not likely remove the
planktonic cells that remain at the site of infection. Therefore, combination with conventional
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anti-fungal compounds would be required for effective clearance. Alternatively, where the
potency of the anti-biofilm activity can be synthetically enhanced through further derivatization,
clearance by the immune system might also be realistic.
The molecular mechanisms through which AHQs and the lead compounds identified in this study
disrupt the formation of biofilms by C. albicans remains to be fully elucidated. Previously we
have shown that HHQ does not affect adhesion, but rather impacts directly on the subsequent
developmental stages in a TUP1-dependent manner (10). In this study we have shown that the
expression of several cell-wall associated genes is increased in response to HHQ during the
switch to hyphal growth. These genes have previously been implicated in the formation of C.
albicans biofilms and have been shown to exhibit increased levels of expression during the
hyphal transition (50, 51, 65). Therefore, anti-biofilm compounds might be expected to suppress
this induction rather than enhance it. However, five of the target genes tested exhibited an
increase in expression relative to control cells under inducing conditions. This may be a
reflection of the previous observation that HHQ interferes with the later stages of biofilm
development (10). Alternatively, this hyper-expression phenotype may affect the capacity of the
cell to engineer a community based biofilm. Future studies will focus on elucidating the
pathways through which C. albicans perceives and responds to challenge with HHQ with the aim
of identifying potential therapeutic targets.
Further work using defined in vivo models of biofilm and infection will be required to progress
the development and evaluation of these small molecules as anti-biofilm compounds. Models are
now available for the investigation of infections involving medical devices such as vascular
catheters, dentures, urinary catheters, and subcutaneous implants, as well as mucosal biofilm
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infections (66). The ongoing development of cell-based or animal models to study in vivo
infections (66-69), whether as single pathogen or co-culture systems (70), has provided a well-
equipped tool-kit for the pre-clinical assessment of these AHQ-based compounds.
Conclusions
In this study, we have functionalized the important microbial signaling molecules HHQ and PQS
in order to exploit their interkingdom role to control biofilm formation in C. albicans. In addition
to deciphering further insights into the molecular mechanism through which these chemical
messages elicit a biofilm suppressive response from C. albicans, the bioactivity of several lead
compounds has provided a viable platform for the development of next generation therapeutics.
Crucially, some of these compounds are non-toxic to mammalian cells and have been rendered
incapable of activating P. aeruginosa virulence systems, thus highlighting their potential utility
as an effective therapy combatting human infection.
Author Contributions
FJR, GPM and FOG conceived and designed the investigation. FJR, JPP, LG, and DW
performed the biological experimentation, while RC, RS, and EOM conducted the chemical
synthesis. FJR, JPP and FOG wrote the manuscript and all authors read and edited the final draft.
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