A structure activity-relationship study of the bacterial signal molecule HHQ reveals swarming motility inhibition in Bacillus atrophaeus

Article abstract of DOI:10.1039/c5ob00315f

The sharp rise in antimicrobial resistance has been matched by a decline in the identification and clinical introduction of new classes of drugs to target microbial infections. Thus new approaches are being sought to counter the pending threat of a post-antibiotic era. In that context, the use of non-growth limiting small molecules, that target virulence behaviour in pathogens, has emerged as a solution with real clinical potential. We have previously shown that two signal molecules (HHQ and PQS) from the nosocomial pathogen Pseudomonas aeruginosa have modulatory activity towards other microorganisms. This current study involves the synthesis and evaluation of analogues of HHQ towards swarming and biofilm virulence behaviour in Bacillus atrophaeus, a soil bacterium and co-inhibitor with P. aeruginosa. Compounds with altered C6-C8 positions on the anthranilate-derived ring of HHQ, display a surprising degree of biological specificity, with certain candidates displaying complete motility inhibition. In contrast, anti-biofilm activity of the parent molecule was completely lost upon alteration at any position indicating a remarkable degree of specificity and delineation of phenotype.

Full text of DOI:10.1039/c5ob00315f

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Biomolecular Chemistry
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A structure activity-relationship study of the
bacterial signal molecule HHQ reveals swarming
motility inhibition in Bacillus atrophaeus†
Cite this: Org. Biomol. Chem., 2015,
13, 5537
F. Jerry Reen,a Rachel Shanahan,b Rafael Cano,b Fergal O’Gara*a,c and
Gerard P. McGlacken*b
The sharp rise in antimicrobial resistance has been matched by a decline in the identification and clinical
introduction of new classes of drugs to target microbial infections. Thus new approaches are being
sought to counter the pending threat of a post-antibiotic era. In that context, the use of non-growth
limiting small molecules, that target virulence behaviour in pathogens, has emerged as a solution with
real clinical potential. We have previously shown that two signal molecules (HHQ and PQS) from the
nosocomial pathogen Pseudomonas aeruginosa have modulatory activity towards other microorganisms.
This current study involves the synthesis and evaluation of analogues of HHQ towards swarming and
biofilm virulence behaviour in Bacillus atrophaeus, a soil bacterium and co-inhibitor with P. aeruginosa.
Compounds with altered C6–C8 positions on the anthranilate-derived ring of HHQ, display a surprising
degree of biological specificity, with certain candidates displaying complete motility inhibition. In contrast,
anti-biofilm activity of the parent molecule was completely lost upon alteration at any position indicating
a remarkable degree of specificity and delineation of phenotype.
Received 13th February 2015,
Accepted 9th April 2015
DOI: 10.1039/c5ob00315f
www.rsc.org/obc
bacteria, biofilms play a key role in allowing the pathogen to
overcome host defences and contribute to its virulence. The
Introduction
Quinolones are fused heterocyclic compounds that form the primary autoinducers of the P. aeruginosa AHQ signalling
active structure of a wide range of potent broad-spectrum anti- pathway are 2-heptyl-4-quinolone (referred to as the Pseudomo-
microbial agents.1 In fact, fluoroquinolones are the most suc- nas quinolone signal, or PQS) and its biological precursor
cessful non-natural product class of antibiotics.2 Another 2-heptyl-4-quinolone (HHQ) (Fig. 1)11 and PQS has been
important class of quinolones are the 2-alkyl-4-quinolones shown to mediate biofilm formation in P. aeruginosa.7
(AHQs) which are used as quorum sensing (QS) signaling
Bacillus atrophaeus (also named B. globigii and previously
molecules by pathogenic Gram-negative bacteria, including B. subtilis) is a Gram positive bacterium which co-inhabits the
Pseudomonas aeruginosa,3,4 and certain Burkholderia,5 and soil environment with P. aeruginosa.14 It is indistinguishable
Alteromonas6 strains. AHQ signaling pathways in these species from the typed B. subtilis strain except for the production of
have been shown to control production of multiple virulence pigment, a feature that marks the organism as particularly
determinants.7–9 This includes biofilm formation10,11 which is useful in the phenotypic analysis described here. Bacillus subti-
a structured community of bacterial cells enclosed in a self- lis species are an excellent and well-utilised model system for
produced polymeric matrix adhering to an inert or living Gram positive bacteria, and a cross-species influence with
surface.12,13 This mode of growth is particularly resistant to P. aeruginosa has been shown.15–17 In light of the co-existence
phagocytosis by white blood cells and to antibodies and anti- of both B. atrophaeus and P. aeruginosa in the soil, it is
biotics compared to planktonic cells.13 In multi-drug resistant perhaps unsurprising that communication mechanisms
aBIOMERIT Research Centre, Department of Microbiology, University College Cork,
Ireland. E-mail: f.ogara@ucc.ie; Fax: +353 21 4903101
bDepartment of Chemistry and Analytical & Biological Chemistry Research Facility
(ABCRF), University College Cork, Ireland. E-mail: g.mcglacken@ucc.ie;
Fax: +353 21 4274097
cSchool of Biomedical Sciences, Curtin University, Perth, WA 6845, Australia
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob00315f
Fig. 1 P. aeruginosa signalling molecules HHQ and PQS.
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Table 1 2-Alkyl-4-quinolones studied
Compound
n
R1
R2
R3
R4
Yielda [%]
1
2
3
4
5
6
7
8
6
6
6
6
6
6
6
6
8
8
8
8
8
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
n-Hex
H
H
H
OMe
Cl
H
H
OMe
F
H
H
H
H
31
35
46
12
33
34
21
19
23
28
16
51
16
–CH–CH–CH–CH–
H
OMe
H
H
H
H
H
H
OMe
H
H
H
Cl
H
9
10
11
12
13
H
H
H
n-Hex
–CH–CH–CH–CH–
H
H
a % yields are isolated yields over two steps.
between both organisms would exist and this type of commu- focused on substitution of the C-3 position.16 HHQ was shown
nity dynamic and microbiome has received considerable atten- to inhibit biofilm formation in species that inhabit the same
tion.18 However, the extent of these networks and molecular environment as P. aeruginosa whereas PQS and 3-halo-ana-
mechanisms remain to be ascertained. From a microbial logues did not. Very recently Steinbach and Hartmann also
control point of view, suppression of key virulence phenotypes reported ‘blocking’ the C-3 position thus preventing hydroxy-
in bacteria, independent of growth inhibition, should be less lation to a PQS analogue.20 Following on from their initial
susceptible to the build-up of resistance than with traditional investigations, a simple quinolone was elegantly designed to
antibiotic treatment as metabolic processes and bacterial target PqsR, and subsequently displayed antivirulence activity
growth are not directly targeted.
in vivo.
In the current work, we generated a diverse range of novel
The SAR analysis presented in this study represents the pre-
liminary steps in delineating the action of AHQs towards Gram HHQ analogues by using derivatized anthranilates and, in
positive bacteria, potentially providing a platform for future some cases, by extension of the alkyl chain at C-2 from seven
therapeutic developments.
to nine carbons (Table 1). HHQ analogues were prepared using
a procedure similar to that described by Somanathan et al.,21
and us,22 involving a Conrad–Limpach type cyclisation. Thus
β-keto-esters were prepared by acylation of Meldrum’s acid
(2,2-dimethyl-1,3-dioxane-4,6-dione) with octanoyl chloride or
decanoyl chloride, followed by methanolysis (Scheme 1). Con-
densation with a variety of anilines in the presence of acid
using a Dean–Stark apparatus and subsequent cyclisation of
the enamine at reflux in diphenyl ether gave the corresponding
2-heptyl- (1–8) or 2-nonyl-4-quinolone (9–13). During the
course of this work, an alternative method for accessing the
enamine intermediate was also used.23 On occasion, this
method provided a more efficient route. As mentioned earlier,
B. atrophaeus was selected due to is co-existence with P. aerugi-
nosa in the soil. Additionally, the organism is widely used as
a surrogate for B. anthracis investigations in biodefense
research,24 as well as being used as a sterilisation control
strain in industry.25 B. atrophaeus also exhibits strong
swarming and biofilm phenotypes that are characteristic of the
multicellular behaviour underpinning virulence in many
Results and discussion
In light of the important roles played by PQS and HHQ in viru-
lence, both molecules have been the subject of a number of
structure–activity relationship (SAR) analyses in recent
years.9,15,16,19 A study carried out by Hodgkinson’s group
showed evidence that the PQS regulator protein (PqsR), iron-
chelating ability (specifically PqsR-independent production of
pyoverdine) and membrane vesicle formation are altered upon
changes to the PQS framework.19 For example, shortening or
lengthening the C2 alkyl chain decreased activation of PqsR
and substitution of the anthranilate ring compromised either
the specific signalling, or membrane vesicle promotion. This
indicates that there are multiple signalling mechanisms by
which HHQ and PQS may possibly act. Antagonism of biofilm
formation or any cross-species effects were not reported.
A structure–activity investigation carried out by Reen et al.
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isms27 and underpinned by both intra and intercellular signal-
ling. In the case of B. atrophaeus, swarming cells are joined in
chain formations and the phenotype is promoted by the
secretion of surfactant ahead of the swarm front.28,29 Pre-
viously, we showed that addition of PQS to swarm plates com-
pletely abolished swarming motility in B. atrophaeus, while
addition of HHQ led to little reduction in this key virulence
phenotype.15 However in the study reported herein, addition of
HHQ analogues (1–13) to swarming media led to a significant
alteration in activity compared to the parent compound HHQ.
Remarkably, some analogues exhibited PQS like inhibition,
while others had the effect of promoting swarming activity in
the model organism (Fig. 2). Of the compounds tested, com-
pounds 1, 3, 4, 6, 8, 12 and 14 led to a phenotype statistically
similar to that observed with the carrier control MeOH. Quino-
lones 2, 5, 9, 11 and 13 gave a moderate decrease in swarming,
whereas quinolones 7 and 10 completely abrogated swarming
activity in B. atrophaeus as shown in Fig. 2. Overall substitution
at the 6-position afforded the most response. In compounds
with a C-2 heptyl chain, an electron withdrawing group (7)
gave a dramatic decrease in swarming. Interestingly, this is
consistent with Hartmann’s observation, where a nitro group
at the 6-position gave antagonistic behaviour in P. aerugi-
nosa.20 When we prepared an analogue possessing an electron
releasing group (6) no decrease in swarming was observed.
Scheme 1 Synthesis of anthranilate-substituted 2-alkyl-4-quinolone.
pathogens.26 Firstly, growth of B. atrophaeus was investigated
in the presence of HHQ, PQS and the suite of analogues. Pre-
pared in honeycomb 100-well plates, in TSB supplemented
with 10 µM of compound or equivalent volume of methanol as
control, all readings were taken on a BioScreen Analyser.
Growth of B. atrophaeus was not significantly altered in the
presence of any of the modified analogues (SM Fig. 1) and
thus any virulence effects were likely to be growth indepen-
dent. Swarming motility is a key multicellular behaviour in
bacteria, considered a virulence phenotype in many organ-
Fig. 2 Swarming motility of B. atrophaeus is modulated by HHQ, PQS and their analogues on 0.3% agar (w/v) TSA plates. Measurements reflect the
total diameter of the swarm mass, and incorporates the point of inoculation which was performed by sterile toothpick. (A) is a visual representation
of swarming motility in the presence of each analogue while (B) is a graphical representation of the combined data. All experimental datapoints rep-
resent the mean of at least 5 independent biological replicates. Error is presented as standard error of the mean and statistical analysis was per-
formed using Student’s t-test and Bootsratio analysis (* p < 0.05, ** p < 0.005, *** p < 0.001).
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(isolated in 37%) led to significant enhancement of swarming
motility, in direct contrast to the parent molecule.
Having established the influence of the analogues on
swarming motility, it was expected that a similar profile would
emerge from an investigation of their impact on biofilm for-
mation. Swarming motility is associated with biofilm for-
mation in many pathogens, sometimes in what is an inverse
relationship. Therefore, each analogue was tested for its
impact on pellicle formation and attachment in B. atrophaeus.
Surprisingly, all analogues had lost their anti-pellicle and anti-
biofilm formation activity with respect to the parent molecule
HHQ. Modification of any part of the quinolone framework
led to loss of activity, even for those where suppression of
swarming motility was enhanced (Fig. 3).
Scheme 2 Synthesis of N- and O-methylated compounds.
Remarkably, extension of the C-2 alkyl chain in the non-inhibi-
tory analogue, afforded a molecule (10) capable of returning
anti-motility behaviour.30
4-Quinolones such as HHQ exist as two interconverting tau-
tomeric forms (a 4-quinolone and a 4-hydroxyquinoline) and
equilibrium tends towards the quinolone form under physio-
logical conditions.31 We were intrigued to delineate these two
structural isomers and elucidate if either structural form main-
tained activity if ‘frozen out’ by methylation at oxygen or
nitrogen.
Preparation of the N- and O-methylated compounds was
carried out by reaction of quinolone substrate 10 with methyl
iodide in acetone and K2CO3. The resulting isomeric mixture
was purified by column chromatography to afford the desired
products 14 (quinolone) and 15 (quinoline) in 14 and 42%
respectively (Scheme 2). When tested on B. atrophaeus, both
compounds had lost anti-swarming activity (Fig. 2) relative to
the parent quinolone (10) but the quinoline form 15 did show
a slight enhancement of anti-swarming activity relative to qui-
nolone 14. We also chose to carry out a similar investigation
using quinolone 2, which displayed enhanced suppression of
swarming motility relative to MeOH (and HHQ itself). In this
case the N–Me derivative 16 (isolated in 10%) again showed
little or no variation. However, addition of the O–Me variant 17
Conclusions
In this study we prepared a number of analogues of known
P. aeruginosa signalling molecule HHQ, present in complex
multi-bacterial and multi-kingdom environments, and tested
them for anti-swarming and anti-biofilm activity towards
B. atrophaeus. Several of the novel analogues show potent sup-
pression of swarming motility, which suggests some degree of
freedom regarding the structural interaction between the qui-
nolone core molecule and the receiving organism. However,
the lack of anti-biofilm activity exhibited by all the analogues
would suggest that the anti-biofilm activity exhibited by HHQ
is highly sensitive to structural modification and may reflect a
highly specific protein–ligand interaction.
Previously, HHQ itself has shown very minor anti-swarming
activity in a range of organisms in contrast to PQS, which com-
pletely abolished swarming activity.15 Strikingly, modification
of the quinolone backbone structure described here, resulted
in quinolones possessing potent suppression of swarming,
similar to that with PQS. While there is no evidence of a pqsH
type-activity encoded in a genome outside of P. aeruginosa, and
thus no known mechanism for the hydroxylation of our HHQ
analogues to PQS analogues,15 other biosynthetic steps could
be at play and our investigations in this area will be published
in due course.
Acknowledgements
The authors would like to thank David Woods for technical
assistance. This research was supported in part by grants
awarded to GMG by Science Foundation of Ireland (SFI) (SFI/
12/TIDA/B2405, SFI/12/IP/1315, SFI/09/RFP/CHS2353 and
SSPC2 12/RC/2275) and the Irish Research Council and to FOG
Fig. 3 Biofilm formation by B atrophaeus in the presence of 10 µM of
compound, compared with an equivalent volume of methanol as
control. A visualisation of the pellicle formed in the presence of each (SFI: SSPC2 12/RC/2275; 13-TIDA-B2625; 07/ IN.1/B948;
compound is presented above the graph. Each datapoint, which is pre-
sented as fold change in Abs595nm relative to methanol control, is the
average of at least 4 independent biological replicates. Error is presented
as standard error of the mean and statistical analysis was performed
12/TIDA/B2411; 12/TIDA/B2405; 09/RFP/BMT 2350); the
Department of Agriculture, Fisheries and Food (DAFF11/F/009
MabS; FIRM/RSF/CoFoRD; FIRM 08/RDC/629); the Environ-
mental Protection Agency (EPA 2008-PhD/S-2); the Irish
Research Council for Science, Engineering and Technology
using Student’s t-test and Bootsratio analysis (* p < 0.05, ** p < 0.005,
*** p < 0.001).
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