Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases

Article abstract of DOI:10.1128/AAC.02410-13

Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy. Copyright

Full text of DOI:10.1128/AAC.02410-13

AAC Accepts, published online ahead of print on 30 December 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.02410-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Disruption of Biofilm Formation by the Human
Pathogen Acinetobacter baumannii using
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Engineered Quorum-quenching Lactonases
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Jeng Yeong Chow , Yuanyong Yang , Song Buck Tay , Kim Lee Chua , and Wen Shan
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Yew
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Department of Biochemistry, Yong Loo Lin School of Medicine, National University of
Singapore, 8 Medical Drive, Singapore 117597.
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To whom correspondence should be addressed: KLC Tel: 65-6516-3684; Fax: 65-6779-
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1453; Email: kim_lee_chua@nuhs.edu.sg; WSY Tel: 65-6516-8624; Fax: 65-6779-1453;
Email: bchyws@nus.edu.sg
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Running Title: AHLase disruption of Acinetobacter baumannii biofilm.
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ABSTRACT
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Acinetobacter baumannii is a major human pathogen associated with multi-drug resistant
nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm
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formation, and disruption of biofilm formation is an attractive anti-virulence strategy. Here,
we report the first successful demonstration of biofilm disruption in a clinical isolate of A.
baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this
engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms,
demonstrating the utility of this anti-virulence strategy.
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Acinetobacter baumannii is a Gram-negative bacterium that has gained global
notoriety due to its rapid emergence as an opportunistic pathogen in nosocomial or hospital-
acquired infections (1). The high morbidity rate associated with A. baumannii-mediated
infections has earned the bacterial pathogen the moniker of the “Gram-negative” methicillin-
resistant Staphylococcus aureus (MRSA) (2). Bacterial transmission between patients in
hospitals has been associated with the use of indwelling medical devices such as catheters
and implants (3,4). The situation is exacerbated by the emergence of a number of A.
baumannii isolates that were found to be resistant to carbapenem, an antibiotic used for the
treatment of infections caused by A. baumannii (5). The persistency of A. baumannii in
hospital-acquired infections has been associated with biofilm formation by the bacteria; the
biofilm provides protection for the bacteria against host immune systems and antibiotic
treatment (6,7).
The process of biofilm formation in many bacteria is mediated through quorum-
sensing pathways. In A. baumannii, biofilm is formed upon the activation of a typical
LuxI/LuxR-type quorum-sensing network that involves an abaI synthase and abaR receptor
(8,9). Although various forms of N-acyl-homoserine lactones (AHLs) were found to be
present in various Acinetobacter spp., a study demonstrated that 3-hydroxy-dodecanoyl-L-
homoserine lactone (3-OH-C12-HSL) is the major quorum signal that is produced by the M2
strain of A. baumannii (9,10). Use of AHL analogues to inhibit the quorum-sensing pathway
of A.baumannii has been proven to be a valid strategy in the attenuation of biofilm formation
in this bacteria (11) . This anti-virulence strategy is therapeutically attractive since it targets
the virulence of the bacteria and hence minimizes the chance for the selection of resistant-
strains.
Quorum-quenching can also be achieved through the enzymatic degradation of the
quorum signal by an AHL-lactonase (AHLase) (12,13). Numerous attempts have been made
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to extend the application of these enzymes in the attenuation of bacterial virulence in human
pathogens. Although it had been demonstrated that the expression level of virulence factors
in Pseudomonas aeruginosa can be attenuated by AHLases (14), there is currently no
evidence to suggest the effective use of quorum-quenching enzymes in the disruption of
biofilm formation in bacterial pathogens. Recently, we reported on the directed evolution of
a thermostable quorum-quenching lactonase from Geobacillus kaustropilus (GKL); a
thermostable engineered mutant of the quorum-quenching enzyme was obtained with
enhanced catalytic activity and broadened substrate range against AHLs (15). This enzyme
belongs to the Phosphotriesterase-like lactonase (PLL) family of the amidohydrolase
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superfamily and possesses the commonly encountered (β/α) -barrel fold (16). Here, we
report the use of this catalytically enhanced mutant enzyme in the disruption of biofilm
formation by A. baumannii. With its inherent thermostability and molecular tractability
(modulability in activity and substrate range through choice mutations in the enzyme scaffold
(17)), we envision the further development of this enzyme (and other quorum-quenching
enzyme scaffolds) for use as anti-virulence therapeutics against A. baumannii-mediated
infections; this demonstration also illustrates the utility of quorum-quenching enzymes in
addressing the increasing therapeutic needs of our generation.
Our previous efforts in enhancing the catalytic activity (and broadening the substrate
range) of a thermostable AHL-lactonase resulted in the development of a number of GKL
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mutants with enhanced catalytic efficiency (kcat/K ) against various forms of AHLs (15).
Although a large panel of AHLs was previously tested for reactivity, past unavailability of C3
hydroxyl-substituted AHLs prevented an assessment of the lactonase activities of our
engineered enzymes against these quorum molecules. In fact, C3-hydroxylated AHLs were
rarely tested as substrates for AHL-lactonases, and hence, very little information is available
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with regards to the effect of hydroxylation at the C3 position of the acyl chains (of the lactone
substrates) on the catalytic efficiency of these enzymes (16,18,19).
Although it has been reported that the M2 strain of A. baumannii uses 3-OH-C12-
HSL as the major quorum molecule, we found that a clinical isolate, A. baumannii S1, uses 3-
hydroxy-decanoyl-L-homoserine lactone (3-OH-C10-HSL) as the major quorum signaling
molecule (Table S1 of the Supplemental Material). Since the lactonase activity of the wild-
type and E101G/R230C mutant of GKL against both 3-OH-C10-HSL and 3-OH-C12-HSL,
respectively, were not known, we sought to determine the hydrolytic activities of our
enzymes against these A. baumannii quorum substrates. Using wild-type GKL, we were
unable to detect lactonase activity against 3-OH-C12-HSL; the unevolved enzyme
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hydrolyzed 3-OH-C10-HSL with a kcat ≤ 0.011 s (Table S2 of the Supplemental Material).
Using the evolved mutant GKL enzyme (E101G/R230C), reasonable kinetic parameters
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against 3-OH-C10-HSL and 3-OH-C12-HSL, respectively, were observed (kcat/K of 180 M
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s and 150 M s , respectively). A more detailed comparison of the kinetic parameters with
different forms of C10-HSL and C12-HSL revealed that the catalytic rates of the wild-type
and mutant GKL varied with chain length and substitution of the AHLs.
Wild-type and AHL-synthase-deficient (ΔabaI) mutant of A. baumannii were cultured
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in a low salt medium at 30 C and biofilm formation was determined using crystal violet
staining. Within expectation, a deletion of the AHL-synthase (ΔabaI) from the genome of A.
baumannii resulted in a reduction in the amount of biofilm formed by the mutant bacteria
(Figure 1). In an attempt to determine the effect of quorum-quenching lactonases on biofilm
formation, both wild-type GKL, the catalytically-inactive E101G/R230C/D266N mutant of
GKL, and the catalytically-enhanced E101G/R230C mutant of GKL were added to a log
phase culture of wild-type A. baumannii. The catalytically-inactive D266N mutant of GKL
(and the equivalent catalytically-inactive E101G/R230C/D266N mutant) served as negative
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controls to rule out any sequestration effects exhibited by the quorum-quenching lactonases.
Significant reduction in the amount of biofilm formed was observed in the presence of the
engineered mutant enzyme (Figures 1 and 2).
Confocal laser scanning microscopy (CLSM) was used to (qualitatively and
quantitatively) access the effect of lactonase treatment on the overall morphology and
architecture of the A. baumannii biofilm. We chose to use the engineered E101G/R230C
mutant based upon the observed enhancement in lactonase activity. As shown in the
differential image contrast (DIC) image, treatment with the enhanced GKL mutant caused a
reduction in the size of the biofilm (Figure 2). The biofilm was stained with Alex Fluo 488-
conjugated wheat germ agglutinin (WGA) to reveal a reduction in the biomass, thickness, and
surface area of the biofilm after treatment with the engineered lactonase (Figure 2, Table 1).
In addition, although it had been shown that AHL analogues could also prevent the
formation of biofilm in A. baumannii (11), the bioavailability (or lack thereof) of these
quorum inhibitors has limited their translational potential in the treatment of quorum-
mediated diseases. The use of catalytic quorum-quenching enzymes, on the other hand, could
circumvent the bioavailability problem. One could envision synergy/complementarity
between these two therapeutic approaches in the treatment of quorum-mediated diseases: a
bioactive, multi-functional biomaterial (involving immobilized quorum-quenching enzymes
and matrix-entrapped quorum inhibitors for controlled release) could be used to functionalize
the surface of catheters or implants to prevent biofilm formation.
The complexity of bacterial quorum-signaling systems has limited the success of
using quorum-quenching enzymes for biofilm disruption of bacterial pathogens: 1)
recombinant AiiA (a type of AHL-lactonase belonging to the metallo-β-lactamase
superfamily) was used to reduce the amount of planktonic cells residing within P.
aeruginosa-mediated biofilm structures (20); 2) immobilized SsoPox, an orthologue of GKL,
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was used to inhibit the production of various virulence factors in P. aeruginosa (18).
However, in both studies, there was no direct evidence for the reduction of the biomass of the
biofilm structures.
In this study, we determined the rates of hydrolysis of two biologically relevant C3-
hydroxylated AHLs (3-OH-C10-HSL and 3-OH-C12-HSL, respectively) by quorum-
quenching lactonases. The rates observed with these substrates revealed that modification via
hydroxylation of the C3 position (in comparison to unmodified decanoyl- and dodecanoyl-
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HSLs) resulted in a decrease in catalytic efficiencies (kcat/K ); nevertheless, the broadened
range of substrate specificities (promiscuities) translated to a serendipitous quenching of
quorum-mediated biofilm formation, resulting in a decrease in biomass (and thickness) of the
biofilm structures. Our observation of the use of an alternate AHL (3-OH-C10-HSL instead
of the previously identified 3-OH-C12-HSL for the M2 strain) by a clinical isolate of A.
baumannii (the S1 strain) further highlighted the utility of engineered quorum-quenching
lactonases with broad spectrum (range) reactivities. In addition, the effect of biofilm
disruption against the S1 strain was also evident in the presence of serum-like conditions
(Figure S5); the translational potential of using the quorum-quenching enzymes to disrupt
biofilm formation was further highlighted by the observed stability of the enzymes in
bacterial cultures (Figure S6). The efficacy of biofilm disruption was also demonstrated
against multiple strains of A. baumannii (S2 and R2 strains, respectively, Figures S7 and S8),
and against pre-formed biofilm (Figure S9).
In summary, we have provided the first demonstration of the use of recombinant
quorum-quenching enzymes in the disruption of biofilm formation (vis a vis reduction in bio-
mass and thickness) by a bacterial human pathogen, A. baumannii. A decrease in the biomass
of biofilm can translate to more effective antibiotic therapies due to the increased
susceptibility of bacteria towards antibiotic treatments. We believe that future development
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of quorum-quenching enzymes will be critical in translating the utility of this therapeutic
route in the treatment of biofilm-mediated bacterial diseases.
ACKNOWLEDGEMENTS
We would like to thank Otsuka Pharmaceuticals for the kind gift of 3-oxo-C12-HSL. We
also thank Dr. Lai Lai Yap for her technical assistance in confocal scanning laser microscopy.
This work was supported by grants from the Academic Research Fund of the Ministry of
Education, and the National Medical Research Council and the National Research
Foundation, Singapore.
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FIGURE LEGENDS
Figure 1. Biofilm disruption assay. Biofilm was quantitated by crystal violet staining. Red
columns represent amount of biofilm formed by A. baumannii (wild-type and ΔabaI mutant,
respectively) without the addition of AHL-lactonases. Blue columns represent the amount of
biofilm formed by wild-type A. baumannii in the presence of different GKL enzymes
(inactive D266N GKL, wild-type GKL, inactive E101G/R230C/D266N GKL, and
E101G/R230C GKL, respectively). ****, p value ≤ 0.0001.
Figure 2. Representative confocal laser scanning microscopy images of A. baumannii
biofilms. A. baumannii were treated with A) inactive E101G/R230C/D266N GKL, and B)
E101G/R230C GKL, and stained with Alex Fluo 488-conjugated WGA. DIC images of the
biofilms (left panels), and fluorescence images of the biofilms (right panels) are shown for
representative xy (center), yz (right) and xz (bottom) sections.
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Table 1: Quantification of untreated and treated A. baumannii biofilm structures.
Treatment with inactive
mutant
Treatment with E101G/R230C
mutant
No Treatment
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Biomass (μm /μm )
2.57 ± 1.65
3.68 ± 2.51
3.39 ± 1.33
3.41 ± 1.31
1.37*** ± 0.20
1.21*** ± 0.21
7.75** ± 1.63
115354.9* ± 7630.3
1.90 ± 0.17
Average thickness (μm)
Maximum thickness (μm)
Surface area (μm)
11.49 ± 4.72
13.82 ± 4.17
235920.59 ± 79456.46
2.74 ± 1.53
209872.6 ± 115094.7
1.57 ± 1.06
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Surface to volume ratio (μm /μm )
Roughness coefficient
0.58 ± 0.17
0.65 ± 0.10
0.59 ± 0.08
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N=10 image stacks, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, compared with treatment with inactive E101G/R230C/D266N mutant.
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Figure 1
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Figure 1. Biofilm disruption assay. Biofilm was quantitated by crystal violet staining. Red
columns represent amount of biofilm formed by A. baumannii (wild-type and ΔabaI mutant,
respectively) without the addition of AHL-lactonases. Blue columns represent the amount of
biofilm formed by wild-type A. baumannii in the presence of different GKL enzymes
(inactive D266N GKL, wild-type GKL, inactive E101G/R230C/D266N GKL, and
E101G/R230C GKL, respectively). ****, p value ≤ 0.0001.
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Figure 2
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Figure 2. Representative confocal laser scanning microscopy images of A. baumannii
biofilms. A. baumannii were treated with A) inactive E101G/R230C/D266N GKL, and B)
E101G/R230C GKL, and stained with Alex Fluo 488-conjugated WGA. DIC images of the
biofilms (left panels), and fluorescence images of the biofilms (right panels) are shown for
representative xy (center), yz (right) and xz (bottom) sections.
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