Background
Quorum sensing (QS) is the term which refers to bacterial cell-cell communication
to coordinate a diverse array of physiological behaviors in the entire communities.
This signaling system allows the bacteria to sense its population density in response
to the concentration of signaling molecules (Miller and Bassler, 2001). Unlike Gram-positive
bacteria which employ oligopeptide/two component-type QS circuit, Gram-negative bacteria
use the diffusible N-acylhomoserine lactones (AHLs) signaling molecules to mediate
expression of many phenotypes and cellular activities (Federle and Bassler, 2003).
AHLs are widely conserved signal molecules; they consist of the same homoserine lactone
moiety but differ in the length and structure of acyl side chain at the C3 carbon
(Fuqua and Greenberg, 2002). With such structural molecules, AHLs are generally amphipathic
in nature. They are water-soluble and able to diffuse freely across the phospholipid
cell membranes (Pearson et al., 1999). AHL-dependent QS system comprises three principal
components: (i) the AHL signaling molecules, (ii) AHL synthase protein, LuxI to produce
AHLs, and (iii) a transcriptional regulator protein, LuxR to bind to AHLs (Shrout
and Nerenberg, 2012). Accumulation of AHLs above a threshold concentration triggers
the formation of signal-receptor protein complexes which in turn activate the expression
of appropriate target genes (McClean et al., 1997; Eberl, 1999). In the past decade,
numerous studies have shown the importance of AHLs in regulation of a range of biological
functions such as biofilm formation, antibiotic production, swarming motility, conjugation,
and sporulation. The basic mechanisms of AHL synthesis and regulation are found to
be conserved in many species of proteobacteria despite expression of different target
phenotypes (Miller and Bassler, 2001; Dong et al., 2002). In addition, each QS-regulated
target gene requires a specific cell density in order to be activated and there is
no such single population density which could trigger expression of all genes (Schuster
et al., 2003).
Aeromonas spp. are Gram-negative, facultative anaerobic, rod-shaped bacteria belonging
to the family Aeromonadaceae. They are ubiquitous bacteria which thrive in many terrestrial
and aquatic environment (Daskalov, 2006). This genus consists of psychrophiles and
mesophiles which are both primary and opportunistic pathogens of cold and warm-blooded
animals. Recently, Aeromonas spp. have gained more clinical recognitions as they are
commonly associated with food and waterborne diseases (Ansari et al., 2011). The most
important pathogens are A. hydrophila, A. caviae, and A. veronii biovar sobria. These
species are primary causative agents of both gastrointestinal and extra-intestinal
infectious diseases (Vila et al., 2002). In fact, A. veronii is often associated with
traveler diarrhea (Gröbner et al., 2007).
Recently, our group has isolated 22 Aeromonas strains from various clinical samples
i.e., bile, blood, peritoneal fluid, pus, stool, and urine, and their QS activities
were investigated. Among the Aeromonas isolates, A. veronii biovar sobria strain 159
(hereafter referred to as strain 159) isolated from a stool sample was found to exhibit
QS activities (Chan et al., 2011). Whole genome sequencing of this bacterium was performed
and it was found that this strain shares a high degree of genome synteny with A. hydrophila
ATCC 7966. Upon analysis and genome annotation, a pair of LuxIR homologs, termed as
AveIR, was found to be located in Contig 47. The AHL profile was then obtained from
the culture supernatant of strain 159 using liquid chromatography mass spectrometry
(LC-MS).
Materials and methods
Bacterial source, isolation and culture
A. veronii strain 159 was isolated from the stool of a patient admitted to University
of Malaya Medical Center, Malaysia. This bacterium was maintained aerobically in LB
(Luria Bertani, Merck) medium at 37°C. Strain 159 was also stored frozen at −70°C
in 50% (v/v) glycerol.
Gene annotation and phylogenetic analysis
Gene annotation was performed using the SEED-based automated annotation system provided
by the Rapid Annotations using Subsystems Technology (RAST) server (version 4.0) (Aziz
et al., 2008) to look for the presence of LuxIR homologs. The protein sequences of
both AveI and AveR were compared with GenBank databases using BLASTX program available
from NCBI website (http://www.ncbi.nlm.nih.gov/). Ten LuxI and LuxR homologs with
the highest similarities were chosen. Redundant sequences or bacteria strains with
ambiguities were omitted. A phylogenetic trees corresponding to both proteins were
constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 5.2 (Tamura
et al., 2011). Neighbor joining algorithm was used with boostrap value of 1000, expressed
as percentage of 1000 replicates.
Extraction of AHL
A. veronii strain 159 was grown in LB medium buffered to pH 6.5 with 50 mM of 3-[N-morpholino]
propaneusulfonic acid (MOPS) to prevent degradation of AHLs (Yates et al., 2002).
The bacterium was grown at 37°C with agitation at 220 rpm. The spent culture supernatant
was extracted thrice with equal volume of acidified ethyl acetate (0.1% v/v glacial
acetic acid in ethyl acetate, Merck, Germany). The ethyl acetate extracts were evaporated
to dryness in fume hood. The dried extracts were then resuspended in 1 mL of acidified
ethyl acetate and allowed to dry again. Then, 1 mL of acetonitrile (HPLC grade, Merck,
Germany) was added to dissolve the extracted AHLs. The mixture was then filtered and
100 μL of aliquot was withdrawn and placed in sample vials for analysis by liquid
chromatography mass spectrometry (LC-MS).
Identification of AHL molecules by mass spectrometry (MS)
The AHL prrofile of strain 159 was obtained by High Resolution Tandem Triple Quadrupole
Mass Spectrometry (LC-MS/MS) System according to previously reported method (Wong
et al., 2012). LC delivery system using Agilent 1290 Infinity system (Agilent Technologies
Inc., Santa Clara, CA, USA) was employed with Agilent ZORBAX Rapid Resolution HT column
(2.1 × 50 mm, 1.8 μm particle size). Both mobile phases A and B were 0.1% v/v formic
acid in water and 0.1% v/v formic acid in acetonitrile, respectively. The parameters
of the gradient profiles were set as followed (time: mobile phase A: mobile phase
B): 0 min: 60:40, 5 min: 20:80, 7 and 10 min: 5:95, and 11 and 13 min: 60:40. Two
microliter of sample was injected and the analysis was performed using a flow rate
of 0.3 mL/min at 37°C. The Agilent 6490 Triple-Quad LC-MS/MS system was used to perform
the high-resolution electrospray ionization mass spectrometry (ESI-MS) in positive
mode. The probe capillary voltage was set at 3 kV, sheath gas at 11 mL/h, nebulizer
pressure at 20 psi and desolvation temperature at 250°C. Nitrogen gas was used as
the collision gas in the collisionally-induced dissociation mode for the MS/MS analysis
and the collision energy was set at 10 eV. The Agilent MassHunter software was used
to analyse the MS data (Yin et al., 2012; How et al., 2015). Known amounts of synthetic
AHLs (Sigma, St. Louis, MO) were injected as standards.
Results
Whole-genome shotgun sequencing of A. veronii strain 159 was performed recently using
Illumina HiSeq 2000 (Illumina, Inc., CA) platform (Chan et al., 2012). The genome
sequence has been deposited at DDBJ/EMBL/GenBank under the accession no. ALOT00000000.
From RAST analysis of the draft genome, a copy of each LuxI and LuxR homologs were
found, termed as AveI and AveR, respectively. Both protein sequences were deposited
in NCBI with GenBank accession number WP_026034966.1 and WP_019445709.1. The phylogenetic
tree illustrated that both AveI and AveR proteins share high homology with their reported
counterparts from other Aeromonas species, indicating that they may share some ancestry
relationship and the proteins are conserved throughout evolution (Figures 1A,B). The
analysis revealed that LuxI homolog from strain 159 is more closely related to LuxI
homologs from A. veronii strains [AAY89629 and AAY54302]. It falls within a clade
that includes A. bestiarum [AAY 89614] as well as A. enteropelogenes [AAY 89610].
On the other hand, transcriptional regulator, AveR of strain 159 shows closer affinity
to A. sobria [WP_042019486] than to A. veronii [ERF62569, AAX12571, and AAX12598].
The high degree of homology suggests that the LuxI/R are conserved throughout evolution
among Aeromonas species and these proteobacteria may possibly share similar basic
QS mechanism and gene regulation in AHL production even though their expression target
genes could be different.
Figure 1
Phylogenetic tree of LuxIR homologs and the organization of the gene cluster. Phylogenetic
tree describing evolutionary distances between (A) LuxI homologs and (B) LuxR homologs
from different Aeromonas species using Neighbor-Joining algorithm. The tree is drawn
to scale, with branch lengths in the same units as those of the evolutionary distances
used to infer the phylogenetic tree. The horizontal bar at the bottom represents mean
number of substitutions per site. The bootstrap values are expressed as percentages
of 1000 replications. (C) The organization of LuxIR homologs in strain 159 and closely
related species. The gene clusters of LuxI/R homologs in strain 159 were compared
with closely-related species, A. hydrophila subsp. hydrophila ATCC 7966, A. veronii
AMC34, A. veronii AER39, and A. hydrophila SNUFPC-A8. Homologous proteins are shown
with the same color and arrows indicate the relative orientations of the genes. GenBank
accession numbers (in parentheses): LuxI-like protein, A. veronii [AAY89629]; quorum
sensing autoinducer synthase, A. veronii [AAY54302]; LuxI-like protein, A. enteropelogenes
[AAY89610]; LuxI-like protein, A. bestiarum [AAY89614]; acyl-homoserine-lactone synthase,
A. sobria [WP_042019484]; LuxI-like protein, A. veronii bv. sobria [AAY89603]; acyl-homoserine-lactone
synthase, A. australiensis [WP_040094882]; acyl-homoserine-lactone synthase, A. allosaccharophila
[WP_042063590]; acyl-homoserine-lactone synthase, A. jandaei [WP_041209189]; acyl-homoserine-lactone
synthase, Aeromonas sp. L_1B5_3 [WP_043849715]; LuxR-type transcriptional regulator,
A. veronii CECT 4246 [AAX12598]; LuxR-like protein, A. veronii bv. sobria [AAY44573];
LuxR-type transcriptional regulator, A. veronii MTCC 3249T [AAX12571]; LuxR-like protein,
A. caviae [AAY44571]; transcriptional regulator, A. veronii strain Hm21 [ERF62569];
transcriptional regulator, A. sobria [WP_042019486]; LuxR-type transcriptional regulator,
A. enteropelogenes [AAX12592]; transcriptional regulator, Aeromonas sp. 4287D [WP_033135665];
transcriptional regulator, A. hydrophila [WP_017785811]; LuxR-like protein, Aeromonas
sp. CCRC 13881 [AAY44569].
From in silico analysis of strain 159 and closely-related species, all Aeromonas spp.
are found to possess single copy of both aveI and aveR, which are 639 bp and 777 bp
genes, which encode for 212 and 258 amino acids, respectively. RAST analysis revealed
that aveIR gene cluster of strain 159 shares high homology with its closest counterpart,
A. hydrophila ATCC7966. Both autoinducer synthesis proteins and transcriptional activator
proteins are found in reversed orientation. In the vicinity of LuxI/R homologs are
genes encoding chromosome initiation inhibitor, arginine exporter protein, and exoribonuclease
II enzyme. Chromosome initiation inhibitor is a DNA-binding protein that inhibits
chromosome replication while exoribonuclease II involves in mRNA degradation. Such
organization of gene cluster is also found in A. hydrophila and A. salmonicida (Swift
et al., 1997). It could be possibly that the regulation of the genes responsible for
cell divisions are regulated by LuxI/R system in these Aeromonas species. In strain
159, aveI and its cognate aveR partner are adjacent genes that are 62 bp apart, similar
to A. hydrophila ATCC7966 (Figure 1C). In between, a region of dyad symmetry known
as the lux box was identified. Surprisingly, the lux box was found to be identical
to the one from A. veronii MTCC 3249 but shorter than A. hydrophila and A. salmonicida
(Jangid et al., 2012).
The amino acid sequence of AveI was analyzed using InterPro (http://www.ebi.ac.uk)
to identify conserved protein domain and molecular function. Results from InterPro
shows that AveI fulfills the criteria as an AHL synthase as it possesses autoinducer
synthesis and acyl-CoA N-acyltransferase conserved sites. Meanwhile, InterPro revealed
that AveR protein consists of both N-terminal autoinducer binding domain and C-terminal
DNA-binding domain of LuxR-like protein which are the fundamental requirements of
a functional LuxR transcriptional receptor (Tsai and Winans, 2010).
In our previous study (Chan et al., 2011), a bioassay using thin layer chromatography
demonstrated that strain 159 secreted C4-HSL into the growth media. Hence, in this
study, we used Triple-Quad LC-MS/MS to determine the AHL profile of extracted culture
supernatant of strain 159. The chromatogram of strain 159 was overlaid with the chromatogram
from the synthetic standard at 0.7 min (Figure 2). The presence of C4-HSL with m/z
value 172.000 was indistinguishable to the corresponding synthetic compounds at their
respective retention times. The product ion of m/z 102 corresponds to the presence
of lactone ring moiety of C4-HSL. Long chain AHL was not secreted by strain 159. This
was in agreement with the recently published data by Chan et al. (2011).
Figure 2
MS analyses showing the AHL profile of strain 159 using LC-MS/MS. (A) Overlaid of
the chromatogram of synthetic standard of C4-HSL with the chromatogram of culture
supernatant from strain 159 at retention time of 0.7 min. (B) Spectrum extracted from
the selected peak of chromatogram for strain 159 at 0.7 min shows the presence of
C4-HSL product ion (m/z: 102) and precursor ion (m/z: 172).
Among Aeromonas species, most of the studies on QS were substantially found from A.
hydrophila (AhyIR) and A. salmonicida (AsaIR) (Swift et al., 1997, 1999). Both Aeromonas
species are well-known pathogens of humans and fish: A. hydrophila is the etiological
agent for aeromonad septicemia while A. salmonicida is responsible for furunculosis
in salmonid fish (Fryer and Bartholomew, 1996). Studies have demonstrated that AhyIR
plays essential roles in biofilm formation, exoprotease production and type IV secretion
system (Swift et al., 1999; Khajanchi et al., 2009) while AsaIR is possibly implicated
to regulation in cell division (Swift et al., 1997). Hence, it is of interest to get
further insights of the roles played by AveIR in strain 159.
According to a study by Jangid et al. (2007), LuxIR homologs are universally present
in the genus Aeromonas. The LuxR homologs in Aeromonas species showed a wide range
of similarity, from 79.28 to 100%. In contrast, the LuxI homologs showed lower sequence
similarity which ranged from 69.34 to 100%. From in silico analysis of strain 159
and closely-related species, all Aeromonas spp. are found to possess single copy of
LuxI homologs (data not shown). In terms of AHL profile, C4-HSL is found to be the
major AHL produced by both AhyI and AsaI. Apart from C4-HSL, minute amount of C6-HSL
was identified from the spent supernatant of both A. hydrophila and A. salmonicida
(Swift et al., 1997). In fact, the production of C4-HSL and C6-HSL by AhyI was detected
to be in approximate ratio of 70:1. In contrast, C6-HSL is not found in the spent
supernatant of A. veronii strain 159. Besides C4-HSL and C6-HSL, C5-HSL is another
AHL significantly detected from many A. hydrophila strains from clinical samples (Chan
et al., 2011). In this study, the production of only a single type of AHL, i.e. C4-HSL
and the presence of a single copy of the QS genes in strain 159 highly suggests a
tight regulation in pathogenesis by a singular type of autoinducer.
In conclusion, the whole genome sequencing of strain 159 enables the prediction of
DNA sequences of target genes, i.e., QS-related genes. The findings of AHL-based QS
system in strain 159 have placed further interest to explore its role in the mechanism
of pathogenesis. This could possibly provide a model for bacterial cell-cell communication
among Aeromonas species and hence a potential antimicrobial target in treating Aeromonas
infections.
Author contributions
XC and KH conceived and designed the experiments; XC performed the experiments and
analyzed the data; KH and WY wrote the paper; KC edited and approved the manuscript.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.