IJBPAS, July, 2014, 3(7): 1084-1091
ISSN: 2277–4998
HOMOSERINE LACTONE PRODUCTION BY CLINICAL ISOLATES OF Proteus
vulgaris FROM PATIENTS WITH URINARY TRACT INFECTIONS
GHAIDAA J. MOHAMMED1* AND MOHAMMED S.ABDUL RAZAQ2
1: Department of Biology, College of Science, Babylon University, Babil, Iraq
2: Department of Microbiology, College of Medicine, Babylon University, Babil, Iraq
*Corresponding Author: E Mail: ghaidaam@ymail.com
ABSTRACT
Proteus vulgaris has been described as an aetiological agent in urinary tract infections.
Quorum sensing regulates the expression of virulence factors in a wide variety of pathogenic
bacteria.Most Gram negative bacteria produce acyl homoserine lactones. In this study,
Homoserine L-lactone was synthesized by Proteus vulgaris through using aspartic acid which
was added to the culture media at a final concentration 1%. Homoserine lactone production
was checked at different intervals of incubation (after 2, 3, 4, 5, 6 and 24 hour) where 0.01%
of KCN is added to show the time of Homoserine L-lactone appearance in the bacterial
growth. When one drop of culture supernatant was added to fresh growth of Proteus vulgaris
on slide, aggregation or quorum sensing was seen as indicator of HSL (Homoserine
lactoneproduction) production.
Keywords: Proteus vulgaris, Quorum Sensing, Homoserine Lactone
INTRODUCTION
Proteus vulgaris is a rod-shaped Gram-
infections, but can also produce severe
negative chemoheterotroph bacterium. The
abscesses [1]. Many different factors may
size
from
influence the evolution and course of
P.vulgaris
pathogenesis; temperature, pH, chemical
of
0.4~0.6μm
individual
by
cells
1.2~2.5μm.
varies
possesses peritrichous flagella, making it
compounds
from the
actively motile. It inhabits the soil, polluted
numerous natural microflora should be
water, raw meat, gastrointestinal tracts of
added to host factors, which may also detect
animals, and dust. In humans, Proteus
signals
species most frequently cause urinary tract
species. During this process, known as
deriving
from
host.
other
Moreover,
bacterial
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IJBPAS, July, 2014, 3(7)
Ghaidaa J. Mohammed* And mohammed S.Abdul Razaq
quorum
sensing
(QS),
which
is
the
Research Article
production, biofilm formation, and virulence
technique in which bacteria regulate their
factor secretion [3, 5, 6].
gene expression to better serve the needs of
Three major quorum-sensing circuits have
the group rather than the individual.
been described: one used primarily by
Numerous species of bacteria employ a
Gram-negative bacteria, one used primarily
mechanism
This
by Gram-positive bacteria, and one that has
cells
been proposed to be universal [7]. Most
comprising a bacterial colony to coordinate
Gram negative bacteria use a certain
their gene expression in a cell-density
molecule called an acyl homoserine lactone´
dependent manner [2].Quorum sensing is
or AHL´ that is used as the signaling
mediated by small diffusible molecules
molecule.
termed autoinducers that are synthesized
oligopeptides as their signaling molecule,
intracellularly (throughout the growth of the
which
bacteria) and released into the surrounding
polypeptide synthesized in the cytoplasm.
milieu. As the number of cells in a bacterial
These two systems are species-specific, and
colony increases, so does the extracellular
each Gram negative or Gram positive
concentration of the autoinducer. Once a
bacteria produce their own specific auto-
threshold concentration is reached (at which
inducer. Thus, these molecules enable intra-
point the population is considered to be
species communication. These are private,
“quorate”),
secret conversations [8].
signaling
of
quorum
process
sensing.
allows
productive
the
binding
of the
Gram positive
have
been cut
bacteria
from a
use
large
autoinducer to cognate receptors within the
Most Gram negative bacteria produce Acyl
bacterial cells occurs, triggering a signal
homoserine lactones (AHLs for short) as the
transduction
signaling
cascade
that
results
in
molecule
of
choice,
whose
depends
on
S-
population wide changes in gene expression
production
[3, 4]. Thus, quorum sensing enables the
adenosylmethionine (SAM) as a substrate
cells within a bacterial colony to act
[9]. Each of these molecules interacts with
cooperatively,
its own specific binding protein, but with no
dependent
facilitating
adaptive
behavior
populationQS
other [8]. These molecules are small and can
controls genes that direct activities that are
easily enter and exit the cellular membrane.
beneficial when performed by groups of
At a certain threshold value of auto-inducer,
bacteria acting in synchrony. Processes
the molecule binds to the binding protein
controlled by QS include bioluminescence,
and group behavior becomes expressed
sporulation,
bacteria produce signal called autoinducer
competence,
[2].
antibiotic
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IJBPAS, July, 2014, 3(7)
Ghaidaa J. Mohammed* And mohammed S.Abdul Razaq
Research Article
(AI), which is secreted to environment.
b) After incubation with P. vulgaris,
Every AI, reaching critical concentration
the flasks were incubated at 37°C for
causes growth of the bacterial population
intervals (2, 3, 4, 5, 6, and 24hr).
and induces changes in the expression of
c) At the end of each interval, KCN
genes resulting from switching life cycle
(0.01%) was added and then after
and bacterial metabolism [6]. In some cases
18hr the media was filtered by
of Gram-negative bacterial QS, AIs are
Millipore filter (0.4mm) and then the
detected by two-component histidine kinase
filtrated
receptors that function analogously to those
sensing detection.
was
used
for
quorum
described in the preceding paragraph for
d) The quorum sensing test was done
Gram-positive QS bacteria [6]. The aim of
on slide through mixing one drop of
this study was to detect quorum sensing in
supernatant and one drop of fresh
Proteus vulgaris by detection the synthesis
bacterial growth , and then stained
of Homoserine lactone.
with gram stain and the slide was
MATERIALS & METHODS
examined by microscope.
e) The positive result was scored due to
Bacterial Isolates
Proteus vulgaris was isolated from a patient
the presence of aggregation bacterial
with UTI who were admitted to four
cell.
hospitals: Babylon Hospital for Maternal
Chemical
and Pediatrics, Al-Hilla Surgical Teaching
Lactone
Hospital, Al-Hashymia hospital and Al-
Homoserine
Qasim hospital during the period from
through the separation of supernatant from
4/2012 to 1/2013.Standard biochemical tests
culture media, and then the supernatant was
were used for detecting P. vulgaris strains
dialyzed verses LB free of KCN. After 24
and by Vitek system (BioMerieux, USA)
hours the dialyzed homoserine containing
[10, 11].
media were inoculated by P.vulgaris again
Detection
of
Quorum
Sensing
in
Detection
of
production
Homoserine
was
checked
for 24 hour and then Brands test was used to
P.vulgaris
detect methionine or homocystein synthesis
The detection of quorum sensing was
through
carried out according to [12, 13].
homocystein [12, 13].
a) LB media were prepared and
supplemented with 1% aspartic acid
and distributed in six flask.
conversion
of
homoserine
to
Brands Test
Reaction of sodium nitroprusside with
sulfhydryl
componud
(e.g
cysteine,
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IJBPAS, July, 2014, 3(7)
Ghaidaa J. Mohammed* And mohammed S.Abdul Razaq
Research Article
homocysteine) yeilds rose or purple red
Bacterial virulence factors are regulated by
complex products [14].
quorum-sensing
molecules
which
are
derivatives of serine substituted by a fatty
RESULTS & DISCUSSION
acid,
To study quorum sensing in P. vulgaris,
abbreviated as acyl-HSLs [15, 16, 17]. The
aspartic acid was used as the main focal
quorum sensing mechanism involves two
metabolites for homoserine synthesis. It was
types of autoinducers: AI-1 based on
observed that homoserine was accumulated
homoserine lactone and AI-2 based on other
in culture media after the addition of KCN
molecules.
in which the later will inhibit threonine
substances in Gram-negative bacteria are
synthesis, through its effect on threonine
substituted by fatty acid derivatives of acyl-
synthase
lactone
HSL (AI-1). There is no evidence that
production was also checked by using
quorum sensing receptors and AI-1 signal
brands test in order to ensure that quorum
molecules are associated with swarming
sensing occurs as a result of homoserine
motility in Proteus [18]. Whereas, Daniels
lactone synthesis.
et al. [19] showed that the QS signal
enzyme.
Homoserine
Quorum sensing
was
carried
out
as
i.e.,
acylatedhomoserine
The
majority
lactones,
of
signal
acylhomoserine lactone enhances swarming
mentioned in paragraph (Detection of
motility
quorum
The
Among the Gram-negative bacteria, the
presence of aggregation of P. vulgaris as in
most well studied quorum-sensing system is
Figure 1 was considered a positive result
the LuxR-LuxI homologous system [20].
versus the negative results in the absence of
This quorum-sensing system is widespread
homoserine lactone.
among
The best interval for accumulation of
involved in the regulation of many host-
homoserine lactone was after 4hours of
associated phenotypes, including production
incubation in which the homoserine lactone
of
became
metabolites
However,
sensing
at
in
P.vulgaris).
maximum
under
concentration.
in
Serratia
Gram-negative
virulence
factors
[20,
21].
liquefaciens.
genera
and
Two
and
is
secondary
distinct
certain condition the
mechanisms of signalling mediated by N-
bacteria can form homocystein as a result of
AHLs have been described. In most Gram-
production of homocystein synthase, and the
negative bacteria, the signal is generated by
later
an N-AHL synthase of the LuxI family of
may be
used
as
homoserine lactone synthesis.
indicator
for
proteins, and is perceived by an N-AHL
receptor protein belonging to the LuxR
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IJBPAS, July, 2014, 3(7)
Ghaidaa J. Mohammed* And mohammed S.Abdul Razaq
Research Article
family of transcriptional regulators. The N-
by
AHL autoinducers bind to their cognate
biomarkers in the management of bacterial
LuxR-type proteins only on reaching a
diseases and, thus, monitoring them in
critical
biological samples may be a significant
threshold
Autoinducer
concentration.
binding
could
serve
as
potential
the
analytical tool for the investigation of such
transcriptional activity of the LuxR protein
diseases [24]. Specifically, altered bacteria-
in regulating the expression of target genes,
host interaction has been implicated in
which can include luxI. This establishes a
several health imbalances and disorders. For
positive
N-AHL
instance, there are several reports referred to
synthesis, although it must be noted that
the role of bacteria in gastrointestinal
positive feedback is not a universal feature
disorders such as inflammatory bowel
of
quorum-sensing
disease and irritable bowel syndrome [25].
systems. In some Gram-negative bacteria
The observation that quorum sensing is
such as Vibrio spp., N-AHL synthesis is
linked to virulence factor production and
directed by a LuxM synthase (unrelated to
biofilm formation suggests that
LuxI) and perception of the signal involves
virulent Gram-negative organisms could
a cytoplasmic membrane-associated sensor
potentially be rendered nonpathogenic by
kinase. To date, N-AHL-dependent quorum-
inhibition of their quorum-sensing systems.
sensing circuits have been identified in a
Research
wide range of Gram-negative bacteria,
inhibition thereof, may provide a means of
where they regulate various functions
treating many common and damaging
including
chronic infections without the use of
feedback
loop
N-AHL-mediated
conjugal
controls
bacteria
for
bioluminescence,
transfer,
biofilm
plasmid
formation,
into
growth-inhibitory
quorum
sensing,
agents,
such
many
and
as
motility, antibiotic biosynthesis, and the
antibiotics, preservatives, and disinfectants,
production of virulence factors in plant and
that
animal pathogens [22, 23]. AHLs produced
organisms [20].
A
unavoidably
select
for
resistant
B
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IJBPAS, July, 2014, 3(7)
Ghaidaa J. Mohammed* And mohammed S.Abdul Razaq
Research Article
Figure (1): Detection of Quorum Sensing in P. vulgaris (100x)
A: control (absence of homoserine); B: positive result ( )
[3] Ng WL and Bassler BL, Bacterial
CONCLUSION
Quorum sensing was carried out through the
quorum-sensing
production
architectures, Ann. Rev. Genet., 43,
of
homoserine
lactone
by
Proteus vulgaris isolates where, it was
found that there was an aggregation of the
bacterial
cells
after
addition
of
the
supernatant that likely occurs as a result for
the
presence
of
homoserine
lactone.
network
2009, 197-222.
[4] Atkinson S and Williams PJR.
Quroum sensing. Soc. Interface, 6,
2009,959.
[5] Novick
RP
and
Geisinger
E,
Quorum sensing in staphylococci.
ACKNOWLEDGMENTS
I am grateful to Biomedical Science
Annu. Rev Genet., 42, 2008, 541-
Department of the Medicine College at
564.
Florida State University especially the
[6] Rutherford ST and Bassler BL,
laboratory of Dr. Wang. We are so grateful
Bacterial Quorum Sensing: It’s Role
to Dr. Wang Yanchang, Dr. Fengshan
in Virulence and Possibilities for Its
Liang, Dr. Fengzhi Jin and Kelly McKnight
Control.
for their assistance. Also, I would like to
Presepectives in Medicine, 4 (3),
express my deep gratitude to Prof. Dr.
2012, 1-25.
Mohammed Sabri for his kind assistance.
Cold
Spring
Harbor
[7] Readind, Nicola C, Sperandio, Vanessa
and Dallas TX, Quorum sensing: the
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