Download The social behaviours of bacterial pathogens

Published Online August 22, 2008
The social behaviours of bacterial pathogens
Roman Popat, Shanika A. Crusz, and Stephen P. Diggle*
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University
Park, University of Nottingham, Nottingham NG7 2RD, UK
Introduction: The term quorum sensing (QS) is used to describe communication
between bacterial cells, whereby a coordinated population response is
controlled by diffusible signal molecules produced by individuals.
Sources of data: Studies on QS-mediated signalling processes in bacteria have
revealed the existence of intricate regulatory networks to enable bacterial
populations to fine tune their responses to environmental changes and increase
their chances of survival, using complex signalling pathways.
Areas of agreement: A population of bacteria invading a host may benefit from
the coordinated release of virulence determinants and in vitro studies have
shown that QS regulates virulence factor production in many species of bacteria.
Areas of controversy: However, the role of QS in vivo is less well understood, but
has been demonstrated to be important in several pathogenic organisms.
Growing points and areas timely for developing research: There is a growing
interest in blocking bacterial cell– cell communication as a means to control
infections. This review discusses QS from a pathogenic perspective and discusses
the potential of QS as an anti-pathogenic target.
Keywords: quorum sensing/Pseudomonas aeruginosa
Introduction
Accepted: July 28, 2008
*Correspondence to:
Stephen P. Diggle,
Institute of Infection,
Immunity and
Inflammation, Centre for
Biomolecular Sciences,
University Park, University
of Nottingham,
Nottingham NG7 2RD, UK.
E-mail: steve.diggle@
nottingham.ac.uk
In 1667, Antonie van Leeuwenhoek was the first person to observe
microorganisms under his handmade microscopes. Those first glimpses
of a tiny microbial world would have enormous implications for the
emerging field of medical sciences when bacteria were exposed as causative agents of disease in the 19th century. Following this, the 20th
century saw incredible strides in the war against infectious diseases
with antibiotics, vaccination and clean water supplies dramatically
reducing death rates, particularly in developed countries. However, the
relentless rise of resistance to antibiotics in many species of bacterium
allows them to continue to threaten human health and welfare. Indeed,
at the beginning of the 21st century, infectious diseases accounted for
41% of the global disease burden.
British Medical Bulletin 2008; 87: 63–75
DOI:10.1093/bmb/ldn030
& The Author 2008. Published by Oxford University Press.
All rights reserved. For permissions, please e-mail: [email protected]
R. Popat et al.
At the heart of tackling, the huge challenge posed by infectious
microorganisms is the overwhelming need to understand their nature.
Although often viewed as simple creatures, the study of microbial
development has shown that they are capable of a multitude of behaviours.1 Crucially, this is not just at the level of the individual cell. One
of the most remarkable discoveries in microbiology in the past 30 years
has been the fact that bacterial cells communicate with each other in a
process commonly known as quorum sensing (QS). This enables the
coordination of a population of cells, resulting in a hitherto unrecognized sophistication in the behaviour of bacteria. Unravelling the complexities of bacterial social behaviours enables a deeper understanding
of their pathogenic mechanisms and offers new and promising opportunities for future antimicrobial agents. The progress made in this field
will be discussed in this review and illustrated using the behaviour of
the important human pathogen Pseudomonas aeruginosa in the cystic
fibrosis (CF) lung environment.
What is quorum sensing?
The history of bacterial cell-to-cell communication dates back several
decades. While working on the marine bioluminescent bacterium
Vibrio fischeri which naturally illuminates the light organs of squid,
Nealson and colleagues noticed that bioluminescence only occurred at
a threshold population density of bacterial cells and that spent culture
supernatants could induce luminescence at lower population densities.
It was hypothesized that small diffusible molecules (autoinducers) were
emitted and that the concentration of autoinducer could be sensed by
bacterial cells and that this conveyed information about population
density. The autoinducer was later purified and identified as
N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL) which is a
member of the N-acyl homoserine lactone (AHL) family of molecules.2
Although initially thought to be a phenomenon restricted to a few
marine Vibrio species, it was later shown that the production of the
b-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (carbapenem)
by the terrestrial plant pathogen Erwinia carotovora was also regulated
by AHLs.3 Since then, many Gram-negative species have been shown
to possess AHL-signalling systems that regulate a wide variety of different phenotypes.2 Signalling is not restricted to Gram-negative bacteria;
a number of Gram-positive organisms have been shown to employ
small, modified oligopeptides as extracellular signalling molecules.
These peptides activate gene expression by interacting with twocomponent histidine protein kinase signal transduction systems. For
example, in Staphylococcus aureus, the expression of a number of cell
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The social behaviours of bacterial pathogens
Fig. 1 The chemical diversity of quorum sensing signalling molecules (– found in Gram – ve
organisms, þ found in Gram þve organisms). (1) 3-oxo-AHL, N-(3-oxoacyl)-homoserine
lactone (–); (2) 3-hydroxy-AHL, N-(3-hydoxyacyl)-homoserine lactone (– ) and (3) AHL,
N-acylhomoserine lactone where R ranges from C1 to C15 (– ); (4) A-factor,
2-isocapryloyl-3-hydroxy-methyl-g-butyrolactone (– ); (5) autoinducing peptide-1 (AIP-1)
from S. aureus (þ); (6) AI-2, autoinducer-2, furanosyl borate ester form (– and þ); (7) PQS,
Pseudomonas quinolone signal, 2-heptyl-3-hydroxy-4(1H )-quinolone (– ); (8) DSF, ‘diffusible
factor’, methyl dodecenoic acid (–) and (9) PAME, hydroxyl-palmitic acid methyl ester (–).
density-dependent virulence factors is regulated by the global regulatory locus agr (accessory gene regulator).2 In fact, a lexicon of compounds have now been described as QS signal molecules in both
Gram-negative and Gram-positive bacteria2 (Fig. 1). For this short
review, we focus primarily on AHL QS systems found in Gramnegative bacteria.
The process of cell-to-cell signalling using small molecules was
termed ‘Quorum sensing’ because it is a similar process to how
decisions in a law court are made only when a threshold number of
members (a quorum) are present.4 Autoinduction by AHL molecules
occurs when an AHL signal binds to its cognate receptor inside the
cell, usually at a threshold concentration of the signal which correlates
to the number of bacterial cells present. Once the signal-receptor
complex is formed, it binds to the promoter region of the signal
synthase gene. This results in an autoinduction of signal synthesis. It is
important to note that the process of signal autoinduction also occurs
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Fig. 2 A simple quorum sensing autoinduction circuit. R represents a receptor/response
protein and I represents a QS synthase protein. The binding of signal to the R protein at
high cell densities leads to autoinduction of signal synthesis and a change in the behaviour
of the bacterial population.
in QS systems where AHLs are not the signal molecule. In addition to
increased signal production, the signal-receptor complex regulates the
expression of a suite of genes, resulting in a change in phenotypic behaviour. An example of a QS-circuit is depicted in Figure 2.
AHL-dependent QS has been shown to regulate a number of diverse
behaviours and phenotypes in Gram-negative bacteria. Although many
of these behaviours can theoretically be performed by individual bacterial cells working in isolation, they become more effective when the
bacteria work as a group. These include antibiotic production, exotoxin production, virulence and motility.2 One of the most complex
social behaviours performed by microbes is the development of biofilms. In the formation of biofilms, cells abandon the isolation of the
planktonic mode of growth and group together to form organized
‘slime-cities’. These complicated structures often contain channels for
the import of nutrients and the disposal of waste products and they
may even contain specialist cells, which appear to have specific roles
within the biofilm.5 Medically, biofilms are of huge importance as they
are capable of forming in the lungs of chronically ill patients and often
colonize medical devices such as catheters and prosthetic valves. This is
especially problematic as they are often resistant to desiccation and
treatment with biocides and antibiotics. QS appears to play a role in
the development of biofilms as in several species of Gram-negative
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The social behaviours of bacterial pathogens
bacteria, disruption of the QS system has been shown to affect biofilm
formation and differentiation.6
Quorum sensing and virulence
QS and virulence factor production
The virulence of an organism often depends on its ability to produce
and release toxins that can damage a host. However, production of
extracellular metabolites may lead to detection of the bacteria and ultimately destruction by the host immune system. Therefore, an effective
strategy for bacterial cells to adopt is to avoid producing and releasing
toxins until their numbers are sufficient when a simultaneous toxin
release can overwhelm a host immune response. By using several
animal host models, including nematodes and mice, QS-dependent
control of virulence determinants and virulence itself has now been
demonstrated in several human pathogenic organisms. These include
Burkholderia pseudomallei, the causative agent of melioidosis,7
B. mallei,8 B. cenocepacia,9 V. cholerae 10 and S. aureus. 11
Perhaps, the most studied QS system is that of P. aeruginosa.
Pseudomonas aeruginosa is a Gram-negative bacterium, capable of
causing disease in plants, animals and humans.12 It is a major source
of nosocomial infections and is a leading cause of mortality in CF
patients.13 As an opportunistic human pathogen, P. aeruginosa can
colonize a wide variety of anatomical sites. This is because the organism produces an arsenal of extracellular virulence factors which are
capable of causing extensive tissue damage and bloodstream invasion
which consequently promotes systemic dissemination. Many of these
exoproducts are regulated in a cell density-dependent manner via QS.
Pseudomonas aeruginosa possesses two AHL-dependent QS systems.
These are termed the las and rhl systems, comprising the LuxRI homologues, LasRI14 and RhlRI,15 respectively. LasI directs the synthesis of
primarily N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL)
and together with the transcriptional regulator LasR regulates the production of virulence factors including elastase, the LasA protease, alkaline protease and exotoxin A.2 RhlI directs the synthesis of
N-butanoyl-L-homoserine lactone (C4-HSL)16 which activates RhlR and
in turn RhlR/C4-HSL induces the production of rhamnolipid, elastase,
LasA protease, hydrogen cyanide, pyocyanin, siderophores and the
cytotoxic lectins PA-I and PA-II.15 – 18 The las and the rhl systems
are organized in a hierarchical manner such that the las system
exerts transcriptional control over both rhlR and rhlI17 (Fig. 3).
In addition to AHL-dependent QS, P. aeruginosa also produces over
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Fig. 3 Pseudomonas aeruginosa QS cell-to-cell signalling. As the cells enter stationary
phase, a critical concentration of the LasR/3O-C12-HSL complex induces the rhl QS system.
Both the las and the rhl QS system are responsible for the regulation of a number of virulence determinants. LasR/3O-C12-HSL also positively regulates the PQS biosynthetic genes
and hence PQS signal levels.
50 2-alkyl-4(1H )-quinolones (AQs), some of which were originally
identified from their antibacterial properties, although the biological
function of many of these is not known.19 One of these compounds,
2-heptyl-3-hydroxy-4(1H )-quinolone was discovered to function as a
diffusible signal molecule and termed the pseudomonas quinolone signal
(PQS; Fig. 1).20 Subsequently, PQS was shown to regulate P. aeruginosa
virulence gene expression and to function as an integral component of
the QS network since PQS production is modulated by both the las
and the rhl systems (Fig. 3).19 This demonstrates the complexity of some
QS systems. The importance of QS for the virulence of P. aeruginosa in
acute infections has been confirmed in a number of animal models
including mice,21,22 nematodes and insects.23
QS in vivo: the cystic fibrosis lung
While P. aeruginosa can cause severe infections in burn wounds and
intubated patients, it is particularly problematic in the lungs of patients
with CF where it causes chronic infections which result in extensive
tissue damage, reduction in lung function and ultimately death.13 It has
been demonstrated that P. aeruginosa QS genes are expressed in the
lungs of CF patients24 and that sputum from the CF lung often contains QS signal molecules suggesting that QS virulence is important
during pathogenesis in CF.25
In CF infections, P. aeruginosa is more resistant to antibiotic treatment than is predicted by susceptibility tests on planktonic cells and it
is thought that the added resistance of P. aeruginosa is due, in part, to
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The social behaviours of bacterial pathogens
a ‘biofilm’ lifestyle within the lung.26 Here, the cells adhere to a
surface and secrete a polymeric matrix that binds and protects them
from antibiotic therapy and the host immune response.27 QS has been
shown to play a role in the development of P. aeruginosa biofilms
in vitro. Inactivation of QS results in the formation of flatter, less structured biofilms than those seen in the corresponding wild type.
Furthermore, when QS is disrupted, the biofilms formed are often
more susceptible to treatment with biocides and antibiotics.28
However, in vivo, the role of QS in biofilm formation is less clear.
Recent observations suggest that QS-deficient P. aeruginosa mutants
are often isolated from sputum samples.29,30 These mutants mostly
carry mutations in the lasR regulator gene which impairs the response
to signal molecules. There are a number of plausible explanations for
this. First, QS may not be important for growth in this environment
and therefore Darwinian selection results in a loss of QS over time.
Secondly, it has been suggested that a lasR mutation confers a growth
advantage on particular carbon and nitrogen sources which may give a
selective advantage over QS-positive strains.31
Another plausible explanation for the prevalence of lasR mutants is
that bacteria can ‘cheat’ or ‘freeload’ on QS cooperating populations.32
In P. aeruginosa, many QS-regulated products are released into the
extracellular environment and benefit not only the producing cell but
also its neighbours. Mutants that do not respond to QS signals do not
incur the cost of producing these ‘public goods’ but gain the benefit of
production by neighbours.33,34 Put another way, lasR cheaters have a
social fitness advantage over QS-positive strains. This cooperation and
cheating principle has been demonstrated for QS in vitro 33,34 but also
for the production of other public goods which in turn influence the
virulence of an infection.35,36 Social cheating could reconcile why lasR
mutants are repeatedly found in clinical isolates from CF patients and
yet QS-deficient mutants of P. aeruginosa have reduced virulence when
grown in monocultures.37 A detailed ecological survey of CF lung
infections investigating the extent of QS-positive and negative strains
will help us to unravel the secrets of this problematic organism within
this environment and may provide clues as to why CF infections
become chronic and persistent over time.
Bacterial cross-talk
Much of the work on QS signalling systems has focused on studying
signalling between single species (intraspecies) bacterial populations
and communication between P. aeruginosa cells using AHLs is a likely
example of intraspecies signalling. Because different bacterial species
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may make similar signal molecules, more recently researchers have
begun to focus on interactions between bacterial species (interspecies)
and even between bacteria and eukaryotic (interkingdom) organisms
sharing a similar environment. This has been commonly called ‘cross
talk’.38
Within a medical context, much of this research has focused on
studying organisms that are commonly found together in the CF lung.
Pseudomonas aeruginosa and B. cenocepacia often occur together in
the lungs of people with CF, where they are associated with high morbidity and mortality.13,39 Burkholderia cenocepacia has been shown
to up-regulate the production of virulence determinants in response to
AHLs produced by P. aeruginosa, although this does not appear to
happen the other way round. The term ‘cross talk’ is suggestive of a
cooperative venture between two or more species; however, we must be
careful with our interpretation of certain behaviours. In this case, it
suggests that B. cenocepacia uses P. aeruginosa AHLs as an environmental cue to alter its behaviour rather than there being true signalling
between the two bacterial species. Pseudomonas aeruginosa pays the
cost of producing AHLs, possibly for intraspecies signalling, but
appears to gain no benefit from B. cenocepacia in return.
Understanding the true nature of these interactions is important if we
are to fully understand the complex ecology of environments such as
the CF lung. Cross-talk using QS signals is not limited to just
Gram-negative bacteria. Pseudomonas aeruginosa and S. aureus also
co-exist in the CF lung, where AHLs from the former can influence the
expression of virulence determinants in the latter40 and AQs can
induce the formation of S. aureus small colony variants which increases
the resistance of S. aureus to antibiotics.41
In addition to controlling gene expression in bacterial populations,
AHLs have also been found to be directly recognized by eukaryotic
cells and even influence the behaviour of eukaryotic organisms. AHLs
have been shown in several different studies to have immunomodulatory effects, influencing the production of cytokines that in turn determines the type of immune response elicited upon infection.42,43
Furthermore, AHLs can also have cardiovascular effects by inducing
relaxation of blood vessels.44 If we put these two effects into the
context of infection, it becomes apparent that bacteria have the power
to influence immune responses, probably to their benefit, and stimulate
the delivery of nutrients for their survival by increasing the blood
supply.
QS signalling systems are quite vulnerable and can be degraded or
‘quenched’ by other bacteria, eukaryotic cells or even eukaryotic organisms. This degradation can be dependent upon conditions in the surrounding environment or due to the action of certain enzymes. AHL
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signals are rendered biologically inactive in alkaline environments and,
therefore, in certain environmental niches, signalling may be ineffective.45 A number of bacteria co-existing with AHL producers have been
found to produce enzymes which can degrade these signal molecules.
This has been shown in soil, where Bacillus strains produce lactonase
enzymes responsible for this activity.46 There are also organisms which
have the ability to synthesize as well as degrade QS molecules. One
such organism is P. aeruginosa in which a number of enzymes have
been shown to have the ability to inactivate AHLs.47 Whether this is
their primary role has yet to be elucidated.
In clinical settings, P. aeruginosa has been shown to co-exist and suppress the growth of the fungus Candida albicans. 48 However, it is reasonable to assume that eukaryotic organisms such as C. albicans will have
developed mechanisms to protect themselves against bacterial damage. In
fact, C. albicans has been shown to down-regulate the production of virulence factors by P. aeruginosa by producing the sesquiterpene compound
farnesol.49 It was demonstrated that addition of farnesol to P. aeruginosa
cultures resulted in a reduction in the levels of the PQS QS signal molecule and subsequently a reduction in the production of the phenazine
pyocyanin.49 Farnesol is not unique to C. albicans and it is likely
that many other organisms are capable of modulating the virulence of
P. aeruginosa using this or related compounds.
It is now known that human cells synthesize enzymes which are able
to degrade AHLs possibly as a strategy to prevent their damage by bacterial pathogens50 and this forms the basis for novel antimicrobial
therapies against problematic organisms such as P. aeruginosa.
Quorum sensing as a target for therapy
It should not be a surprise that bacteria acquire resistance to antibiotics
as most antimicrobial agents are derived from microorganisms themselves, the products of microbial warfare in a competitive unicellular
environment. Treatment with antibiotics creates a natural selection for
resistant genotypes, which consequently spread through a population.
Often the genes for antibiotic resistance are carried on mobile plasmids
that can be horizontally transferred from resistant to non-resistant
cells, increasing the spread of resistance through a population. In the
medical arms race with microbes, the supply of novel antibiotics has
been plentiful but is starting to run dangerously low and the scientific
challenge is compounded by social problems. General Practitioners
must be sparing with the prescription of antibiotics in order to reduce
the emergence of resistance. However, resistance can spread between
bacterial taxa, between patients and between nations, so global
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inconsistencies in treatment practices can lead to a widespread resistance on a global scale. There is now, therefore, a clear need to design
novel antimicrobial treatments. Progressive routes to antimicrobial
therapy include approaches that do not kill the cells or impair their
essential functions but more specifically inhibit the mechanisms of
pathogenesis (i.e. disabling rather than destroying). Such antipathogenic agents are less likely to pose harsh selective pressures for
the development of resistant mutants. The basic strategy is to downregulate virulence, which will assist the immune system in eradicating
the invading bacteria.
QS systems regulate bacterial pathogenesis and therefore represent
novel targets for such therapies. There has recently been much interest
in the discovery of QS inhibitors (QSI). The first naturally occurring
agents found to possess such QS antagonistic activity were brominated
furanones produced by the Australian macro-alga Delisea pulchra. 51
Biofilms grown in the presence of synthetic furanone derivatives were
rendered susceptible to antimicrobial killing with the antibiotic tobramycin and detergent SDS in contrast to the untreated biofilms.52
Further reports of QSI compounds derived from food, herbal and
fungal sources have been published and their potential therapeutic role
investigated in biofilm and pulmonary murine models.53,54 A recent
study identified three compounds that efficiently inhibited the synthesis
of molecules required for the activation of the P. aeruginosa AQ regulatory pathway.22 This limited P. aeruginosa infection in mice, without
affecting bacterial viability and such compounds provide a starting
point for the design and development of novel anti-pathogenic agents
targeted at important human pathogens.
Conclusions
Microbes display an extraordinary level of social differentiation, organization and coordination. Much of this is orchestrated by QS communication systems which allow cells to sense their population density and
coordinate an appropriate response. There are several species of bacteria where it has been demonstrated that QS is important for the full
virulence of the organism. The molecular mechanisms governing QS
systems are being increasingly understood, but there are still many
questions that need to be addressed. For example, what is the role of
QS in vivo? In only a handful of well-studied organisms (e.g. P. aeruginosa)
has this been partially addressed and the results suggest a role for QS
in virulence. Given this, does QS provide an attractive target for novel
antibacterial agents? Proof of principle studies in animal models has
been encouraging and a future challenge will be to take these studies
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The social behaviours of bacterial pathogens
into human clinical trials. Another area that is growing in maturity is
understanding social evolution in microorganisms. As a population of
pathogenic bacteria may evolve during an infection, cooperation and
conflict among bacterial cells may ultimately have a profound influence
on the outcome of an infection. Therefore, understanding the conditions that favour cooperation will enable us to better understand the
evolution of virulence over the course of an infection. Only by understanding the complicated ecology of bacterial environments can we
continue to develop successful treatments against these problematic
organisms.
Funding
We gratefully acknowledge BBSRC (R.P.), The Wellcome Trust (S.A.C.)
and The Royal Society (S.P.D.) for funding.
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