Emerging Infectious Diseases
Volume 4 No. 4 / October-December 1998



Synopses

Cell-to-Cell Signaling and Pseudomonas aeruginosa Infections

Christian Van Delden and Barbara H. Iglewski
University of Rochester School of Medicine and Dentistry, Rochester, New
York, USA

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      Pseudomonas aeruginosa is a bacterium responsible for severe
      nosocomial infections, life-threatening infections in
      immunocompromised persons, and chronic infections in cystic
      fibrosis patients. The bacterium's virulence depends on a large
      number of cell-associated and extracellular factors.
      Cell-to-cell signaling systems control the expression and allow
      a coordinated, cell-densitydependent production of many
      extracellular virulence factors. We discuss the possible role
      of cell-to-cell signaling in the pathogenesis of P. aeruginosa
      infections and present a rationale for targeting cell-to-cell
      signaling systems in the development of new therapeutic
      approaches.

Pseudomonas aeruginosa as a Human Pathogen

Pseudomonas aeruginosa, an increasingly prevalent opportunistic human pathogen,
is the most common gram-negative bacterium found in nosocomial infections.            
 P. aeruginosa is responsible for 16% of nosocomial pneumonia cases (1), 12%    
 of hospital-acquired urinary tract infections (2), 8% of surgical wound      
infections (3), and 10% of bloodstream infections (4). Immunocompromised 
patients, such as alginate/biofilm, neutropenic cancer and bone marrow        
transplant patients, are particularly susceptible to opportunistic infections.
In this group of patients, P. aeruginosa is responsible for pneumonia and
septicemia with attributable deaths reaching 30% (5,6). P. aeruginosa is
also one of the most common and lethal pathogens responsible for
ventilator-associated pneumonia in intubated patients (7), with
directly attributable death rates reaching 38% (8). In burn patients,
P. aeruginosa bacteremia has declined as a result of better wound
treatment and dietary changes (removal of raw vegetables, which can
be contaminated with P. aeruginosa, from the diet) (3). However, P.
aeruginosa outbreaks in burn units are still associated with high (60%)
death rates (9). In the expanding AIDS population, P. aeruginosa bacteremia
is associated with 50% of deaths (10). Cystic fibrosis (CF) patients are
characteristically susceptible to chronic infection by P. aeruginosa, which
is responsible for high rates of illness and death in this population (11).
The capacity of P. aeruginosa to produce such diverse, often overwhelming
infections is due to an arsenal of virulence factors (Figure 1). Many
extracellular virulence factors secreted by P. aeruginosa have been shown
to be controlled by a complex regulatory circuit involving cell-to-cell
signaling systems that allow the bacteria to produce these factors in a
coordinated, cell-densitydependent manner (12). In this article we
describe major virulence factors of P. aeruginosa and the possible
involvement of cell-to-cell signaling in the pathogenesis of acute P.
aeruginosa infection. We also summarize data suggesting that these
regulatory systems could be exploited for the design of therapeutic
interventions.

[Figure 1.] Virulence factors of Pseudomonas aeruginosa. P.
aeruginosa has both cell-associated (flagellum,
pilus, nonpilus adhesins, lipopolysaccharide [LPS]) and
extracellular virulence factors proteases, hemolysins, exotoxin
A, exoenzyme S, pyocyanin).


Pathogenesis of P. aeruginosa Infections

P. aeruginosa (family Pseudomonadaceae), an aerobic, motile, gram-negative rod
able to grow and survive in almost any environment, lives primarily in water,
soil, and vegetation. However, despite abundant opportunities for spread, P.
aeruginosa rarely causes community-acquired infections in
immunocompetent patients. As a result, the pathogen is viewed as opportunistic.
The different phases of P. aeruginosa infection are shown in Figure 2.

[Figure 2.]  Model of the different phases of Pseudomonas aeruginosa
infection. After an initial colonization phase, mostly       
dependent on cell-associated virulence factors, the 
infectious process evolves either to a chronic infection
characterized by low production of extracellular virulence
factors or to an acute infection characterized by high production 
of cell-to-cell signaling dependent virulence factors.
During acute exacerbation of chronic infection, the
production of cell-to-cell factors is highly increased.     

Colonization: The Predominant Role of Cell-Associated Virulence Factors
 
To initiate infection, P. aeruginosa usually requires a substantial break in
first-line defenses. Such a break can result from breach or bypass of normal
cutaneous or mucosal barriers (e.g., trauma, surgery, serious burns, or
indwelling devices), disruption of the protective balance of normal mucosal
flora by broad-spectrum antibiotics, or alteration of the immunologic
defense mechanisms (e.g., in chemotherapy-induced neutropenia, mucosal
clearance defects from cystic fibrosis, AIDS, and diabetes mellitus).

The first step in P. aeruginosa infections is colonization of altered
epithelium. The pathogen colonizes the oropharynx of up to 6% and is
recovered from the feces of 3% to 24% of healthy persons (2). In contrast,
up to 50% of hospitalized patients are at high risk for P. aeruginosa
colonization (2). Adherence of P. aeruginosa to epithelium is probably
mediated by type 4 pili similar to those of Neisseria gonorrhoeae (13).
Several other nonpilus adhesins responsible for the binding to mucin have
been described, but their role in the infection process remains unclear
(14). Flagella, which are primarily responsible for motility, may also act
as adhesins to epithelial cells (15).

From Colonization to Acute Infection: The Role of Extracellular Virulence
Factors

P. aeruginosa produces several extracellular products that after
colonization can cause extensive tissue damage, bloodstream invasion, and
dissemination (Figure 1). In vivo studies have shown that mutants defective
in the production of exotoxin A, exoenzyme S, elastase, or alkaline
protease are essential for maximum virulence of P. aeruginosa; however, the
relative contribution of a given factor may vary with the type of infection
(16). Many of these factors are controlled by regulatory systems involving
cell-to-cell signaling. We will summarize the known biologic effects of the
most-studied extracellular virulence factors associated with acute P.
aeruginosa infection.

Exotoxin A is produced by most P. aeruginosa strains that cause clinical
infections. Like diphtheria toxin, P. aeruginosa exotoxin A catalyzes
ADP-ribosylation and inactivation of elongation factor 2, leading to
inhibition of protein biosynthesis and cell death (17). Exotoxin A is
responsible for local tissue damage, bacterial invasion (18), and
(possibly) immunosuppression (19). Purified exotoxin A is highly lethal for
mice which supports its role as a major systemic virulence factor of P.
aeruginosa (18).

Exoenzyme S is also an ADP-ribosyl transferase, but unlike exotoxin A, it
preferentially ribosylates GTP-binding proteins such as Ras (20). This
exoproduct is responsible for direct tissue destruction in lung infection
(21) and may be important for bacterial dissemination (22).

Two hemolysins, phospholipase C and rhamnolipid, produced by P. aeruginosa,
may act synergistically to break down lipids and lecithin. Both may
contribute to tissue invasion by their cytotoxic effects. Rhamnolipid, a
rhamnose-containing glycolipid biosurfactant, has a detergentlike structure
and is believed to solubilize the phospholipids of lung surfactant, making
them more accessible to cleavage by phospholipase C (23). The resulting
loss of lung surfactant may be responsible for the atelectasis associated
with chronic and acute P. aeruginosa lung infection (24). Rhamnolipid also
inhibits the mucociliary transport and ciliary function of human
respiratory epithelium (25). However, the relative role of rhamnolipid in
acute or chronic infection is not known.

Proteases are assumed to play a major role during acute P. aeruginosa
infection. P. aeruginosa produces several proteases including LasB
elastase, LasA elastase, and alkaline protease (12). The role of alkaline
protease in tissue invasion and systemic infections is unclear; however,
its role in corneal infections may be substantial (26,27). The ability of
P. aeruginosa to destroy the protein elastin is a major virulence
determinant during acute infection. Elastin is a major part of human lung
tissue and is responsible for lung expansion and contraction. Moreover,
elastin is an important component of blood vessels, which rely on it for
their resilience. The concerted activity of two enzymes, LasB elastase and
LasA elastase, is responsible for elastolytic activity (28). Elastolytic
activity is believed to destroy elastin-containing human lung tissue and
cause the pulmonary hemorrhages of invasive P. aeruginosa infections. LasB
elastase is a zinc metalloprotease that acts on a number of proteins
including elastin. LasB elastase is highly efficient, with a proteolytic
activity approximately 10 times that of P. aeruginosa alkaline protease and
an activity toward casein approximately four times that of trypsin (28).
The LasA elastase is a serine protease that acts synergistically with LasB
elastase to degrade elastin. LasA elastase nicks elastin, rendering it
sensitive to degradation by other proteases such as LasB elastase, alkaline
protease, and neutrophil elastase (28). Both LasB elastase and LasA
elastase have been found in the sputum of CF patients during pulmonary
exacerbation (29). However, the role of LasB elastase in tissue destruction
during the chronic phase of CF is less clear. It has been postulated that
during this phase, antibodies present in high titers neutralize LasB
elastase, and elastin damaged by minute amounts of LasA is degraded mostly
by neutrophil elastase (28). LasB elastase degrades not only elastin but
also fibrin and collagen (30). It can inactivate substances such as human
immunoglobulins G and A (31), airway lysozyme (32), complement components
(33), and substances involved in protecting the respiratory tract against
proteases such as a-1-proteinase inhibitor (34) and bronchial mucus
proteinase inhibitor (35). Therefore, LasB elastase not only destroys
tissue components but also interferes with host defense mechanisms. Studies
in animal models show that mutants defective in LasB elastase production
are less virulent than their parent strains (16,36,37), which supports the
role of LasB elastase as a virulence factor.

Cell-to-Cell Signaling: A Global Regulation System of P. aeruginosa
Extracellular Virulence Factors

Cell-to-Cell Signaling Systems

P. aeruginosa appears to control the production of many of its
extracellular virulence factors by a mechanism that monitors bacterial cell
density and allows communication between bacteria by cell-to-cell
signaling. Bacteria are able to sense their environment, process
information, and react appropriately; however, their ability to sense their
own cell density, to communicate with each other, and to behave as a
population instead of individual cells has only recently been understood
(38,39). This phenomenon, called quorum-sensing or cell-to-cell signaling,
is a generic phenomenon described in many gram-negative (40) and
gram-positive bacteria (41).

The first cell-to-cell signaling system described is the lux system,
responsible for the cell-densitydependent control of bioluminescence genes
by the marine bacteria Vibrio fischeri (38). Cell-to-cell signaling systems
of gram-positive bacteria involve peptide pheromones as signals (41). In
contrast, cell-to-cell signaling systems of gram-negative bacteria, with
the exception of Ralstonia solanacearum, in which an endogenous fatty acid
derivative has been suggested as a cell-to-cell signal (42,43), are
composed of a small molecule called an autoinducer, which is synthesized by
a LuxI-type autoinducer synthase and a LuxR-type transcriptional activator
protein (R-protein) (Figure 3) (38). The various autoinducers described in
gram-negative bacteria are homoserine lactone-based molecules that differ
between one another in length and substitutions on their acyl side chains.
At low cell density, autoinducer is synthesized at basal levels and is
thought to diffuse into the surrounding media, where it becomes diluted.
With increasing cell density, the intracellular concentration of
autoinducer increases until it reaches a threshold concentration. At this
critical concentration, the autoinducer has been proposed to bind to a
specific R-protein (40). The R-protein itself is not active without the
corresponding autoinducer, and it is the R-protein/autoinducer complex that
is proposed to bind to specific DNA sequences upstream of target genes
enhancing their transcription (44,45). The resulting increase in expression
of these genes can reach 1,000-fold. The autoinducer, therefore, allows the
bacteria to communicate with each other (cell-to-cell signaling), to sense
their own density (quorum-sensing), and together with a transcriptional
activator to express specific genes as a population instead of individual
cells.

[Figure 3.] Cell-to-cell signaling systems. Cell-to-cell signaling
systems are composed of two genes. The I gene encodes an
autoinducer synthase and the R gene encodes a transcriptional
activator protein (R-protein). The autoinducer synthase is
responsible for the synthesis of an autoinducer molecule (AI),
which crosses the cell membrane. With increasing cell-density the
intracellular concentration of AI reaches a threshold level, and
the AI then binds to the transcriptional activator. The
complex R-protein/AI activates the expression of specific target genes.


The las Cell-to-Cell Signaling System of P. aeruginosa
                                         
described in P. aeruginosa was shown to regulate expression of LasB elastase
and was therefore named the las system (46). The las cell-to-cell signaling     
system is composed of lasI, the autoinducer synthase gene 3-oxo-C12-HSL 
N-[3-oxododecanoyl]-L-homoserine lactone, previously named PAI-1 or 
OdDHL), and the lasR gene that codes for a transcriptional activator protein  
(Figure 4) (47,48). The las cell-to-cell signaling system regulates  
lasB expression and is required for optimal production of other 
extracellular virulence factors such as LasA protease and exotoxin A (49).
LasI  is the most sensitive gene to activation by LasR/3-oxo-C12-HSL (50).   
The preference for the lasI promoter allows an initial rapid rise in
autoinducer synthesis, which increases the amount of 3-oxo-C12-HSL
available to bind to LasR. This autoinduction hierarchy is responsible for
a dramatic increase of expression of virulence genes (such as lasB) once a
critical cell density has been reached. Recently, the las system has also
been shown to activate the xcpP and xcpR genes that encode proteins of the
P. aeruginosa secretory pathway (51). 3-oxo-C12-HSL alone has been
suggested to contribute to the virulence of P. aeruginosa because it has
some immunomodulatory activity (52). The las cell-to-cell signaling system
is positively controlled by GacA (53), as well as by Vfr, which is required
for the transcription of lasR (54). An inhibitor, RsaL, that represses the
transcription of lasI, has also been described (de Kievit et al., unpub.
data). The multiple regulatory levels of the las cell-to-cell signaling
system and the various genes under its control highlight the importance of
this system for P. aeruginosa.

[Figure 4.] The cell-to-cell signaling circuitry of P.   
aeruginosa. The las cell-to-cell signaling system
controls the rhl cell-to-cell signaling system in a
hierarchy cascade. The LasR/3-oxo-C12-HSL complex
activates the transcription of rhlR, and 3-oxo-C12-HSL     
blocks the activation of RhlR by C4-HSL. The las system
itself is controlled positively by Vfr and GacA,
and negatively by RsaL. 3-oxo-C12-HSL is required for
biofilm differentiation and has immunomodulatory
activity. Both cell-to-cell signaling systems regulate
the expression of numerous genes (lasB: LasB elastase,
lasA: LasA elastase, toxA: exotoxin A, aprA: alkaline
protease, xcpP and xcpR: genes of the xcp secretory
pathway, rhlAB: rhamnosyltransferase required
for rhamnolipid production, rpoS: stationary phase sigma
factor).                      
     
The rhl Cell-to-Cell Signaling System of P. aeruginosa

P. aeruginosa has a second cell-to-cell signaling system, named the rhl system
because of its ability to control the production of rhamnolipid. This system is
composed of rhlI, the C4-HSL N-butyrylhomoserine lactone, previously
named PAI-2 or BHL) autoinducer synthase gene, and the rhlR gene encoding a
transcriptional activator protein (Figure 4) (55-59). This system regulates the
expression of the rhlAB operon that encodes a rhamnosyltransferase required for
rhamnolipid production (60). The rhl system is also necessary for optimal production of
LasB elastase, LasA protease, pyocyanin, cyanide, and alkaline protease
(53,57,61,62). Therefore, like the las cell-to-cell signaling system, the rhl
system, sometimes referred to as vsm (virulence secondary metabolites),
regulates the expression of various extracellular virulence factors of P.
aeruginosa. Interestingly, the rhl system also regulates the expression of rpoS,
which encodes a stationary sigma factor [sigma] dS) involved in the regulation of various
stress-response genes (63,64).
 
 The Cell-to-Cell Signaling Hierarchy in P. aeruginosa
 
Recent data have shown that the las and rhl cell-to-cell
signaling systems of P. aeruginosa interact. Both systems are
highly specific in that their respective autoinducers are unable to
activate the transcriptional activator protein of the other system (i.e.,
3-oxo-C12-HSL cannot activate RhlR, and C4-HSL cannot activate LasR)
(57,62). It has also been shown that the R-protein/autoinducer complexes
prefer certain promoters that they will activate; LasR/3-oxo-C12-HSL
preferentially activates lasB over rhlA, and RhlR/C4-HSL preferentially
activates rhlA over lasB (57,62). However, neither system is completely
independent of the other. The LasR/3-oxo-C12-HSL complex activates the
expression of rhlR placing the las system in a cell-to-cell signaling
hierarchy above the rhl system (Figure 4) (63,65). Moreover, 3-oxo-C12-HSL
can bind to RhlR, blocking the binding of C4-HSL to its transcriptional
activator rhlR (65). The las system therefore controls the rhl system at
both a transcriptional and posttranslational level. Another yet
unidentified regulatory mechanism directly influencing the expression of
rhlRI has been suggested (63). Rhl system regulation of such important
genes as rpoS could explain why multiple levels of controls are required
for its tight regulation.

Cell-to-Cell Signaling: A Powerful System to Overcome Host Defenses during
Acute Infection

Cell-to-cell signaling systems might enable P. aeruginosa to overcome host
defense mechanisms. Isolated production of extracellular virulence factors
by a small number of bacteria would probably lead to an efficient host
response neutralizing these compounds. However, the coordinated expression
of virulence genes by an entire bacterial population once a certain density
has been reached might allow P. aeruginosa to secrete extracellular factors
only when they can be produced at high enough levels to overcome host
defenses. These factors could alter the precarious balance between host
defenses and production of bacterial toxins, leading to invasion of blood
vessels, dissemination, systemic inflammatory-response syndrome, and
finally death. Even appropriate antibiotic therapies are often unable to
stop this course; therefore, the process must be blocked early, before
virulence gene expression can be coordinated.

Biofilms and Cell-to-Cell Signaling

The most striking feature of persistent P. aeruginosa infections in CF
patients is the selection of mucoid mutants producing the exopolysaccharide
alginate (a polymer of mannuronic and guluronic acid) (11). These mutant
bacteria grow inside a biofilm (defined as microcolonies surrounded by an
exopolysaccharide) as a survival strategy because the surrounding matrix
protects bacteria from phagocytes and complement activity (11). P.
aeruginosa growing in an alginate "slime matrix" are resistant to
antibiotics (e.g., aminoglycosides, -lactam antibiotics, fluoroquinolones)
and disinfectants (11). The exact nature of the increased resistance is
unclear but has been attributed to slow growth, penetration barriers,
-lactamase production, and other factors. P. aeruginosa also produces
other less well-defined biofilms essential in the colonization of
indwelling devices such as catheters. Recently, the las cell-to-cell
signaling system has been shown to be involved in the differentiation of P.
aeruginosa biofilms (66). A mutant defective in the production of
3-oxo-C12-HSL formed an abnormal biofilm that, in contrast to the wild type
biofilm, was sensitive to low concentrations of the detergent sodium
dodecyl sulfate (SDS). Furthermore, the addition of 3-oxo-C12-HSL to the
culture media restored production of a differentiated, SDS-resistant
biofilm by the mutant (66). Whether the formation of an undifferentiated
biofilm renders this mutant more sensitive to antibiotics is still unknown.
Also unclear is whether 3-oxo-C12-HSL is required for the differentiation
of alginate biofilms. The link between 3-oxo-C12-HSL and biofilm
differentiation highlights the broad range of systems controlled by
cell-to-cell signaling in P. aeruginosa.

Interference with Cell-to-Cell Signaling: A Potential Therapeutic Approach
against P. aeruginosa

The increasing emergence of bacterial strains resistant to antibacterial
drugs is a major challenge. P. aeruginosa is one of the most problematic
human pathogens, showing intrinsic resistance to many structurally
unrelated antibiotics. Resistance mechanisms include low outer membrane
permeability or multidrug efflux pumps (tetracycline, imipenem,
fluoroquinolones, aminoglycosides) or production of antibiotic modifying
enzymes (aminoglycosides, -lactams) (67,68). Previous exposure to
antibiotics often leads to multidrug-resistant P. aeruginosa strains.
Moreover, the eradication of colonization with P. aeruginosa is almost
impossible in CF patients because of the emergence of multidrug-resistant
strains and the protective effects of alginate (69). Therefore, major
efforts have been made to find new therapeutic interventions against P.
aeruginosa. A tempting approach is to reduce the production of
extracellular virulence factors. In this regard, the interference with
cell-to-cell signaling systems is very attractive. Experimental evidence
for effective interference with cell-to-cell signaling systems has been
recently provided by the use of autoinducer analogs (70-72) or furanone
compounds (73). The LasR/3-oxo-C12-HSL complex, which plays a central role
in controlling virulence gene expression in P. aeruginosa, is essential not
only for the production of elastase and other proteases required for tissue
invasion but also for the control of the rhl cell-to-cell signaling system
in a hierarchical cascade and for the differentiation of biofilm. Mutants
with a nonfunctional las cell-to-cell signaling system are unable to
produce several virulence factors (including elastase) and have
significantly reduced virulence in a neonatal mouse pneumonia model (37).
Therefore, the LasR/3-oxo-C12-HSL complex has become a potential target for
new therapeutic interventions. Analogs of 3-oxo-C12-HSL (74) are under
investigation for their ability to act as 3-oxo-C12-HSL antagonists, which
block activation of LasR and thereby interfere with virulence gene
expression. The fact that 3-oxo-C12-HSL can block the bioactivity of C4-HSL
(65) suggests that it might be possible to interfere efficiently with
cell-to-cell signaling in P. aeruginosa. As elements essential for the
synthesis of autoinducers have recently been described, drugs inhibiting
the biosynthesis process could also be designed (75-80). These approaches
could reduce the production of virulence factors, and by interfering with
biofilm differentiation they could also render colonizing P. aeruginosa
more susceptible to antibiotics and biocides. Combination therapies,
including antibiotics and drugs reducing virulence factor production, might
have an important role in the treatment of CF and intensive-care patients.

As with any therapeutic approach against P. aeruginosa, the possible
emergence of mutations that would prevent the desired effect is a concern.
We have recently described spontaneous mutants that were restored in the
production of extracellular virulence factors (elastase and rhamnolipid) in
the absence of a functional las cell-to-cell signaling system (81). We were
unable to isolate mutants restored in virulence factor production when both
the las and the rhl cell-to-cell signaling systems were deficient. These
results suggest that therapeutic interventions directed only against the
las cell-to-cell signaling system are likely to fail because of the
emergence of resistant strains able to produce virulence factors despite an
inactive las system (81). However, if both the las and the rhl cell-to-cell
signaling systems are blocked, P. aeruginosa might be unable to restore the
production of cell-to-cell signaling dependent virulence factors; this
approach may efficiently reduce virulence factor production and high death
rates associated with P. aeruginosa.

Cell-to-Cell Signaling Systems in Other Human Gram-Negative Pathogens

Other gram-negative human pathogens including Serratia marcescens, S.
liquefaciens, Aeromonas hydrophila, Vibrio cholerae, V. parahaemoliticus,
Yersinia enterocolitica, Enterobacter agglomerans, Hafnia alvei, and
Rahnella aquatis have been shown to produce homoserine lactone-based
autoinducer molecules. These organisms are thought to contain cell-to-cell
signaling systems analogous to the las and rhl systems of P. aeruginosa
(39,40). Burkholderia cepacia (formerly known as Pseudomonas cepacia) is an
opportunistic pathogen responsible for fatal pulmonary infections in CF
patients (11). Virulence factor production of this pathogen can be enhanced
by P. aeruginosa exoproducts, and this interspecies communication may be
due to homoserine-lactone signals (82). Whether B. cepacia contains
cell-to-cell signaling systems is still unknown. As the number of bacteria
known to use cell-to-cell signaling systems increases, therapeutic
interference with cell-to-cell signaling might expand to other human
pathogens.

Conclusions

The pathogenesis of P. aeruginosa is clearly multifactorial as underlined
by the large number of virulence factors and the broad spectrum of diseases
the bacterium causes. Many of the extracellular virulence factors required
for tissue invasion and dissemination are controlled by cell-to-cell
signaling systems involving homoserine lactone-based signal molecules and
specific transcriptional activator proteins. These regulatory systems
enable P. aeruginosa to produce virulence factors in a coordinated,
cell-densitydependent manner that could allow the bacteria to overwhelm
the host defense mechanisms. Interference with cell-to-cell signaling
dependent virulence factor production is a promising therapeutic approach
for reducing illness and death associated with P. aeruginosa colonization
and infection. The growing number of human pathogens found to contain
cell-to-cell signaling systems highlights the importance of exploring
interference with bacterial cell-to-cell signaling for new therapeutic
interventions.

Acknowledgment

      We thank E.C. Pesci, T. deKievit, and J.P. Pearson for helpful
discussions.

      Supported by NIH grant R01A133713-04 (to B.H.I.), a Wilmot
Foundation, and a Swiss Research Foundation grant (to C.V.D.).

      Dr. van Delden is consulting physician, Division of Infectious
Diseases, and investigator, Swiss National Science Foundation, Department
of Genetics and Microbiology, University Hospital, Geneva, Switzerland. His
research interests include bacterial pathogenesis and the development of
new antibacterial approaches.

      Dr. Iglewski is professor and chair, Department of Microbiology and
Immunology, University of Rochester, Rochester, New York. Her research
focuses on microbial pathogenesis, cell-to-cell signaling, and gene
regulation in bacteria.

      Address for correspondence: Barbara H. Iglewski, Department of
Microbiology and Immunology, University of Rochester, 601 Elmwood Avenue,
Rochester, NY 14642, USA; fax: 716-473-9573; e-mail:
BIGL@uhura.cc.rochester.edu.

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