The diversity of roles that phospholipases C (PLCs)
play in biology and medicine is extraordinary. In
the past decade this class of phospholipid
hydrolyzing enzymes has been shown to be
considerably more complex than initially perceived
and their impact on a wide range of basic cellular
processes in eukaryotes, including oncogenesis,
apoptosis, and inflammation has been increasingly
appreciated. Likewise, there are many sundry and
important functions for PLCs in microbial
pathogenesis.
We identified and characterized the first member of
a novel class of homologous PLCs, the hemolytic
phospholipase C (PlcH) of Pseudomonas aeruginosa.
Members of this class of PLCs are produced by an
array of opportunistic and frank pathogens,
including potential bioterrorist agents. We, as well
as others, provided cogent evidence that members of
this novel class of PLCs play significant and
diverse roles in the infectious diseases caused by
those agents. Although these
PLCs share considerable amino acid homology, each
member has distinct properties. There are some
important differences in their substrate
specificities, and many members have unique
structural features that probably play a specific
functional role in the pathogenesis of the organisms
that produce them. During the past five years we
identified and characterized some noteworthy
properties of PlcH. The substrate specificity of
PlcH is remarkable by comparison with other
microbial PLCs and even eukaryotic PLCs. PlcH only
hydrolyzes phospholipids with choline phosphate (CP)
head groups, which include phosphatidylcholine (PC),
sphingomyelin (SM), platelet activating factor (PAF)
and certain classes of plasmalogens (PM).
Additionally, we recently discovered that PlcH is an
enzyme with dual functions. That is, PlcH not only
cleaves the CP head group from PC (a PC-PLC) or SM
(a sphingomyelinase), but if SM is not available as
a substrate, PlcH will hydrolyze PC and transfer the
CP moiety to ceramide (CM), thereby synthesizing SM.
This is the first prokaryotic or eukaryotic protein
yet identified that is able to synthesize SM (SM
Synthase). The substrates (e.g. PC & SM) of PlcH or
the products (e.g. DAG, CM or SM) that it generates
could have profound biological effects, particularly
with respect to signaling processes in eukaryotic
cells and the host responses to this infectious
agent. PlcH is highly cytotoxic, but its
cytotoxicity is not merely associated with its
ability to attack PC or SM in eukaryotic membranes
and lyse cells. Supporting this view are our data
presented in this application demonstrating a wide
range of susceptibilities of eukaryotic cells to
PlcH. That is, some eukaryotic cells are readily
killed by picomolar concentrations of purified PlcH
while others are remarkably resistant to high
concentrations (micromolar) of purified PlcH.
Moreover, we identified an
Arginine-Glycine-Aspartate (RGD) motif in PlcH. RGD
motifs are found in an array of proteins highly
implicated for their role in microbial pathogenesis,
including proteins associated with foot and mouth
disease virus, adenovirus, HIV (TAT protein),
adhesions of Bordetella pertussis and Group A
Streptococcal proteases. The RGD motifs of these
proteins bind to members of a family of eukaryotic
cell receptors known as integrins. The interaction
of these RGD proteins with their specific subclass
of integrin receptors ultimately triggers an
intracellular signal leading to significant
biological consequences including entry of the
ligand (e.g. protein, virus) and apoptotic cell
death. We also have data demonstrating that PlcH
induced signaling (i.e.
Ca2+ intracellular levels) and
cytotoxicity in susceptible cells are inhibited by
RGD peptides, but not by scrambled peptides
containing these residues. We propose that PlcH
binds and enters susceptible cells via RGD mediated
interactions with integrin receptors. We propose
that it alters the normal pattern of phospholipid
mediated signaling events through its ability to
generate DAG or CM or through its ability to
synthesize SM in inappropriate cellular
compartments, or at inappropriate times.
The dynamic control of intracellular iron
concentrations is paramount to all biological
systems. One aspect of this issue is that,
especially in an aerobic environment, biologically
useful iron (i.e. Fe2+) is extremely
limiting or it is highly insoluble (i.e.Fe3+).
Accordingly, biological entities have evolved
efficient mechanisms to acquire this nutrient from
the insoluble form, which is generally in plentiful
quantities. On the other hand, further acquisition
of iron above biologically useful concentrations can
have dire consequences for a cell. Excess free iron
will catalyze the generation of highly reactive
oxygen and nitrogen intermediates that will damage
all known biological macromolecules. This conflict,
in a major way is dealt with in a diverse array of
pathogenic and commensal prokaryotic microbes, by
repressor proteins, which play the key role in
controlling iron homeostasis at the level of
transcription. The ferric uptake regulator
(Fur) serves this function in many bacteria. In
fact, in the opportunistic pathogen Pseudomonas
aeruginosa Fur (PA-Fur) is an essential protein that
controls the expression of genes involved in the
acquisition of environmental iron, including those
that contribute to its virulence. For example,
PA-Fur controls the production of: (i) extracellular
proteinases, which degrade host iron binding
proteins (e.g. lactoferrin) (ii) low molecular
weight, high affinity, iron binding compounds (i.e.
siderophores) (iii) iron storage proteins (e.g.
bacterioferritin) and (iv) a potent extracellular
toxin (exotoxin A). We have determined the
crystalline structure of PA-Fur and we are currently
evaluating how it interacts with its operator
sequence. Moreover, we identified two small RNA
transcripts (sRNA) whose expression is directly
controlled by PA-Fur. These sRNA transcripts control
the expression of a set of genes at the
post-transcriptional level. These genes are induced
under iron-replete conditions and are involved in
iron storage and defense against oxidative stress.
Based on these and other data, there are also
compelling reasons to believe that Fur functions in
defense against oxidative stress as well in P.
aeruginosa. This characteristic of Fur is not yet
well understood.
Additionally we recently provided intriguing data
supporting the hypothesis that a siderophore of P.
aeruginosa (i.e. pyoverdine) has a noteworthy
biological function beyond its ability to scavenge
iron in response to its sequestration by an infected
host. That is, remarkably pyoverdine is capable of
transducing an intercellular signal to other cells
in a P. aeruginosa population. This signal also
requires expression of the ferripyoverdine receptor
protein (FpvA) that recognizes pyoverdine and
transduces an intracellular signal to stimulate
further production of pyoverdine. Moreover, this
signaling process also induces the expression of
genes encoding at least two known extracellular
virulence determinants of this opportunist (exotoxin
A and a powerful extracellular endoprotease that
cleaves lactoferrin and decorins). While this novel
signaling process may occur in other microbial
pathogens, to our knowledge ours is the first report
that provides direct evidence of such a dual
function for a siderophore.
A novel secretion pathway originally found in plants
has recently been discovered in bacteria and termed
TAT, for ''twin-arginine translocation,'' with
respect to the presence of an Arg-Arg motif in the
signal sequence of TAT-secreted products. However,
it is unknown whether the TAT system contributes in
any way to virulence through the secretion of
factors associated with pathogenesis or stress
response. We found that the opportunistic pathogen
Pseudomonas aeruginosa produces several virulence
factors that depend on the TAT system for proper
export to the periplasm, outer membrane, or
extracellular milieu. We identified at least 18 TAT
substrates of P. aeruginosa and characterized the
pleiotropic phenotypes of a tatC deletion mutant.
The TAT system proved essential for the export of
phospholipases, proteins involved in pyoverdine-mediated
iron uptake, anaerobic respiration, osmotic stress
defense, motility, and biofilm formation. Because
all these traits have been associated with
virulence, we studied the role of TAT in a rat lung
model. A tatC mutant did not cause the typical
multifocal pulmonary abscesses and did not evoke a
heavy inflammatory host response compared with wild
type, indicating that tatC mutant cells are
attenuated for virulence. Because the TAT apparatus
is well conserved among important bacterial
pathogens yet absent in mammalian cells, it
represents a potential target for novel
antimicrobial compounds. We are currently exploring
the potential use of Tat mutants as vaccines against
a variety of bacterial pathogens.
Selected Publications
Vasil, M.L. and Ochsner, U.A. (1999). The response of
Pseudomonas aeruginosa to iron: Genetics, biochemistry
and virulence. Molec. Microbiol. 34, 399-413.
Terada, L.S. Johansen, K.A. Nowbar, S. Vasil, A.I. and
Vasil, M.L. (1999) Pseudomonas aerugniosa hemolytic
phospholipase C suppresses neutrophil respiratory burst
activity. Infect. & Immun. 67: 2371-2376.
Wilderman, P.J. Vasil, A.I., Johnson, Z. and Vasil, M.L.
(2001) Genetic and Biochemical Analyses of a Eucaryotic-like
Phospholipase D of Pseudomonas aeruginosa Suggest
Horizontal Acquisition and a Role for Persistence in a
Chronic Pulmonary Infection Model. Molecular
Microbiology 39:291-303.
Wilderman, P.J. Vasil, A.I., Johnson, Z. Wilson,M.,
Cunliffe, H.E. Lamont, I. and Vasil, M.L. (2001)
Characterization of an Endoprotease (PrpL) Encoded by a
PvdS-Regulated Gene in Pseudomonas aeruginosa Infect.
Immun. 69:5385-5389.
Wilderman, P.J., Vasil, A.I., Martin, W.E., Murphy, R.C.,
and Vasil, M.L. (2002) Pseudomonas aeruginosa
synthesizes phosphatidylcholine by use of the
phosphatidylcholine synthase pathway. J Bacteriol 184:
4792-4799.
Lamont, I.L., Beare, P.A., Ochsner, U.A., Vasil, A.I.,
and Vasil, M.L. (2002) Siderophore-mediated signaling
regulates virulence factor production in Pseudomonas
aeruginosa: Proc. Natl. Acad. Sci. USA, 99, 7072-7077
Ochsner, U.A, Snyder, A., Vasil, A.I. and Vasil, M.L.
(2002) Effects of the Twin-Arginine Translocase on
Secretion of Virulence Factors, Stress Response, and
Pathogenesis (2002) Proc. Natl. Acad. Sci.
99:8312-8317.
Stonehouse M.J., Cota-Gomez, A. Parker, S.K.
Martin, W.A., .Hankin, J.A.,. Murphy, R.C. Chen, W.,
Hackett, M., Vasil, A.I. and Vasil, M.L. (2002) A Novel
Class of Phosphocholine-specific Phospholipases C Molec.
Microbiol. 46:661-676.
Ochsner, U.A. Wilderman, P.J. Vasil, A.I. and Vasil, M.L.
(2002) GeneChip® Expression Analysis of the Iron
Starvation Response in Pseudomonas aeruginosa:
Identification of Novel Pyoverdine Biosynthesis Genes
Molec. Microbiol. 45:1277-1287.
Pohl, E. Haller, J.C. Mijovilovich, A. Meyer-Klaucke, W.
Garman, E. and Vasil, M.L. (2003) Architecture of a
Protein Central to Iron homeostasis:Crystal Structure
and Spectroscopic Analysis of the Ferric Uptake
Regulator. Molec. Microbiol. 47:903-915.
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