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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.