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pathogenesis. The three genes also are required for effective killing of C. elegans (6, 7). The hrpM gene of P. syringae pv. syringae was identified as a homologue of the Escherichia coli mdoH gene, which is part of an operon involved in the synthesis of membrane-derived oligosaccharides (MDO) (53). Although MDO have been found to have different functions in a variety of Gram-negative bacteria, the role of MDO in bacterial pathogenesis is not well understood. Mutation in the hrpM locus of the plant pathogen P. siryngae pv. syringae abolishes both the development of disease symptoms on host plants as well as the hypersensitivity response in nonhost plants (54). The GacS protein is a sensor kinase of a two-component bacterial family of regulators (55). GacA, the cognate response regulator of GacS, initially was identified as a global regulator of secondary metabolites in P. fluorescens (56, 57). It has been shown that mutations in both the gacA and gacS genes in P. siryngae lead to decreased lesion formation in beans and no production of the toxin syringomycin (46, 55, 58).

The requirement of the lasR, gacS, and gacA gene products for pathogenicity in plants, nematodes, and mice provides evidence that quorum sensing and regulated export of proteins are general features of pathogenesis for all three hosts. The lasR, gacS, and gacA genes are present in numerous plant and animal bacterial pathogens as well as in saprophytes. It is likely that LasR, GacS, and GacA initially served as master regulators, enabling ancestral Gram-negative organisms to adapt to their environment. Subsequently, these proteins evolved to regulate a variety of genes that allowed prokaryotes to invade and establish their presence in eukaryotic hosts.

Another class of conserved virulence factors important in plant and nematode pathogenesis is the genes involved multidrug efflux pumps. The mexA gene corresponding to mutant 23A2 encodes a component of a multidrug efflux pump; it recently has been shown to be involved in active efflux of P. aeruginosa autoinducers that are not freely diffusable (59). However, mutant 23A2 was only marginally compromised in the mouse burn model. Further studies using a lower inoculum need to be conducted to ascertain its role as a virulence factor in mammalian pathogenesis. An additional protein relevant to plant pathogenesis, but not to mammalian pathogenesis, is a homologue of the putative E. coli integral membrane protein AefA (Proposite: PS01246). The function of this protein is yet unknown.

Other known proteins identified in our screens, but not previously shown to be involved in pathogenesis, include a PtsP homologue of A. vinelandii required for poly-β-hydroxybutyrate accumulation. The ptsP gene is predicted to encode enzyme INtr, a presumptive transcriptional regulator of RpoN-dependent operons (60).

One of the novel virulence factors identified in the plant screen (Pho34B12) affects hemolytic and elastolytic activity as well as pyocyanin production (refs.5 and 7; H.C. and L.G.R., unpublished work), all of which are under quorum-sensing regulation. The protein encoded by pho34B12 contains a helixturn–helix DNA binding motif similar to that found in the LysR family of transcriptional regulators (ref.5; H.C. and L.G.R., unpublished work). This class of proteins includes regulators involved in both mammalian and plant pathogenesis (1).

Our multihost pathogenesis study results indicate that phenazines are an important class of virulence-related effector molecules. Despite intensive in vitro analyses of phenazines, the physiological significance of their role in P. aeruginosa pathogenesis in mammals has been controversial (61). Before our studies, there had been no demonstration of their role in vivo. Mutants 3E8 and 6A6 correspond to the previously identified phzB gene in P. fluorescens and phzY gene in P. aureofaciens. Both phzB and phzY are present in operons known to regulate production of phenazine-1-carboxylate (62). Additional effector molecules that play a role in multihost pathogenesis include the ToxA protein and the previously unidentified virulence gene encoding the 34H4 protein. Current work indicates that 34H4 contains a bi-partite nuclear localization signal required for translocation of the protein into the nucleus of mammalian cells (G.W.L. and L.G.R., unpublished work).

A common phenotype of at least two of the mutants, 33A9 and 3E8, is reduced motility and altered surface attachment ability (Table 2). Mutant 3E8 exhibits reduced attachment to abiotic surfaces, such as polyvinilchloride plastic surfaces (ref.63; S.M.-M. and F.M.A., unpublished work) whereas 33A9 exhibited increased surface attachment ability (E.D., G. O'Toole, F.M.A., and L.G.R., unpublished work). Bacterial adhesion is an essential step in biofilm formation and consequently, in bacterial virulence. No significant similarity to any known genes was found in the gene corresponding to mutant 33A9. Interestingly, DNA sequence analysis of the region containing the 33A9 gene revealed that this gene is not present in the recently sequenced genome of the P. aeruginosa strain PAO1.

Conclusions

In summary, we can draw the following conclusions from our studies of P. aeruginosa multihost pathogenesis. First, the variety of virulence-associated genes described in our experiments indicates that the multihost strategy has few limitations with regard to the categories of virulence-related functions that can be identified. Second, the fact that most of the genes identified were not previously known to be involved in pathogenesisrelated functions demonstrates that the multihost strategy is particularly efficient in identifying novel P. aeruginosa virulence factors. Third, and perhaps most importantly, even though many pathogens cause disease in a single or limited number of host species these studies provide strong evidence that there exists several universal bacterial virulence mechanisms highly conserved across phylogeny.

Although some of the virulence-associated genes identified have homologues in other pathogenic bacteria, the exact role of these genes in pathogenesis remains unclear in most cases. The use of genetically tractable host systems, such as plants, nematodes, and insects, will generate important information about host responses and lead to a better understanding of the fundamental molecular mechanisms that underlie bacterial pathogenesis.

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