The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ⌬bifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ⌬bifA mutant but does not contribute to the observed swarming defect. The ⌬bifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.The gram-negative bacterium Pseudomonas aeruginosa is an important model organism for the study of bacterial surface interactions, including biofilm formation and surface-mediated twitching and swarming motilities. However, the precise molecular mechanisms required for transition from a planktonic mode of existence to that of a surface-associated lifestyle are only beginning to come to light.In the case of biofilm formation, microscopic studies, as well as genetic analyses, have shown that the initial surface attachment phase by P. aeruginosa proceeds in two distinct steps (8,20). In the first step, known as reversible attachment, cells are loosely attached via a single cell pole and may readily detach and return to the planktonic phase. In the second step, cells that are tethered by a pole become attached via the long axis of the cell body. Such cells, deemed irreversibly attached, are more firmly attached to the surface. Genetic studies of initial attachment have led to the identification of SadB, a key component required for this transition from reversible to irreversible attachment in P. aeruginosa (8).The ability to form a robust biofilm also requires the production of an exopolysaccharide (EPS) component of the biofilm matrix. Recent studies have identified genetic loci that are important for synthesis of an EPS component of biofilm matrix in several P. aeruginosa strains. In PA14, the pel genes are required for ...

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methylaccepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.Pseudomonas aeruginosa is an important model organism for the study of gram-negative biofilm development, yet little is known about the molecular mechanisms underlying the initial events leading to the surface interactions that characterize the early steps in bacterial biofilm formation. Microscopic observations (23,26,40,51) and genetic analyses (2) revealed two sequential events that lead to stable surface interactions. First, a bacterial cell pole contacts the surface in a process referred to as reversible attachment. This is a relatively unstable interaction, as reversibly attached bacteria can readily return to a planktonic existence. The second event is a transition from the polar association to one that is mediated by the long axis of the cell body, referred to as irreversible attachment. In P. aeruginosa, the only mutation known to block the transition from reversible to irreversible attachment is in the sadB gene (2).Another key aspect of biofilm formation by P. aeruginosa is the production of an extracellular matrix. In pseudomonads, this matrix is thought to be comprised of exopolysaccharides (EPS), DNA, and protein (19). The biofilm matrix has typically been credited with structuring the mature biofilm (4). Studies have identified the pel and psl loci as two sets of genes predicted to be involved in the production of the polysaccharide component of the matrix required for biofilm maturation by P. aeruginosa on abiotic surfaces, although only the pel gene cluster is found in P. aeruginosa strain PA14 (7,8,15,27), the focus of study in this report. Interestingly, recent studies suggest that the pel locus also plays a role in early biofilm formation. A pel mutant of P. aeruginosa PAK shows a strong attachment defect in a strain lacking type IV pili (48) and P. aeruginosa PAO1 with a mutation in the psl locus has a block in biofilm initiat...

Candida albicans is a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. Although Candida can cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature of Candida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation of C. albicans filamentation in vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growth in vivo.Candida albicans is a human commensal that causes lifethreatening invasive infections in immunocompromised patients. Disseminated candidiasis is the 4th leading infection in hospitalized patients in the United States, and despite antifungal therapy, the mortality associated with candidemia can reach 30 to 40% (62). Innate immunity is a key mediator of resistance to disseminated infection in both mice and humans, and defects in professional innate immune cells predispose individuals to invasive candidemia (5,26,68).The zebrafish larva is a unique and powerful model for noninvasively visualizing and understanding the interactions of pathogens with the innate immune system (17,48,56). Notably, zebrafish have similar signaling through Toll-like receptors to that in humans, express similar cytokines, and have macrophages, neutrophils, dendritic cells, mast cells, eosinophils, T cells, and B cells (78). The delayed development of T cells and B cells, which do not mature until approximately 30 days postfertilization, permits a natural focus on innate immunity in embryonic and larval infection models. A larval model of candidemia offers several advantages compared to other recently described models of zebrafish infection with C. albicans. Specifically, the adult zebrafish candidemia model does not permit real-time visualization of infection or morpholino (MO)-directed gene knockdown, both of which are techniques available with the larval host (19). Also, while others have described a localized embry...

Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.

The epithelium provides a crucial barrier to infection, and its integrity requires efficient wound healing. Bacterial cells and secretomes from a subset of tested species of bacteria inhibited human and porcine corneal epithelial cell migration in vitro and ex vivo. Secretomes from 95% of Serratia marcescens, 71% of Pseudomonas aeruginosa, 29% of Staphylococcus aureus strains, and other bacterial species inhibited epithelial cell migration. Migration of human foreskin fibroblasts was also inhibited by S. marcescens secretomes indicating that the effect is not cornea specific. Transposon mutagenesis implicated lipopolysaccharide (LPS) core biosynthetic genes as being required to inhibit corneal epithelial cell migration. LPS depletion of S. marcescens secretomes with polymyxin B agarose rendered secretomes unable to inhibit epithelial cell migration. Purified LPS from S. marcescens, but not from Escherichia coli or S. marcescens strains with mutations in the waaG and waaC genes, inhibited epithelial cell migration in vitro and wound healing ex vivo. Together these data suggest that S. marcescens LPS is sufficient for inhibition of epithelial wound healing. This study presents a novel host-pathogen interaction with implications for infections where bacteria impact wound healing and provides evidence that secreted LPS is a key factor in the inhibitory mechanism.