Nicole T. Liberati

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

BackgroundPseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.ResultsThe PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.ConclusionGenes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein.

Members of the Smad family of proteins are thought to play important roles in transforming growth factor beta (TGF-beta)-mediated signal transduction. In response to TGF-beta, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-beta on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-beta-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-beta-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-beta-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-beta. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.

Smad3-Smad4 and AP-1 Complexes Synergize in Transcriptional Activation of the c-Jun Promoter by Transforming Growth Factor β

ABSTRACT Transcriptional regulation by transforming growth factor β (TGF-β) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-β responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-β-induced transcriptional activation of the c-Jun promoter.

BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14

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 DeltabifA 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 DeltabifA mutant but does not contribute to the observed swarming defect. The DeltabifA 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.

Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

ABSTRACT Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

Ligand-dependent ubiquitination of Smad3 is regulated by casein kinase 1 gamma 2, an inhibitor of TGF-beta signaling.

Transforming growth factor-beta (TGF-β) elicits a variety of cellular activities primarily through a signaling cascade mediated by two key transcription factors, Smad2 and Smad3. Numerous regulatory mechanisms exist to control the activity of Smad3, thereby modulating the strength and specificity of TGF-β responses. In search for potential regulators of Smad3 through a yeast two-hybrid screen, we identified casein kinase 1 gamma 2 (CKIγ2) as a novel Smad3-interacting protein. In mammalian cells, CKIγ2 selectively and constitutively binds Smad3 but not Smad1, -2 or -4. Functionally, CKIγ2 inhibits Smad3-mediated TGF-β responses including induction of target genes and cell growth arrest, and this inhibition is dependent on CKIγ2 kinase activity. Mechanistically, CKIγ2 does not affect the basal levels of Smad proteins or activity of the receptors. Rather, CKIγ2 preferentially promotes the ubiquitination and degradation of activated Smad3 through direct phosphorylation of its MH2 domain at Ser418. Importantly, mutation of Ser418 to alanine or aspartic acid causes an increase or decrease of Smad3 activity, respectively, in the presence of TGF-β. CKIγ2 is the first kinase known to mark activated Smad3 for destruction. Given its negative function in TGF-β signaling and its reported overexpression in human cancers, CKIγ2 may act as an oncoprotein during tumorigenesis.

Combining Genomic Tools to Dissect Multifactorial Virulence in Pseudomonas aeruginosa

The growing number of sequenced bacterial genomes, including those of important pathogenic isolates, has made a significant impact on the field of bacterial pathogenesis (Raskin et al., 2006). Combined with other technical advances in the laboratory, this wealth of information has made possible the development and widespread adoption of genomic tools for the study of infectious disease. This chapter focuses on the sequencing and functional analysis of PA14, a clinical isolate of the ubiquitous environmental bacterium and important opportunistic human pathogen, Pseudomonas aeruginosa. By comparing PA14 to the sequence of the less virulent P. aeruginosa isolate, PAO1, we have identified genomic sequences absent in one or the other strain in order to examine the relationship between genomic content and pathogenicity. To assess the importance of strain-specific genes and virulence, we tested 20 diverse P. aeruginosa strains, including PA14 and PAO1, in a Caenorhabdidits elegans pathogenesis model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of genes present in PA14 and absent in PAO1 did not correlate with the virulence of these strains. To further examine the roles in virulence of PA14 genes absent in PAO1, we utilized a full-genome non-redundant mutant library of PA14 to identify five ORFs (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. Genes required for pathogenicity in one strain are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is a complex process that is both multifactorial and combinatorial. Not only are multiple pathogenicity determinants acting in parallel within a given strain, but also, when comparing different strains, different combinations of pathogenicity factors may be selected to determine the ultimate virulence

Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.

Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.

Using PATIMDB to Create Bacterial Transposon Insertion Mutant Libraries

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software. Curr. Protoc. Mol. Biol. 86:19.7.1‐19.7.34.