Pil Jung Kang

PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital‐acquired pneumonia. We identified a 73 kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. Secretion occurred without cleavage of a signal peptide and in low‐calcium environments in the presence of a divalent cation chelator, as is the case for characterized P. aeruginosa type III secreted proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino‐terminal peptide sequence analysis indicated that the amino‐terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A–X–K–S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5′ of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5′ of the PepA translational start site. Analysis of G + C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa‐induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon‐tagged non‐cytotoxic mutants of a cytotoxic and lung‐virulent strain of P. aeruginosa (PA103). The transposon‐insertion site was determined by using an inverse polymerase chain reaction followed by DNA‐sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt−). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein‐secretion apparatus appears to be involved in this process.

Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways

Virulence of Pseudomonas aeruginosa involves the co‐ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)‐mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N‐terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface‐assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild‐type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.

The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast

Bimolecular fluorescence complementation assays allow the visualization of the homotypic and heterotypic GTPase interactions in vivo. The Rsr1 homotypic interaction involves its polybasic region and depends on its GDP-GTP exchange factor. Dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.

The Rho5 GTPase is necessary for oxidant-induced cell death in budding yeast

In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H2O2). Here, we report that the Rho5 GTPase of budding yeast is necessary for H2O2-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Δ) exhibits little cell death, whereas the constitutively active rho5G12V mutant exhibits excess ROS accumulation and increased cell death upon H2O2 treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H2O2 but also in the vacuolar lumen after H2O2 treatment. Trr1 levels are elevated in rho5Δ cells but are elevated only slightly in WT and not in the rho5G12V cells after H2O2 treatment. Taken together, these data suggest that Rho5 mediates H2O2-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.

Coupling of septins to the axial landmark by Bud4 in budding yeast

Summary Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern that depends on their cell type. Haploid a and &agr; cells bud in the axial budding pattern, which requires assembly of a landmark that includes the Bud4 protein. To understand how an axial bud site is established, we performed a structure–function analysis of Bud4. Bud4 contains DUF1709 (domain of unknown function), which is similar to a part of the anillin-homology domain, and a putative Pleckstrin homology (PH) domain near to its C terminus. Although its localization depends on septins, a conserved family of GTP-binding proteins, Bud4 is necessary for the stable inheritance of septin rings during cell division. Although some anillins interact directly with septins, we find that neither DUF1709 nor the PH domain is necessary for targeting Bud4 to the mother-bud neck. Instead, this C-terminal region is crucial for association of Bud4 with Bud3 and other components of the axial landmark. Remarkably, septins colocalize with Bud4 mutant proteins that lack these C-terminal domains, forming an arc or a single ring instead of a double ring during and after cytokinesis. Interestingly, overexpression of Bud4 also induces formation of extra Bud4 rings and arcs that are associated with septins. Analyses of a series of bud4 truncation mutants suggest that at least two domains in the central region play a redundant role in targeting Bud4 to the mother-bud neck and are thus likely to interact with septins. Taken together, these results indicate that Bud4 functions as a platform that links septins to the axial landmark.

Bud3 activates Cdc42 to establish a proper growth site in budding yeast

Biphasic activation of Cdc42 by Bud3 and then Cdc24 during G1 of the yeast cell cycle is necessary for assembly of a proper bud site.

Bud4 mediates the cell-type-specific assembly of the axial landmark in budding yeast.

Summary Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and &agr; cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here, we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1 phase. Whereas Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP-binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and &agr; cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.

Specific Residues of the GDP/GTP Exchange Factor Bud5p Are Involved in Establishment of the Cell Type-specific Budding Pattern in Yeast

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and α cells bud in an axial pattern whereas diploid a/α cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/α cells without affecting Bud5p localization in haploid α cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.

Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein

In vivo and in vitro analyses reveal how a Cdc42 GTPase-activating protein (GAP) interacts with other negative polarity cues at old division sites in budding yeast. Mathematical modeling suggests that spatial distribution of a Cdc42 GAP in coordination with G1 progression is critical for fine-tuning the orientation of the polarity axis.