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The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm posttranscriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the posttranscriptional level. Previous work found that RsmA activity is controlled by at least three small, noncoding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in silico approach to identify additional small RNAs (sRNAs) that might function in the sequestration of RsmA and/or RsmF (RsmA/RsmF) and identified RsmV, a 192-nucleotide (nt) transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1 , a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contributes to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from those of RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play a distinct role in controlling RsmA and RsmF activity. IMPORTANCE The members of the CsrA/RsmA family of RNA-binding proteins play important roles in posttranscriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small noncoding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, and RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal and absolute expression levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues., (Copyright © 2018 American Society for Microbiology.)

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA. IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA., (Copyright © 2018 American Society for Microbiology.)

Unlabelled: CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent., Importance: The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Bacterial chemosensory signal transduction systems that regulate motility by type IV pili (T4P) can be markedly more complex than related flagellum-based chemotaxis systems. In T4P-based systems, the CheA kinase often contains numerous potential sites of phosphorylation, but the signaling mechanisms of these systems are unknown. In Pseudomonas aeruginosa, the Pil-Chp system regulates T4P-mediated twitching motility and cAMP levels, both of which play roles in pathogenesis. The Pil-Chp histidine kinase (ChpA) has eight "Xpt" domains; six are canonical histidine-containing phosphotransfer (Hpt) domains and two have a threonine (Tpt) or serine (Spt) in place of the histidine. Additionally, there are two stand-alone receiver domains (PilG and PilH) and a ChpA C-terminal receiver domain (ChpArec). Here, we demonstrate that the ChpA Xpts are functionally divided into three categories as follows: (i) those phosphorylated with ATP (Hpt4-6); (ii) those reversibly phosphorylated by ChpArec (Hpt2-6), and (iii) those with no detectable phosphorylation (Hpt1, Spt, and Tpt). There was rapid phosphotransfer from Hpt2-6 to ChpArec and from Hpt3 to PilH, whereas transfer to PilG was slower. ChpArec also had a rapid rate of autodephosphorylation. The biochemical results together with in vivo cAMP and twitching phenotypes of key ChpA phosphorylation site point mutants supported a scheme whereby ChpArec functions both as a phosphate sink and a phosphotransfer element linking Hpt4-6 to Hpt2-3. Hpt2 and Hpt3 are likely the dominant sources of phosphoryl groups for PilG and PilH, respectively. The data are synthesized in a signaling circuit that contains fundamental features of two-component phosphorelays., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in avfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (PexsA) located immediately upstream of exsA PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA promoter, which is internal to the previously characterized exsCEBA operon., Importance: Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS., Importance: Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 β-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a major cause of healthcare-associated infections. While the organism's intrinsic and acquired resistance to most antibiotics hinders treatment of P. aeruginosa infections, the regulatory networks controlling its virulence provide novel targets for drug development. CyaB, a key regulator of P. aeruginosa virulence, belongs to the Class III adenylyl cyclase (AC) family of enzymes that synthesize the second messenger cyclic adenosine 3',5'-monophosphate. These enzymes consist of a conserved catalytic domain fused to one or more regulatory domains. We describe here the biochemical and structural characterization of CyaB and its inhibition by small molecules. We show that CyaB belongs to the Class IIIb subfamily, and like other subfamily members, its activity is stimulated by inorganic carbon. CyaB is also regulated by its N-terminal MASE2 (membrane-associated sensor 2) domain, which acts as a membrane anchor. Using a genetic screen, we identified activating mutations in CyaB. By solving the crystal structure of the CyaB catalytic domain, we rationalized the effects of these mutations and propose that CyaB employs regulatory mechanisms similar to other Class III ACs. The CyaB structure further indicates subtle differences compared to other Class III ACs in both the active site and the inhibitor binding pocket. Consistent with these differences, we observed a unique inhibition profile, including identification of a CyaB selective compound. Overall, our results reveal mechanistic details of the physiological and pharmacological regulation of CyaB and provide the basis for its exploitation as a therapeutic drug target., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5× to 8× the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5× to 4× the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute, invasive infections in immunocompromised individuals and chronic, persistent respiratory infections in individuals with cystic fibrosis (CF). The differential progression of acute or chronic infections involves the production of distinct sets of virulence factors. P. aeruginosa strains isolated from patients with acute respiratory infection are generally nonencapsulated and express a variety of invasive virulence factors, including flagella, the type III secretion system (T3SS), type IV pili (TFP), and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to the production of an alginate capsule. The mucoid phenotype results from loss-of-function mutations in mucA, which encodes an anti-sigma factor that normally prevents alginate synthesis. Here, we report that the cyclic AMP/Vfr-dependent signaling (CVS) pathway is defective in mucA mutants and that the defect occurs at the level of vfr expression. The CVS pathway regulates the expression of multiple invasive virulence factors, including T3SS, exotoxin A, protease IV, and TFP. We further demonstrate that mucA-dependent CVS inhibition involves the alternative sigma factor AlgU (AlgT) and the response regulator AlgR but does not depend on alginate production. Our findings show that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in acute and persistent infections. These results suggest that mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa in the CF lung.

Vfr is a global regulator of virulence factor expression in the human pathogen Pseudomonas aeruginosa. Although indirect evidence suggests that Vfr activity is controlled by cyclic AMP (cAMP), it has been hypothesized that the putative cAMP binding pocket of Vfr may accommodate additional cyclic nucleotides. In this study, we used two different approaches to generate apo-Vfr and examined its ability to bind a representative set of virulence gene promoters in the absence and presence of different allosteric effectors. Of the cyclic nucleotides tested, only cAMP was able to restore DNA binding activity to apo-Vfr. In contrast, cGMP was capable of inhibiting cAMP-Vfr DNA binding. Further, we demonstrate that vfr expression is autoregulated and cAMP dependent and involves Vfr binding to a previously unidentified site within the vfr promoter region. Using a combination of in vitro and in vivo approaches, we show that cAMP is required for Vfr-dependent regulation of a specific subset of virulence genes. In contrast, we discovered that Vfr controls expression of the lasR promoter in a cAMP-independent manner. In summary, our data support a model in which Vfr controls virulence gene expression by distinct (cAMP-dependent and -independent) mechanisms, which may allow P. aeruginosa to fine-tune its virulence program in response to specific host cues or environments.

Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O2 environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surface-dependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of the hcnA promoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that the P. aeruginosa hcnA promoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

Pseudomonas aeruginosa poses a serious risk in individuals suffering from cystic fibrosis (CF). Strains colonizing the CF lung are generally motile but frequently convert to a nonmotile phenotype as the disease progresses. In many cases, this is coordinately regulated with the overproduction of the exopolysaccharide alginate. Both the expression of alginate (mucoidy) and the loss of flagellum synthesis may provide the bacterium with a selective advantage in the CF lung. Previously published data showed that the regulation of alginate production and flagellum biosynthesis in the CF isolate FRD1 is inversely controlled by the alternative sigma factor AlgT. In this study, we observed that in CF isolates, the mucoid and the nonmotile phenotypes occur predominantly together. Using microarrays, we compared the transcriptomes of isogenic AlgT(+) and AlgT(-) P. aeruginosa and discovered that AlgT significantly downregulated the majority of flagellar genes. A pronounced inhibitory effect was observed in several genes essential for proper flagellum expression, including fleQ, which encodes an essential flagellar regulator. The microarray data were confirmed by reverse transcriptase PCR analysis and promoter fusion assays in isogenic AlgT(+) and AlgT(-) strains. Transmission electron microscopy, motility assays, and Western blots showed that ectopic expression of FleQ in mucoid, nonmotile CF isolates restored flagellum biosynthesis and motility. Together, these data show that AlgT mediates the negative control of flagellum expression by inhibiting the expression of the flagellar regulator fleQ.

The type III secretion system (TTSS) is a dedicated bacterial pathogen protein targeting system that directly affects host cell signalling and response pathways. Our goal was to identify host responses to the Pseudomonas aeruginosa effectors, introduced into target cells utilizing the TTSS. We carried out expression profiling of a human lung pneumocyte cell line A549 exposed to isogenic mutants of P. aeruginosa PAK lacking individual or a combination of TTSS components. We then devised a data analysis method to isolate the key responses to specific secreted bacterial effector proteins as well as components of the TTSS machinery. Individually, the effector proteins elicited host responses consistent with their known functions, many of which were cell cycle-related. However, our analysis has shown that the effector proteins elicit a distinct host transcriptional response when present in combination, suggesting a synergistic effect. Furthermore, the pattern of host transcriptional responses is consistent with the pore forming ability of the TTSS needle complex. This study shows that the individual components of the TTSS define an integrated system and that a systems biology approach is required to fully understand the complex interplay between pathogen and host.

A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin. DNA microarray hybridization analysis of a number of P. aeruginosa strains from diverse origins showed that this island is polymorphic. PCR and sequence analysis confirmed that many P. aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent. To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P. aeruginosa strains were determined. Two distinct flagellin subtypes, designated A1 and A2, were apparent. Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1- and A2-type flagellins. These findings indicate that P. aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.

The type III secretion system (TTSS) of the opportunistic pathogen Pseudomonas aeruginosa enables the bacterium to deliver exoenzymes directly into the eukaryotic cell. In this study we have investigated the role of key factors involved in this process. We could demonstrate that the translocators PopB, PopD and PcrV are absolutely required for delivery of Exoenzyme S into host cells. By analyzing different Tfp (type IV pili) mutants we could establish a correlation between the frequency of bacteria binding to the host cell and the levels of translocated ExoS, thereby verifying that the process is contact dependent. However, there was no absolute requirement for the Tfp per se, since the pilus could be substituted with a different type of adhesin, the non-fimbrial adhesin pH6 antigen of Yersinia pestis. Taken together, our results demonstrate that binding to establish close contact between the type III secretion organelle and the host cell is essential for translocation, while the additional activities of Tfp are not essential for the delivery of TTSS proteins.

Many respiratory pathogens, including Hemophilus influenzae, Streptococcus pneumoniae, and Pseudomonas aeruginosa, express neuraminidases that can cleave alpha2,3-linked sialic acids from glycoconjugates. As mucosal surfaces are heavily sialylated, neuraminidases have been thought to modify epithelial cells by exposing potential bacterial receptors. However, in contrast to neuraminidase produced by the influenza virus, a role for bacterial neuraminidase in pathogenesis has not yet been clearly established. We constructed a mutant of P. aeruginosa PAO1 by deleting the PA2794 neuraminidase locus (Delta2794) and tested its virulence and immunostimulatory capabilities in a mouse model of infection. Although fully virulent when introduced i.p., the Delta2794 mutant was unable to establish respiratory infection by i.n. inoculation. The inability to colonize the respiratory tract correlated with diminished production of biofilm, as assessed by scanning electron microscopy and in vitro assays. The importance of neuraminidase in biofilm production was further demonstrated by showing that viral neuraminidase inhibitors in clinical use blocked P. aeruginosa biofilm production in vitro as well. The P. aeruginosa neuraminidase has a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. Inhibiting bacterial neuraminidases could provide a novel mechanism to prevent bacterial pneumonia. [ABSTRACT FROM AUTHOR]