Species-specific recruitment of transcription factors dictates toxin expression.

Tight and coordinate regulation of virulence determinants is essential for bacterial biology and involves dynamic shaping of transcriptional regulatory networks during evolution. The horizontally transferred two-partner secretion system ExlB-ExlA is instrumental in the virulence of different Pseudomonas species, ranging from soil- and plant-dwelling biocontrol agents to the major human pathogen Pseudomonas aeruginosa. Here, we identify a Cro/CI-like repressor, named ErfA, which together with Vfr, a CRP-like activator, controls exlBA expression in P. aeruginosa. The characterization of ErfA regulon across P. aeruginosa subfamilies revealed a second conserved target, the ergAB operon, with functions unrelated to virulence. To gain insights into this functional dichotomy, we defined the pan-regulon of ErfA in several Pseudomonas species and found ergAB as the sole conserved target of ErfA. The analysis of 446 exlBA promoter sequences from all exlBA+ genomes revealed a wide variety of regulatory sequences, as ErfA- and Vfr-binding sites were found to have evolved specifically in P. aeruginosa and nearly each species carries different regulatory sequences for this operon. We propose that the emergence of different regulatory cis-elements in the promoters of horizontally transferred genes is an example of plasticity of regulatory networks evolving to provide an adapted response in each individual niche.

Transcriptional Responses of Pseudomonas aeruginosa to Inhibition of Lipoprotein Transport by a Small Molecule Inhibitor.

Lipoprotein transport from the inner to the outer membrane, carried out by the Lol machinery, is essential for the biogenesis of the Gram-negative cell envelope and, consequently, for bacterial viability. Recently, small molecule inhibitors of the Lol system in Escherichia coli have been identified and shown to inhibit the growth of this organism by interfering with the function of the LolCDE complex. Analysis of the transcriptome of E. coli treated with one such molecule (compound 2) revealed that a number of envelope stress response pathways were induced in response to LolCDE inhibition. However, Pseudomonas aeruginosa is refractory to inhibition by the same small molecule, but we could demonstrate that E. colilolCDE could be substituted for the P. aeruginosa orthologues, where it functions in the correct transport of Pseudomonas lipoproteins, and the cells are inhibited by the more potent compound 2A. In the present study, we took advantage of the functionality of E. coli LolCDE in P. aeruginosa and determined the P. aeruginosa transcriptional response to LolCDE inhibition by compound 2A. We identified key genes that responded to LolCDE inhibition and also demonstrated that the same genes appeared to be affected by genetic depletion of the native P. aeruginosa LolCDE proteins. Several of the major changes were in an upregulated cluster of genes that encode determinants of alginate biosynthesis and transport, and the levels of alginate were found to be increased either by treatment with the small molecule inhibitor or upon depletion of native LolCDE. Finally, we tested several antibiotics with differing mechanisms of action to identify potential specific reporter genes for the further development of compounds that would inhibit the native P. aeruginosa Lol system.IMPORTANCE A key set of lipoprotein transport components, LolCDE, were inhibited by both a small molecule as well as genetic downregulation of their expression. The data show a unique signature in the Pseudomonas aeruginosa transcriptome in response to perturbation of outer membrane biogenesis. In addition, we demonstrate a transcriptional response in key genes with marked specificity compared to several antibiotic classes with different mechanisms of action. As a result of this work, we identified genes that could be of potential use as biomarkers in a cell-based screen for novel antibiotic inhibitors of lipoprotein transport in P. aeruginosa.

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive ß-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.

Complexity of Complement Resistance Factors Expressed by Acinetobacter baumannii Needed for Survival in Human Serum.

Acinetobacter baumannii is a bacterial pathogen with increasing impact in healthcare settings, due in part to this organism's resistance to many antimicrobial agents, with pneumonia and bacteremia as the most common manifestations of disease. A significant proportion of clinically relevant A. baumannii strains are resistant to killing by normal human serum (NHS), an observation supported in this study by showing that 12 out of 15 genetically diverse strains of A. baumannii are resistant to NHS killing. To expand our understanding of the genetic basis of A. baumannii serum resistance, a transposon (Tn) sequencing (Tn-seq) approach was used to identify genes contributing to this trait. An ordered Tn library in strain AB5075 with insertions in every nonessential gene was subjected to selection in NHS. We identified 50 genes essential for the survival of A. baumannii in NHS, including already known serum resistance factors, and many novel genes not previously associated with serum resistance. This latter group included the maintenance of lipid asymmetry genetic pathway as a key determinant in protecting A. baumannii from the bactericidal activity of NHS via the alternative complement pathway. Follow-up studies validated the role of eight additional genes identified by Tn-seq in A. baumannii resistance to killing by NHS but not by normal mouse serum, highlighting the human species specificity of A. baumannii serum resistance. The identification of a large number of genes essential for serum resistance in A. baumannii indicates the degree of complexity needed for this phenotype, which might reflect a general pattern that pathogens rely on to cause serious infections.

cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers.

The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [Keq] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ, resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers.IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Phage Morons Play an Important Role in Pseudomonas aeruginosa Phenotypes.

The viruses that infect bacteria, known as phages, play a critical role in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria but also from the formation of lysogens. In this state, the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell, since increasing the survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons," as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa, PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed an inhibition of the production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons.IMPORTANCE Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons." In this study, we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviors. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases.

Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility.

During environmental adaptation bacteria use small regulatory RNAs (sRNAs) to repress or activate expression of a large fraction of their proteome. We extended the use of the in vivo RNA proximity ligation method toward probing global sRNA interactions with their targets in Pseudomonas aeruginosa and verified the method with a known regulon controlled by the PrrF1 sRNA. We also identified two sRNAs (Sr0161 and ErsA) that interact with the mRNA encoding the major porin OprD responsible for the uptake of carbapenem antibiotics. These two sRNAs base pair with the 5' UTR of oprD leading to increase in resistance of the bacteria to meropenem. Additional proximity ligation experiments and enrichment for Sr0161 targets identified the mRNA for the regulator of type III secretion system. Interaction between the exsA mRNA and Sr0161 leads to a block in the synthesis of a component of the T3SS apparatus and an effector. Another sRNA, Sr006, positively regulates, without Hfq, the expression of PagL, an enzyme responsible for deacylation of lipid A, reducing its pro-inflammatory property and resulting in polymyxin resistance. Therefore, an analysis of global sRNA-mRNA interactions can lead to discoveries of novel pathways controlling gene expression that are likely integrated into larger regulatory networks.

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, including a major regulator of biofilm formation and antibiotic-resistance traits. PAPI-1 is horizontally transferable into recipient strains lacking this island via conjugation mediated by the specialized type IV pilus. The PAPI-1 encodes a cluster of ten genes associated with the synthesis and assembly of the type IV pilus. The PAPI-1 acquisition mechanism is currently not well understood. RESULTS: In this study, we performed a series of conjugation experiments and identified determinants of PAPI-1 acquisition by analyzing transfer efficiency between the donor and a series of mutant recipient strains. Our data show that common polysaccharide antigen (CPA) lipopolysaccharide (LPS), a homopolymer of D-rhamnose, is required for initiating PAPI-1 transfer, suggesting that this structure acts as a receptor for conjugative type IV pilus in recipient strains. These results were substantiated by experimental evidence from PAPI-1 transfer assay experiments, in which outer membrane or LPS preparations from well-defined LPS mutants were added to the transfer mix to assess the role of P. aeruginosa LPS in PAPI-1 transfer and in vitro binding experiments between pilin fusion protein GST-pilV2' and immobilized LPS molecules were performed. Our data also showed that P. aeruginosa strains that had already acquired a copy of PAPI-1 were unable to import additional copies of the island, and that such strains produced proportionally lower amounts of CPA LPS compared to the strains lacking PAPI-1. CONCLUSIONS: These results suggest that a PAPI-1 exclusion mechanism exists in P. aeruginosa that might serve to regulate the avoidance of uncontrolled expansions of the bacterial genome.

Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen.

The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS-GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk.

Transcriptional Responses of Escherichia coli to a Small-Molecule Inhibitor of LolCDE, an Essential Component of the Lipoprotein Transport Pathway.

In Gram-negative bacteria, a dedicated machinery consisting of LolABCDE components targets lipoproteins to the outer membrane. We used a previously identified small-molecule inhibitor of the LolCDE complex of Escherichia coli to assess the global transcriptional consequences of interference with lipoprotein transport. Exposure of E. coli to the LolCDE inhibitor at concentrations leading to minimal and significant growth inhibition, followed by transcriptome sequencing, identified a small group of genes whose transcript levels were decreased and a larger group whose mRNA levels increased 10- to 100-fold compared to those of untreated cells. The majority of the genes whose mRNA concentrations were reduced were part of the flagellar assembly pathway, which contains an essential lipoprotein component. Most of the genes whose transcript levels were elevated encode proteins involved in selected cell stress pathways. Many of these genes are involved with envelope stress responses induced by the mislocalization of outer membrane lipoproteins. Although several of the genes whose RNAs were induced have previously been shown to be associated with the general perturbation of the cell envelope by antibiotics, a small subset was affected only by LolCDE inhibition. Findings from this work suggest that the efficiency of the Lol system function may be coupled to a specific monitoring system, which could be exploited in the development of reporter constructs suitable for use for screening for additional inhibitors of lipoprotein trafficking. IMPORTANCE: Inhibition of the lipoprotein transport pathway leads to E. coli death and subsequent lysis. Early significant changes in the levels of RNA for a subset of genes identified to be associated with some periplasmic and envelope stress responses were observed. Together these findings suggest that disruption of this key pathway can have a severe impact on balanced outer membrane synthesis sufficient to affect viability.

Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction.

Apolipoprotein N-acyltransferase (Lnt) is an essential membrane-bound enzyme that catalyzes the third and last step in the post-translational modification of bacterial lipoproteins. In order to identify essential residues implicated in substrate recognition and/or binding we screened for non-functional variants of Lnt obtained by error-prone polymerase chain reaction in a complementation assay using a lnt depletion strain. Mutations included amino acid substitutions in the active site and of residues located on flexible loops in the catalytic periplasmic domain. All, but one mutation, led to the formation of the thioester acyl-enzyme intermediate and to the accumulation of apo-Lpp, suggesting that these residues are involved in the second step of the reaction. A large cytoplasmic loop contains a highly conserved region and two hydrophobic segments. Accessibility analysis to alkylating reagents of substituted cysteine residues introduced in this region demonstrated that the hydrophobic segments do not completely span the membrane. Two residues in the highly conserved cytoplasmic region were shown to be essential for Lnt function. Together, our data suggest that amino acids located on flexible cytoplasmic and periplasmic loops, predicted to be membrane embedded, are required for efficient N-acylation of lipoproteins.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

The secretome of Acinetobacter baumannii ATCC 17978 type II secretion system reveals a novel plasmid encoded phospholipase that could be implicated in lung colonization.

Acinetobacter baumannii infections are compounded with a striking lack of treatment options. In many Gram-negative bacteria, secreted proteins play an important early role in avoiding host defences. Typically, these proteins are targeted to the external environment or into host cells using dedicated transport systems. Despite the fact that medically relevant species of Acinetobacter possess a type II secretion system (T2SS), only recently, its significance as an important pathway for delivering virulence factors has gained attention. Using in silico analysis to characterize the genetic determinants of the T2SS, which are found clustered in other organisms, in Acinetobacter species, they appear to have a unique genetic organization and are distributed throughout the genome. When compared to other T2SS orthologs, individual components of the T2SS apparatus showed the highest similarity to those of Pseudomonas aeruginosa. A mutant of Acinetobacter baumannii strain ATCC 17978 lacking the secretin component of the T2SS (ΔgspD), together with a trans-complemented mutant, were tested in a series of in vitro and in vivo assays to determine the role of T2SS in pathogenicity. The ΔgspD mutant displayed decreased lipolytic activity, associated with attenuated colonization ability in a murine pneumonia model. These phenotypes are linked to LipAN, a novel plasmid-encoded phospholipase, identified through mass spectroscopy as a T2SS substrate. Recombinant LipAN showed specific phospholipase activity in vitro. Proteomics on the T2-dependent secretome of ATCC 17978 strain revealed its potential dedication to the secretion of a number of lipolytic enzymes, among others which could contribute to its virulence. This study highlights the role of T2SS as an active contributor to the virulence of A. baumannii potentially through secretion of a newly identified phospholipase.

Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response.

Pathogenicity of Pseudomonas aeruginosa, a major cause of many acute and chronic human infections, is determined by tightly regulated expression of multiple virulence factors. Quorum sensing (QS) controls expression of many of these pathogenic determinants. Previous microarray studies have shown that the AmpC ß-lactamase regulator AmpR, a member of the LysR family of transcription factors, also controls non-ß-lactam resistance and multiple virulence mechanisms. Using RNA-Seq and complementary assays, this study further expands the AmpR regulon to include diverse processes such as oxidative stress, heat shock and iron uptake. Importantly, AmpR affects many of these phenotypes, in part, by regulating expression of non-coding RNAs such as rgP32, asRgsA, asPrrF1 and rgRsmZ. AmpR positively regulates expression of the major QS regulators LasR, RhlR and MvfR, and genes of the Pseudomonas quinolone system. Chromatin immunoprecipitation (ChIP)-Seq and ChIP-quantitative real-time polymerase chain reaction studies show that AmpR binds to the ampC promoter both in the absence and presence of ß-lactams. In addition, AmpR directly binds the lasR promoter, encoding the QS master regulator. Comparison of the AmpR-binding sequences from the transcriptome and ChIP-Seq analyses identified an AT-rich consensus-binding motif. This study further attests to the role of AmpR in regulating virulence and physiological processes in P. aeruginosa.

Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing.

An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport.

A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.

High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.

Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR.

Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC ß-lactamase is a common mechanism of ß-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (PampC) and ampR (PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5' rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ(54) and σ(70) consensus sequences were identified at PampR and PampC, respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.

The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth.

Bacterial virulence determinants can be identified, according to the molecular Koch's postulates, if inactivation of a gene associated with a suspected virulence trait results in a loss in pathogenicity. This approach is commonly used with genetically tractable organisms. However, the current lack of tools for targeted gene disruptions in obligate intracellular microbial pathogens seriously hampers the identification of their virulence factors. Here we demonstrate an approach to studying potential virulence factors of genetically intractable organisms, such as Chlamydia. Heterologous expression of Chlamydia pneumoniae CopN in yeast and mammalian cells resulted in a cell cycle arrest, presumably owing to alterations in the microtubule cytoskeleton. A screen of a small molecule library identified two compounds that alleviated CopN-induced growth inhibition in yeast. These compounds interfered with C. pneumoniae replication in mammalian cells, presumably by 'knocking out' CopN function, revealing an essential role of CopN in the support of C. pneumoniae growth during infection. This work demonstrates the role of a specific chlamydial protein in virulence. The chemical biology approach described here can be used to identify virulence factors, and the reverse chemical genetic strategy can result in the identification of lead compounds for the development of novel therapeutics.