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© 2014 Tran et al.Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studie

The first step in attachment of Chlamydia to host cells is thought to involve reversible binding to host heparan sulfate proteoglycans (HSPGs), polymers of variably sulfated repeating disaccharide units coupled to diverse protein backbones. However, the key determinants of HSPG structure that are involved in Chlamydia binding are incompletely defined. A previous genome-wide Drosophila RNAi screen suggested that the level of HSPG 6-O sulfation rather than the identity of the proteoglycan backbone maybe a critical determinant for binding. Here, we tested in mammalian cells whether SULF1 or SULF2, human endosulfatases, which remove 6-O sulfates from HSPGs, modulate Chlamydia infection. Ectopic expression of SULF1 or SULF2 in HeLa cells, which decreases cell surface HSPG sulfation, diminished C. muridarum binding and decreased vacuole formation. ShRNA depletion of endogenous SULF2 in a cell line that primarily expresses SULF2 augmented binding and increased vacuole formation. C. muridarum infection of diverse cell lines resulted indownregulation of SULF2 mRNA. In a murine model of acute pneumonia, mice genetically deficient in both endosulfatases or in SULF2 alone demonstrated increased susceptibility to C. muridarum lung infection. Collectively, these studies demonstrate that the level of HSPG 6-O sulfation is a critical determinant of C. muridarum infection in vivo and that 6-O endosulfatases are previously unappreciated modulators of microbial pathogenesis.

Chlamydia trachomatis, a leading cause of bacterial sexually transmitted infections, creates a specialized intracellular replicative niche by translocation and insertion of a diverse array of effectors (Incs [inclusion membrane proteins]) into the inclusion membrane. Here, we characterize IncE, a multifunctional Inc that encodes two non-overlapping short linear motifs (SLiMs) within its short cytosolic C terminus. The proximal SLiM, by mimicking just a small portion of an R-N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE) motif, binds and recruits syntaxin (STX)7- and STX12-containing vesicles to the inclusion. The distal SLiM mimics the sorting nexin (SNX)5 and SNX6 cargo binding site to recruit SNX6-containing vesicles to the inclusion. By simultaneously binding two distinct vesicle classes, IncE brings these vesicles in close apposition with each other at the inclusion to facilitate C. trachomatis intracellular development. Our work suggests that Incs may have evolved SLiMs to enable rapid evolution in a limited protein space to disrupt host cell processes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The opportunistic pathogen Pseudomonas aeruginosa adapts to solid surfaces to enhance virulence and infect its host. Type IV pili (T4P), long and thin filaments that power surface-specific twitching motility, allow single cells to sense surfaces and control their direction of movement. T4P distribution is polarized to the sensing pole by the chemotaxis-like Chp system via a local positive feedback loop. However, how the initial spatially resolved mechanical signal is translated into T4P polarity is incompletely understood. Here, we demonstrate that the two Chp response regulators PilG and PilH enable dynamic cell polarization by antagonistically regulating T4P extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not strictly required for twitching reversals, it becomes activated upon phosphorylation and breaks the local positive feedback mechanism established by PilG, allowing forward-twitching cells to reverse. Chp thus uses a main output response regulator, PilG, to resolve mechanical signals in space and employs a second regulator, PilH, to break and respond when the signal changes. By identifying the molecular functions of two response regulators that dynamically control cell polarization, our work provides a rationale for the diversity of architectures often found in non-canonical chemotaxis systems., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans 1-6 . However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a membranous compartment, the inclusion, in host cells. Its intracellular life cycle requires host sphingolipids, which are in part acquired through the ER-Golgi localized ceramide transport protein (CERT). The Chlamydia-encoded inclusion membrane protein IncD is composed of two closely linked long hydrophobic domains with their N- and C-termini exposed to the host cytosol. IncD binds directly to the pleckstrin homology (PH) domain of CERT, likely redirecting ceramide to the inclusion. The precise regions of IncD required for this interaction have not been delineated. Using co-transfection studies together with phylogenetic studies, we demonstrate that both the IncD N- and C-terminal regions are required for binding to the CERT PH domain and define key interaction residues. Native gel electrophoresis analysis demonstrates that the transmembrane region of IncD forms SDS-resistant but dithiothreitol-sensitive homodimers, which in turn can assemble to form higher order oligomers through additional N- and C-terminal domain contacts. IncD oligomerization may facilitate high affinity binding to CERT, allowing C. trachomatis to efficiently redirect host ceramide to the inclusion., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The mucosal barrier is composed of polarized epithelial cells with distinct apical and basolateral surfaces separated by tight junctions and serves as both a physical and immunological barrier to incoming pathogens. Specialized polarity proteins are critical for establishment and maintenance of polarity. Many human pathogens have evolved virulence mechanisms that target the polarity network to enhance binding, create replication niches, move through the barrier by transcytosis, or bypass the barrier by disrupting cell-cell junctions. This review summarizes recent advances and compares and contrasts how three important human pathogens that colonize mucosal surfaces, Pseudomonas aeruginosa , Helicobacter pylori , and Neisseria meningitidis , subvert the host cell polarization machinery during infection., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Chlamydia trachomatis is an obligate intracellular pathogen that resides in a membrane-bound compartment, the inclusion. The bacteria secrete a unique class of proteins, Incs, which insert into the inclusion membrane and modulate the host-bacterium interface. We previously reported that IncE binds specifically to the Sorting Nexin 5 Phox domain (SNX5-PX) and disrupts retromer trafficking. Here, we present the crystal structure of the SNX5-PX:IncE complex, showing IncE bound to a unique and highly conserved hydrophobic groove on SNX5. Mutagenesis of the SNX5-PX:IncE binding surface disrupts a previously unsuspected interaction between SNX5 and the cation-independent mannose-6-phosphate receptor (CI-MPR). Addition of IncE peptide inhibits the interaction of CI-MPR with SNX5. Finally, C. trachomatis infection interferes with the SNX5:CI-MPR interaction, suggesting that IncE and CI-MPR are dependent on the same binding surface on SNX5. Our results provide new insights into retromer assembly and underscore the power of using pathogens to discover disease-related cell biology.

Infection of human cells with Yersinia pseudotuberculosis expressing a functional type III secretion system (T3SS) leads to activation of host NF-κB. We show that the Yersinia T3SS activates distinct NF-κB pathways dependent upon bacterial subcellular localization. We found that wildtype Yersinia able to remain extracellular triggered NF-κB activation independently of the non-canonical NF-κB kinase NIK in HEK293T cells. In contrast, Yersinia lacking the actin-targeting effectors YopEHO, which become internalized into host cells, induce a NIK-dependent response and nuclear entry of the non-canonical NF-κB subunit p52. Blocking actin polymerization and uptake of effector mutant bacteria using cytochalasin D shifted the host NF-κB response from NIK-independent to primarily NIK-dependent. We observed similar results using Pseudomonas aeruginosa, which expresses a related T3SS and the actin-targeting effector ExoT. As the NF-κB response of HEK293T cells to effectorless Yersinia has been used both as a screening tool for chemical inhibitors of the T3SS and for bacterial forward genetic screens, a better understanding of this response is important for tool optimization and interpretation., Competing Interests: The authors have declared that no competing interests exist.

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens., (© 2017 Ruch et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Type IV pili (TFP) function as mechanosensors to trigger acute virulence programs in Pseudomonas aeruginosa. On surface contact, TFP retraction activates the Chp chemosensory system phosphorelay to upregulate 3', 5'-cyclic monophosphate (cAMP) production and transcription of virulence-associated genes. To dissect the specific interactions mediating the mechanochemical relay, we used affinity purification/mass spectrometry, directed co-immunoprecipitations in P. aeruginosa, single cell analysis of contact-dependent transcriptional reporters, subcellular localization and bacterial two hybrid assays. We demonstrate that FimL, a Chp chemosensory system accessory protein of unknown function, directly links the integral component of the TFP structural complex FimV, a peptidoglycan binding protein, with one of the Chp system output response regulators PilG. FimL and PilG colocalize at cell poles in a FimV-dependent manner. While PilG phosphorylation is required for TFP function and mechanochemical signaling, it is not required for polar localization or binding to FimL. Phylogenetic analysis reveals other bacterial species simultaneously encode TFP, the Chp system, FimL, FimV and adenylate cyclase homologs, suggesting that surface sensing may be widespread among TFP-expressing bacteria. We propose that FimL acts as a scaffold enabling spatial colocalization of TFP and Chp system components to coordinate signaling leading to cAMP-dependent upregulation of virulence genes on surface contact., (© 2016 John Wiley & Sons Ltd.)

Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Treatment of acute and chronic pulmonary infections caused by opportunistic pathogen Pseudomonas aeruginosa is limited by the increasing frequency of multidrug bacterial resistance. Here, we describe a novel adjunctive therapy in which administration of a mix of simple sugars-mannose, fucose, and galactose-inhibits bacterial attachment, limits lung damage, and potentiates conventional antibiotic therapy. The sugar mixture inhibits adhesion of nonmucoid and mucoid P. aeruginosa strains to bronchial epithelial cells in vitro. In a murine model of acute pneumonia, treatment with the sugar mixture alone diminishes lung damage, bacterial dissemination to the subpleural alveoli, and neutrophil- and IL-8-driven inflammatory responses. Remarkably, the sugars act synergistically with anti-Pseudomonas antibiotics, including β-lactams and quinolones, to further reduce bacterial lung colonization and damage. To probe the mechanism, we examined the effects of sugars in the presence or absence of antibiotics during growth in liquid culture and in an ex vivo infection model utilizing freshly dissected mouse tracheas and lungs. We demonstrate that the sugar mixture induces rapid but reversible formation of bacterial clusters that exhibited enhanced susceptibility to antibiotics compared with individual bacteria. Our findings reveal that sugar inhalation, an inexpensive and safe therapeutic, could be used in combination with conventional antibiotic therapy to more effectively treat P. aeruginosa lung infections.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and the causative agent of blinding trachoma. Although Chlamydia is protected from humoral immune responses by residing within remodeled intracellular vacuoles, it still must contend with multilayered intracellular innate immune defenses deployed by its host while scavenging for nutrients. Here we provide an overview of Chlamydia biology and highlight recent findings detailing how this vacuole-bound pathogen manipulates host-cellular functions to invade host cells and maintain a replicative niche.

Chlamydia species are obligate intracellular pathogens that are important causes of human genital tract, ocular and respiratory infections. The bacteria replicate within a specialized membrane-bound compartment termed the inclusion and require host-derived lipids for intracellular growth and development. Emerging evidence indicates that Chlamydia has evolved clever strategies to fulfil its lipid needs by interacting with multiple host cell compartments and redirecting trafficking pathways to its intracellular niche. In this review, we highlight recent findings that have significantly expanded our understanding of how Chlamydia exploit lipid trafficking pathways to ensure the survival of this important human pathogen., (© 2012 Blackwell Publishing Ltd.)

Pseudomonas aeruginosa, an important opportunistic pathogen of man, exploits numerous factors for initial attachment to the host, an event required to establish bacterial infection. In this paper, we rigorously explore the role of two major bacterial adhesins, type IV pili (Tfp) and flagella, in bacterial adherence to distinct host receptors at the apical (AP) and basolateral (BL) surfaces of polarized lung epithelial cells and induction of subsequent host signaling and pathogenic events. Using an isogenic mutant of P. aeruginosa that lacks flagella or utilizing beads coated with purified Tfp, we establish that Tfp are necessary and sufficient for maximal binding to host N-glycans at the AP surface of polarized epithelium. In contrast, experiments utilizing a P. aeruginosa isogenic mutant that lacks Tfp or using beads coated with purified flagella demonstrate that flagella are necessary and sufficient for maximal binding to heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPGs) at the BL surface of polarized epithelium. Using two different cell-free systems, we demonstrate that Tfp-coated beads show highest binding affinity to complex N-glycan chains coated onto plastic plates and preferentially aggregate with beads coated with N-glycans, but not with single sugars or HS. In contrast, flagella-coated beads bind to or aggregate preferentially with HS or HSPGs, but demonstrate little binding to N-glycans. We further show that Tfp-mediated binding to host N-glycans results in activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway and bacterial entry at the AP surface. At the BL surface, flagella-mediated binding to HS activates the epidermal growth factor receptor (EGFR), adaptor protein Shc, and PI3K/Akt, and induces bacterial entry. Remarkably, flagella-coated beads alone can activate EGFR and Shc. Together, this work provides new insights into the intricate interactions between P. aeruginosa and lung epithelium that may be potentially useful in the development of novel treatments for P. aeruginosa infections.

The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread.

The strain designated Chlamydia trachomatis serovar that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. [corrected]. The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.

Src family kinases (SFKs) regulate key cellular processes and are emerging as important targets for intracellular pathogens. In this commentary, we briefly review the role of SFKs in bacterial pathogenesis and highlight new work on the role of SFKs during the intracellular cycle of Chlamydia species.

Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3', 5'-cyclic adenosine monophosphate, (cAMP), which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP), the Type III secretion system (T3SS), the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA), suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB), fimL, and cpdA, we show that ΔfimL mutants resemble ΔcyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections.

Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of many virulence factors, including type IV pili, which are required for colonization of host tissues and for twitching motility. Type IV pilus function is controlled in part by the Chp chemosensory system, which includes a histidine kinase, ChpA, and two CheY-like response regulators, PilG and PilH. How the Chp components interface with the type IV pilus motor proteins PilB, PilT, and PilU is unknown. We present genetic evidence confirming the role of ChpA, PilG, and PilB in the regulation of pilus extension and the role of PilH and PilT in regulating pilus retraction. Using informative double and triple mutants, we show that (i) ChpA, PilG, and PilB function upstream of PilH, PilT, and PilU; (ii) that PilH enhances PilT function; and (iii) that PilT and PilB retain some activity in the absence of signaling input from components of the Chp system. By site-directed mutagenesis, we demonstrate that the histidine kinase domain of ChpA and the phosphoacceptor sites of both PilG and PilH are required for type IV pilus function, suggesting that they form a phosphorelay system important in the regulation of pilus extension and retraction. Finally, we present evidence suggesting that pilA transcription is regulated by intracellular PilA levels. We show that PilA is a negative regulator of pilA transcription in P. aeruginosa and that the Chp system functionally regulates pilA transcription by controlling PilA import and export.

In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum- or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.

The purposes of this paper were to identify and compare goals and objectives addressed by music therapists that are contained in the SCERTS Model, for use with children at risk or diagnosed with a communication impartment including Autism Spectrum Disorder (ASD). A video analysis of music therapists working with clients at risk or diagnosed with ASD (N = 33) was conducted to: (a) identify the areas of the SCERTS assessment model that music therapists are currently addressing within their sessions for clients with ASD, and (b) compare the frequency of SCERTS domains and goals addressed by music therapists within sessions. Results of the analysis revealed that all three domains of social communication, emotional regulation, and transactional support were addressed within music therapy sessions. Within each domain both broad goals were all addressed including joint attention and symbol use for social communication, self-regulation and mutual regulation for emotional regulation, and interpersonal support and learning support for transactional support. Overall, music therapists addressed transactional support goals and subgoals more often than social communication and emotional regulation goals and subgoals. The highest frequency goal area addressed was interpersonal support (73.96%) and the lowest goal area addressed was joint attention (35.96%). For the social partner and language partner language stages, 58 of the 320 possible subgoals were addressed with 90% frequency or higher, while 13 of the same subgoals were never addressed. The SCERTS Model is designed for use by a multidisciplinary team of professionals and family members throughout a client's treatment and contains an ongoing assessment tool with resulting goals and objectives. This analysis indicates that many SCERTS goals and objectives can be addressed in music therapy interventions. Additionally, goals and subgoals not previously recognized in music therapy treatment can be generated by the use of the SCERTS Model.

When grown as a biofilm in laboratory flow chambers Pseudomonas aeruginosa can develop mushroom-shaped multicellular structures consisting of distinct subpopulations in the cap and stalk portions. We have previously presented evidence that formation of the cap portion of the mushroom-shaped structures in P. aeruginosa biofilms occurs via bacterial migration and depends on type IV pili (Mol Microbiol 50: 61-68). In the present study we examine additional factors involved in the formation of this multicellular substructure. While pilA mutants, lacking type IV pili, are deficient in mushroom cap formation, pilH and chpA mutants, which are inactivated in the type IV pili-linked chemosensory system, showed only minor defects in cap formation. On the contrary, fliM mutants, which are non-flagellated, and cheY mutants, which are inactivated in the flagellum-linked chemotaxis system, were largely deficient in cap formation. Experiments involving DNase treatment of developing biofilms provided evidence that extracellular DNA plays a role in cap formation. Moreover, mutants that are deficient in quorum sensing-controlled DNA release formed microcolonies upon which wild-type bacteria could not form caps. These results constitute evidence that type IV pili, flagellum-mediated motility and quorum sensing-controlled DNA release are involved in the formation of mature multicellular structures in P. aeruginosa biofilms.

Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis). This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP [corrected]. To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA), we identified approximately 226 host genes, including two tyrosine kinases, Abelson (Abl) kinase and PDGF- and VEGF-receptor related (Pvr), a homolog of the Platelet-derived growth factor receptor (PDGFR). We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRbeta may function as a receptor, as inhibition of PDGFRbeta by RNA interference or by PDGFRbeta neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRbeta or through independent activation of Abl kinase, culminating in phosphorylation of the Rac guanine nucleotide exchange factor (GEF), Vav2, and two actin nucleators, WAVE2 and Cortactin. Finally, we show that TARP, a bacterial type III secreted actin nucleator implicated in entry, is a target of Abl kinase. Together, our results demonstrate that PDGFRbeta and Abl kinases function redundantly to promote efficient uptake of this obligate intracellular parasite.

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

Chlamydia spp. are major causes of important human diseases, but dissecting the host-pathogen interactions has been hampered by the lack of bacterial genetics and the difficulty in carrying out forward genetic screens in mammalian hosts. RNA interference (RNAi)-based methodologies for gene inactivation can now be easily carried out in genetically tractable model hosts, such as Drosophila melanogaster, and offer a new approach to identifying host genes required for pathogenesis. We tested whether Chlamydia trachomatis infection of D. melanogaster S2 cells recapitulated critical aspects of mammalian cell infections. As in mammalian cells, C. trachomatis entry was greatly reduced by heparin and cytochalasin D. Inclusions were formed in S2 cells, acquired Golgi-derived sphingolipids, and avoided phagolysosomal fusion. Elementary body (EB) to reticulate body (RB) differentiation was observed, however, no RB to EB development or host cell killing was observed. RNAi-mediated inactivation of Rac, a Rho GTPase recently shown to be required for C. trachomatis entry in mammalian cells, inhibits C. trachomatis infection in S2 cells. We conclude that Drosophila S2 cells faithfully mimic early events in Chlamydia host cell interactions and provides a bona fide system to systematically dissect host functions important in the pathogenesis of obligate intracellular pathogens.

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.

Using pharmacologic and biochemical criteria, we evaluated whether uptake of four different Chlamydia trachomatis serovars, D, E, K, and L2, was dependent upon lipid rafts. Our data suggest that lipid raft-mediated entry is not required for C. trachomatis infection of cultured epithelial cells.

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.

ExoT is a type III secreted effector protein found in almost all strains of Pseudomonas aeruginosa and is required for full virulence in an animal model of acute pneumonia. It is comprised of an N-terminal domain with GTPase activating protein (GAP) activity towards Rho family GTPases and a C-terminal ADP ribosyltransferase (ADPRT) domain with minimal activity towards a synthetic substrate in vitro. Consistent with its activity as a Rho family GTPase, ExoT has been shown to inhibit P. aeruginosa internalization into epithelial cells and macrophages, disrupt the actin cytoskeleton through a Rho-dependent pathway, and inhibit wound repair in a scrape model of injured epithelium. We have previously shown that mutation of the invariant arginine of the GAP domain to lysine (R149K) results in complete loss of GAP activity in vitro but only partially inhibits ExoT anti-internalization and cell rounding activity. We have constructed in-frame deletions and point mutations within the ADPRT domain in order to test whether this domain might account for the residual activity observed in ExoT GAP mutants. Deletion of a majority of the ADPRT domain (residues 234 to 438) or point mutations of the ADPRT catalytic site (residues 383 to 385) led to distinct changes in host cell morphology and substantially reduced the ability of ExoT to inhibit in vitro epithelial wound healing over a 24-h period. In contrast, only subtle effects on the efficiency of ExoT-induced bacterial internalization were observed in the ADPRT mutant forms. Expression of each domain individually in Saccharomyces cerevisiae was toxic, whereas expression of each of the catalytically inactive mutant domains was not. Collectively, these data demonstrate that the ADPRT domain of ExoT is active in vivo and contributes to the pathogenesis of P. aeruginosa infections.

Modulation of cytosolic (intracellular) Ca(2+) concentration (Ca(i)) may be an important host response when airway epithelial cells are exposed to Pseudomonas aeruginosa. We measured Ca(i) in Calu-3 cells exposed from the apical or basolateral surface to cytotoxic and noncytotoxic strains of P. aeruginosa. Apical addition of either noncytotoxic strains or cytotoxic strains failed to affect Ca(i) over a 3-h time period, nor were changes observed after basolateral addition of noncytotoxic strains. In contrast, basolateral addition of cytotoxic strains caused a slow increase in Ca(i) from 100 nM to 200 to 400 nM. This increase began after 20 to 50 min and persisted for an additional 30 to 75 min, at which time the cells became nonviable. P. aeruginosa-induced increases in Ca(i) were blocked by the addition of the Ca channel blocker La(3+) to the basolateral but not to the apical chamber. Likewise, replacing the basolateral but not the apical medium with Ca-free solution prevented P. aeruginosa-mediated changes in Ca(i). With isogenic mutants of PA103, we demonstrated that the type III secretion apparatus, the type III-secreted effector ExoU, and type IV pili were necessary for increased Ca(i). We propose that translocation of ExoU through the basolateral surface of polarized airway epithelial cells via the type III secretion apparatus leads to release of Ca stored in the endoplasmic reticulum and activation of Ca channels in the basolateral membranes of epithelial cells.

The Pseudomonas aeruginosa protein ExoT is a bacterial GTPase-activating protein (GAP) that has in vitro activity toward Rho, Rac, and Cdc42 GTPases. Expression of ExoT both inhibits the internalization of strain PA103 by macrophages and epithelial cells and is associated with morphological changes (cell rounding and detachment) of infected cells. We find that expression of ExoT leads to the loss of GTP-bound RhoA, Rac1, and Cdc42 in transfected HeLa cells, demonstrating that ExoT has GAP activity in vivo toward all three GTPases. GAP activity is absolutely dependent on the presence of arginine at position 149 but is not affected by whether ExoT is expressed in the absence or presence of other P. aeruginosa type III secreted proteins. We also demonstrate that expression of ExoT in epithelial cells is sufficient to cause stress fiber disassembly by means of ExoT's GAP activity toward RhoA.

Objective: Pseudomonas aeruginosa is a frequent cause of ventilator-associated pneumonia. Recent evidence suggests that production of type III secretion proteins is correlated with increased pathogenicity in both cellular and animal models of infection. The objective of this study was to determine whether this system contributes to disease severity in humans with ventilator-associated pneumonia., Design: Retrospective pilot cohort study., Setting: University hospital., Patients: Thirty-five mechanically ventilated patients with bronchoscopically confirmed ventilator-associated pneumonia caused by P. aeruginosa., Measurements and Main Results: Ventilator-associated pneumonia was categorized as severe (patients died or had a recurrence of their pneumonia despite appropriate antibiotic therapy) or mild (patients uneventfully recovered from their pneumonia). The type III secretion genotypes and phenotypes of isolates cultured from the patients with ventilator-associated pneumonia were determined. Whereas every examined isolate harbored type III secretion genes, only 27 (77%) were capable of secreting detectable amounts of type III proteins in vitro. Twenty-two (81%) of the patients infected with these 27 isolates had severe disease. Of the eight isolates that did not secrete type III proteins, only three (38%) were cultured from patients with severe disease. Thus, infection with a type-III-secreting isolate correlated with severe disease (p < .05). In vitro assays indicated that ExoU, the type III effector protein most closely linked to mortality in animal models, was secreted in detectable amounts in vitro by 10 (29%) of the 35 examined isolates. Nine (90%) of these 10 isolates were cultured from patients with severe disease (p < .05 when compared with the nonsecreting isolates). In contrast, ExoS was secreted by 16 (46%) of the 35 examined isolates. Twelve (75%) of these 16 isolates were cultured from patients with severe disease (p = .14 when compared with the nonsecreting isolates)., Conclusions: In patients with ventilator-associated pneumonia, type-III-secreting isolates were associated with worse clinical outcomes, suggesting that this secretion system plays an important role in human disease. Our findings support the hypothesis that antibodies targeted against these proteins may be useful as adjunctive therapy in intubated patients with P. aeruginosa colonization or infection.

The nosocomial pathogen Pseudomonas aeruginosa causes clinical infection in the setting of pre-existing epithelial tissue damage, an association that is mirrored by the increased ability of P. aeruginosa to bind, invade and damage injured epithelial cells in vitro. In this study, we report that P. aeruginosa inhibits the process of epithelial wound repair in vitro through the type III-secreted bacterial protein ExoT, a GTPase-activating protein (GAP) for Rho family GTPases. This inhibition primarily targets cells at the edge of the wound, and causes actin cytoskeleton collapse, cell rounding and cell detachment. ExoT-dependent inhibition of wound repair is mediated through the GAP activity of this bacterial protein, as mutations in ExoT that alter the conserved arginine (R149) within the GAP domain abolish the ability of P. aeruginosa to inhibit wound closure. Because ExoT can also inhibit P. aeruginosa internalization by phagocytes and epithelial cells, this protein may contribute to the in vivo virulence of P. aeruginosa by allowing organisms both to overcome local host defences, such as an intact epithelial barrier, and to evade phagocytosis by immune effector cells.