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Bacterial competition within host-associated polymicrobial communities shapes their composition, often with far-reaching consequences for human health. In this issue of Cell Host & Microbe, Perault et al. reveal how competition between two opportunistic pathogens could account for the epidemiology of chronic lung infections in people with cystic fibrosis.

Surface sensing is a fundamental yet poorly understood behavior of swimming bacteria. In this issue of Cell Host & Microbe, Laventie et al. (2019) describe a cyclic-di-GMP-dependent pathway used by the opportunistic pathogen Pseudomonas aeruginosa to respond to surface binding on a several second timescale.

The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa’s outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae. This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic. IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica. The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S. Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S. Typhimurium. IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S. Typhimurium.

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them., Author summary Attachment to a surface often triggers programs of gene expression that alter the behavior, virulence and fitness of bacteria. Initial contact is usually mediated by surface exposed adhesins, such as flagella or pili/fimbriae, and there is much interest in how these structures might sense and respond to surface attachment. The human bacterial pathogen Pseudomonas aeruginosa can initially contact surfaces via its polar flagellum, the structure that also powers bacterial swimming. We observed that wild-type bacteria quickly stopped rotating their flagellum after surface attachment, but that a mutant lacking the flagellar-associated protein FlhF did not. Using a combination of genetic approaches, we demonstrated that FlhF interacts with a component of the flagellar rotor (FliG) and with a polar scaffolding protein that positively regulates cAMP production (FimV) to stop flagellar rotation and thereby favor bacterial persistence at a surface. We provide evidence that the second messenger cAMP is the likely signal generated by flagellar-mediated surface attachment and show that cAMP is sufficient to alter the behavior of the flagellar motor.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals., Pseudomonas aeruginosa can cause chronic infections on medical devices by forming biofilms, a process regulated by quorum sensing (QS). Here, the authors show that P. aeruginosa is unable to perceive QS signals in the presence of either host urine or urea, and clinical isolates are often defective in QS.

We previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronic Pseudomonas aeruginosa infections, enables bacteria to evade association and ingestion of P. aeruginosa by phagocytes both in vitro and in vivo . Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation by P. aeruginosa , we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotile P. aeruginosa . Nonmotile P. aeruginosa elicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cells in vitro . Importantly, nonmotile P. aeruginosa also elicits reduced IL-1β levels in vivo in comparison to those elicited by wild-type P. aeruginosa . This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

This chapter reviews the observations supporting the idea that acute and chronic Pseudomonas aeruginosa infections represent distinct modes of host-pathogen interaction. The virulence factors associated with acute infections and chronic infections are discussed, with a focus on data obtained from human subject-based studies, when possible. P. aeruginosa expresses many virulence factors that can damage host cells and which contribute to infection in both humans and animal models. Ectopic expression of virulence factor regulator (Vfr) in mucA22 strains restored expression of ExoS, type IV pilus (TFP), and elastase, confirming that downregulation of Vfr is responsible for decreased virulence factor expression in mucoid strains under the conditions evaluated in this study. While it is clear that regulators of TFP biogenesis and function are intimately associated with the control of Vfr and cyclic AMP (cAMP) expression, the mechanism that links twitching motility and Vfr remains to be elucidated. rsmZ and rsmY are two sRNAs that interact with the RNA binding protein RsmA. The paper by Bordi and coworkers also provides the first data that rsmY and rsmZ are not functionally redundant. Increased expression of T6SS genes, whose translation is negatively regulated by RsmA, specifically requires increased expression of the rsmZ sRNA. Most bacterial pathogens, P. aeruginosa included, use their virulence factors for the primary purpose of gaining access to nutrients rather than for causing damage to specific hosts. The chapter focuses on a few examples of regulatory networks that broadly impact virulence factor expression.

Pseudomonas aeruginosa causes hospital-acquired pneumonia and is associated with high mortality. An effective response to such an infection includes efficient clearance of pathogenic organisms while limiting collateral damage from the host inflammatory response, known as host resistance and host tolerance, respectively. P. aeruginosa expresses a type III secretion system (T3SS) needle complex that induces NLRC4 (NOD-like receptor C4) activation, interleukin-1β (IL-1β) production, and host tissue damage. Chitinase 3-like-1 (Chil1) is expressed during infection and binds to its receptor, IL-13 receptor α2 (IL-13Rα2), to regulate the pathogen-host response during Streptococcus pneumoniae infection, but the role Chil1 plays in balancing the host resistance and host tolerance during P. aeruginosa pneumonia is not known. We conducted experiments using C57BL/6 mice with or without a genetic deficiency of Chil1 and demonstrated that Chil1-deficient mice succumb to P. aeruginosa infection more rapidly than the wild type (WT). The decreased survival time in infected Chil1-deficient mice is associated with more neutrophils recruited to the airways, more lung parenchymal damage, and increased pulmonary consolidation while maintaining equivalent bacterial killing compared to WT mice. Infected Chil1-deficient mice and bone marrow-derived macrophages (BMDMs) from Chil1-deficient mice have increased production of tumor necrosis factor alpha (TNF-α) and IL-1β compared to infected WT mice and macrophages. Infection of Chil1-deficient BMDMs with non-NLRC4-triggering P. aeruginosa , which is deficient in the T3SS needle complex, did not alter the excessive IL-1β production compared to BMDMs from WT mice. The addition of recombinant Chil1 decreases the excessive IL-1β production but only partially rescues stimulated BMDMs from IL-13Rα2-deficient mice. Our data provide mechanistic insights into how Chil1 regulates P. aeruginosa -induced host responses.

Innate immune responses play a critical role in controlling acute infections due to Pseudomonas aeruginosa in both mice and in humans. In this review we focus on innate immune recognition and clearance mechanisms that are important for controlling P. aeruginosa in the mammalian lung, with particular attention to those that influence the outcome of in vivo infection in murine models.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by either biofilm formation or swarming motility. The choice of behaviour is influenced by the physical properties of the surface and specific nutrient availability, and subject to regulatory networks that also govern type 2 and type 3 protein secretion. Biofilm formation by clinical isolates has been well-studied. However, the swarming behaviour of human isolates has not been extensively analysed. We collected isolates from 237 hospitalized patients without cystic fibrosis and analysed motility and secretion phenotypes of each isolate. We found biofilm formation and swarming to be negatively associated, while swarming was positively associated with the secretion of both proteases and type 3 exoenzymes. Most isolates were capable of type 3 secretion and biofilm formation, even though these traits are considered to favour distinct modes of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm and secretion phenotypes, many of the predicted relationships between swarming motility and other phenotypes observed in laboratory strains also hold true for bacteria isolated from human patients.

Oxidative stress is one of the main challenges bacteria must cope with during infection. Here, we identify a new oxidative stress sensing and response ospR (oxidative stress response and pigment production Regulator) gene in Pseudomonas aeruginosa. Deletion of ospR leads to a significant induction in H(2)O(2) resistance. This effect is mediated by de-repression of PA2826, which lies immediately upstream of ospR and encodes a glutathione peroxidase. Constitutive expression of ospR alters pigment production and beta-lactam resistance in P. aeruginosa via a PA2826-independent manner. We further discovered that OspR regulates additional genes involved in quorum sensing and tyrosine metabolism. These regulatory effects are redox-mediated as addition of H(2)O(2) or cumene hydroperoxide leads to the dissociation of OspR from promoter DNA. A conserved Cys residue, Cys-24, plays the major role of oxidative stress sensing in OspR. The serine substitution mutant of Cys-24 is less susceptible to oxidation in vitro and exhibits altered pigmentation and beta-lactam resistance. Lastly, we show that an ospR null mutant strain displays a greater capacity for dissemination than wild-type MPAO1 strain in a murine model of acute pneumonia. Thus, OspR is a global regulator that senses oxidative stress and regulates multiple pathways to enhance the survival of P. aeruginosa inside host.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa–induced cell death and release of the proinflammatory cytokine interleukin (IL)-1β. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1–driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response.

Chronic infection with Pseudomonas aeruginosa is a leading cause of morbidity and mortality in individuals with cystic fibrosis despite the aggressive use of antibiotics. P. aeruginosa employs a number of strategies that promote chronic pulmonary colonization instead of acute infection. These include biofilm formation, evasion of the host immune system, and conversion to a mucoid phenotype. This review discusses recent advances regarding P. aeruginosa pathogenesis and biofilm behavior in the setting of chronic pulmonary disease.Biofilm formation in the cystic fibrosis lung likely occurs under anaerobic conditions, is controlled by bacterial quorum-sensing mechanisms, and is enhanced by environmental components in the cystic fibrosis airway. P. aeruginosa possesses regulatory pathways that recognize environmental cues to favor either acute infection or chronic colonization. P. aeruginosa that inhabit the respiratory tract accumulate mutations favoring chronic colonization. Azithromycin, a macrolide with clinical benefit in cystic fibrosis, alters P. aeruginosa biofilm formation. Promising new therapies that target biofilm formation include molecules that disrupt quorum sensing.Eradication of P. aeruginosa in cystic fibrosis patients remains problematic. As more information emerges about P. aeruginosa behavior in vivo, potential therapeutics directed against biofilms and mucoid P. aeruginosa are being developed.

Pseudomonas aeruginosa navigates using two distinct forms of motility, swimming and twitching. A polar flagellum and Type 4 pili power these movements, respectively, allowing P. aeruginosa to attach to and colonize surfaces. Single cell imaging and particle tracking algorithms have revealed a wide range of bacterial surface behaviors which are regulated by second messengers cyclic-di-GMP and cAMP; the production of these signals is, in turn, responsive to the engagement of motility organelles with a surface. Innate immune defense systems, long known to recognize structural components of flagella, appear to respond to motility itself. The association of motility with both upregulation of virulence and induction of host defense mechanisms underlies the complex contributions of flagella and pili to P. aeruginosa pathogenesis.

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa , plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.

The bacterial pathogen Pseudomonas aeruginosa causes acute infections associated with significant morbidity and mortality. P. aeruginosa elicits strong innate immune responses in immunocompetent hosts, and the resulting recruitment of neutrophils to the site of infection is necessary for bacterial clearance. P. aeruginosa lipopolysaccharide and flagellin are recognized by extracellular Toll-like receptors, but the most rapid responses to infection occur when cytosolic receptors sense flagellin or type 3 secretion system (T3SS) structural proteins. The subsequent activation of the NLRC4 inflammasome and caspase-1 generates an interleukin-1β (IL-1β) signal that is required for the rapid neutrophilic response. A T3SS effector, exotoxin U (ExoU), can inhibit activation of the NLRC4 inflammasome and caspase-1. Thus, our observation that IL-1 receptor (IL-1R)-mediated signals were still required to initiate a response to ExoU-producing bacteria was unexpected. As both IL-1α and IL-1β signal via the IL-1R, we examined immune responses in mice lacking either of these cytokines. IL-1β-deficient mice responded to ExoU-producing P. aeruginosa bacteria similarly to wild-type animals; however, IL-1α-deficient mice had an attenuated immune response. The situation was reversed following infections by ExoU-negative bacteria: here, IL-1α was dispensable for neutrophil recruitment, while IL-1β was required. IL-1α secretion by macrophages infected with ExoU-producing P. aeruginosa isolates was independent of both caspase-1 and caspase-11. This study documents distinct roles for IL-1α and IL-1β in the response to P. aeruginosa infection as a function of the T3SS effectors produced by the infecting strain. The redundancy of these two cytokines nonetheless allows the infected host to mount a response to ExoU-positive and -negative bacterial isolates.

The opportunistic pathogen Pseudomonas aeruginosa expresses a type 3 secretion system (T3SS) strongly associated with bacterial virulence in murine models and human patients. T3SS effectors target host innate immune mechanisms, and T3SS-defective mutants are cleared more efficiently than T3SS-positive bacteria by an immunocompetent host. Nonetheless, T3SS-negative isolates are recovered from many patients with documented P. aeruginosa infections, leading us to test whether T3SS-negative strains could have a selective advantage during in vivo infection. Mice were infected with mixtures of T3SS-positive WT P. aeruginosa plus isogenic T3SS-OFF or constitutively T3SS-ON mutants. Relative fitness of bacteria in this acute pneumonia model was reflected by the competitive index of mutants relative to WT. T3SS-OFF strains outcompeted WT PA103 in vivo, whereas a T3SS-ON mutant showed decreased fitness compared with WT. In vitro growth rates of WT and T3SS-OFF bacteria were determined under T3SS-inducing conditions and did not differ significantly. Increased fitness of T3SS-OFF bacteria was no longer observed at high ratios of T3SS-OFF to WT, a feature characteristic of bacterial cheaters. Cheating by T3SS-OFF bacteria occurred only when T3SS-positive bacteria expressed the phospholipase A2 effector Exotoxin U (ExoU). T3SS-OFF bacteria showed no fitness advantage in competition experiments carried out in immunodeficient MyD88-knockout mice or in neutrophil-depleted animals. Our findings indicate that T3SS-negative isolates benefit from the public good provided by ExoU-mediated killing of recruited innate immune cells. Whether this transient increase in fitness observed for T3SS-negative strains in mice contributes to the observed persistence of T3SS-negative isolates in humans is of ongoing interest.

Flagellar-based motility plays a critical role in Pseudomonas aeruginosa pathogenesis, influencing both the establishment of bacterial infection and the host's response to the pathogen. Nonetheless, aflagellate clinical strains are often isolated from acutely and chronically infected patients and include the virulent laboratory strain PA103. We determined that PA103's aflagellate phenotype is the result of a single amino acid change (G240V) in the master flagellar regulator, FleQ. This mutation, which lies just outside the Walker B box of FleQ, abrogates the ability of FleQ to positively regulate flagellar gene expression. Reversal of this seemingly conservative amino acid substitution is sufficient to restore swimming motility to PA103, despite the presence of mutations in other flagellar genes of PA103. We also investigated the consequences of restoring flagellar assembly on PA103 virulence. Although a negative correlation between flagellar assembly and Type 3 secretion system (T3SS) expression has been reported previously, we did not observe downregulation of T3SS expression or function in Fla+ PA103. Restoration of flagellar assembly did, however, amplify IL-1 signals measured during murine pulmonary infection and was associated with increased bacterial clearance. These experiments suggest that loss of flagellar motility may primarily benefit PA103 by attenuating pathogen recognition and clearance during acute infection.

The opportunistic human pathogen Pseudomonas aeruginosa causes rapidly progressive and tissue-destructive infections, such as hospital-acquired and ventilator-associated pneumonias. Innate immune responses are critical in controlling P. aeruginosa in the mammalian lung, as demonstrated by the increased susceptibility of MyD88−/− mice to this pathogen. Experiments conducted using bone marrow chimeric mice demonstrated that radio-resistant cells participated in initiating MyD88-dependent innate immune responses to P. aeruginosa. In this study we used a novel transgenic mouse model to demonstrate that MyD88 expression by epithelial cells is sufficient to generate a rapid and protective innate immune response following intranasal infection with P. aeruginosa. MyD88 functions as an adaptor for many TLRs. However, mice in which multiple TLR pathways (e.g., TLR2/TLR4/TLR5) are blocked are not as compromised in their response to P. aeruginosa as mice lacking MyD88. We demonstrate that IL-1R signaling is an essential element of MyD88-dependent epithelial cell responses to P. aeruginosa infection.

Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has emerged as a major public health threat. Here we report that the cell wall of S. aureus can be covalently re-engineered to contain non-native small molecules. This process makes use of endogenous levels of the bacterial enzyme sortase A (SrtA), which ordinarily functions to incorporate proteins into the bacterial cell wall. Thus, incubation of wild-type bacteria with rationally designed SrtA substrates results in covalent incorporation of functional molecular handles (fluorescein, biotin, and azide) into cell wall peptidoglycan. These conclusions are supported by data obtained through a variety of experimental techniques (epifluorescence and electron microscopy, biochemical extraction, and mass spectrometry), and cell-wall-incorporated azide was exploited as a chemical handle to perform an azide-alkyne cycloaddition reaction on the bacterial cell surface. This report represents the first example of cell wall engineering of S. aureus or any other pathogenic Gram-positive bacteria and has the potential for widespread utility.

Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence.

Background The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium. Methods We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells. Results Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates. Conclusion The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.