Alan R. Hauser

The Type III Secretion System of Pseudomonas aeruginosa: Infection by Injection

The Gram-negative bacterium Pseudomonas aeruginosa uses a complex type III secretion apparatus to inject effector proteins into host cells. The configuration of this secretion machinery, the activities of the proteins that are injected by it and the consequences of this process for infection are now being elucidated. This Review summarizes our current knowledge of P. aeruginosa type III secretion, including the secretion and translocation machinery, the regulation of this machinery, and the associated chaperones and effector proteins. The features of this interesting secretion system have important implications for the pathogenesis of P. aeruginosa infections and for other type III secretion systems.

Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa.

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.

Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

BackgroundPseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.ResultsWe developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.ConclusionsThe P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa

The type III secretion system of Pseudomonas aeruginosa transports four known effector proteins: ExoS, ExoT, ExoU and ExoY. However, the prevalence of the type III secretion system genes or the effector-encoding genes in clinical and environmental isolates of P. aeruginosa has not been well studied. Southern hybridization analyses and PCR were performed on over 100 P. aeruginosa isolates to determine the distribution of these genes. Clinical isolates were obtained from urine, endotracheal, blood and wound specimens, from the sputum of cystic fibrosis (CF) patients, and from non-hospital environmental sites. The popB gene was used as a marker for the presence of the large chromosomal locus encoding the type III secretion machinery proteins. Each isolate contained the popB gene, indicating that at least a portion of this large chromosomal locus was present in all isolates. Likewise, each isolate contained exoT-like sequences. In contrast, the exoS, exoU and exoY genes were variable traits. Overall, 72% of examined isolates contained the exoS gene, 28% contained the exoU gene, and 89% contained the exoY gene. Interestingly, an inverse correlation was noted between the presence of the exoS and exoU genes in that all isolates except two contained either exoS or exoU but not both. No significant difference in exoS, exoU or exoY prevalence was observed between clinical and environmental isolates or between isolates cultured from different disease sites except for CF respiratory isolates. CF isolates harboured the exoU gene less frequently and the exoS gene more frequently than did isolates from some of the other sites of infection, including the respiratory tract of patients without CF. These results suggest that the P. aeruginosa type III secretion system is present in nearly all clinical and environmental isolates but that individual isolates and populations of isolates from distinct disease sites differ in their effector genotypes. The ubiquity of type III secretion genes in clinical isolates is consistent with an important role for this system in human disease.

Relative Contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to Virulence in the Lung

ABSTRACT Pseudomonas aeruginosa uses a type III secretion system to promote development of severe disease, particularly in patients with impaired immune defenses. While the biochemical and enzymatic functions of ExoU, ExoS, and ExoT, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood. Since ExoU and ExoS are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection. In the work described here, several isogenic mutants of a bacterial strain that naturally secretes ExoU, ExoS, and ExoT were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia. Measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of ExoU had the greatest impact on virulence while secretion of ExoS had an intermediate effect and ExoT had a minor effect. It is of note that these results conclusively show for the first time that ExoS is a virulence factor. Infection with isogenic mutants secreting wild-type ExoS, ExoS defective in GTPase-activating protein (GAP) activity, or ExoS defective in ADP-ribosyltransferase activity demonstrated that the virulence of ExoS was largely dependent on its ADP-ribosyltransferase activity. The GAP activity of this protein had only a minor effect in vivo. The relative virulence associated with each of these type III effector proteins may have important prognostic implications for patients infected with P. aeruginosa.

Clinical Significance of Microbial Infection and Adaptation in Cystic Fibrosis

SUMMARY A select group of microorganisms inhabit the airways of individuals with cystic fibrosis. Once established within the pulmonary environment in these patients, many of these microbes adapt by altering aspects of their structure and physiology. Some of these microbes and adaptations are associated with more rapid deterioration in lung function and overall clinical status, whereas others appear to have little effect. Here we review current evidence supporting or refuting a role for the different microbes and their adaptations in contributing to poor clinical outcomes in cystic fibrosis.

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

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

The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage

Mature Pseudomonas aeruginosa biofilms undergo specific developmental events. Using a bacteriophage mutant, generated by deletion of the entire filamentous Pf4 prophage, we show that the phage is essential for several stages of the biofilm life cycle and that it significantly contributes to the virulence of P. aeruginosa in vivo. Here, we show for the first time that biofilms of the Pf4 phage-deficient mutant did not develop hollow centres or undergo cell death, typical of the differentiation process of wild-type (WT) P. aeruginosa PAO1 biofilms. Furthermore, microcolonies of the Pf4 mutant were significantly smaller in size and less stable compared with the WT biofilm. Small colony variants (SCVs) were detectable in the dispersal population of the WT biofilm at the time of dispersal and cell death, whereas no SCVs were detected in the effluent of the Pf4 mutant biofilm. This study shows that at the time when cell death occurs in biofilms of the WT, the Pf4 phage converts into a superinfective form, which correlates with the appearance of variants in the dispersal population. Unexpectedly, mice infected with the Pf4 mutant survived significantly longer than those infected with its isogenic WT strain, showing that Pf4 contributes to the virulence of P. aeruginosa. Hence, a filamentous prophage is a major contributor to the life cycle and adaptive behaviour of P. aeruginosa and offers an explanation for the prevalence of phage in this organism.

Secretion of the Toxin ExoU Is a Marker for Highly Virulent Pseudomonas aeruginosa Isolates Obtained from Patients with Hospital-Acquired Pneumonia

Overall, hospital-acquired pneumonia (HAP) caused by Pseudomonas aeruginosa is associated with high attributable mortality. Although the intrinsic virulence of P. aeruginosa undoubtedly contributes to this phenomenon, it is unclear whether all strains share this property or whether only a subpopulation of strains are capable of causing such severe disease. In this study, the virulence of 35 P. aeruginosa isolates obtained from patients with HAP by use of a cytolytic cell-death assay, an apoptosis assay, and a mouse model of pneumonia. The virulence of individual isolates differed significantly from one to another in each of these assays. Increased virulence was associated with the secretion of ExoU, a toxin transported by the P. aeruginosa type III secretion system. Secretion of ExoS or ExoY, 2 other proteins transported by this system, was not consistently associated with increased virulence. Together, these findings suggest that secretion of ExoU is a marker for highly virulent strains of P. aeruginosa.

The arginine finger domain of ExoT contributes to actin cytoskeleton disruption and inhibition of internalization of Pseudomonas aeruginosa by epithelial cells and macrophages

ABSTRACT Pseudomonas aeruginosa, an important nosocomial pathogen of humans, expresses a type III secretion system that is required for virulence. Previous studies demonstrated that the lung-virulent strain PA103 has the capacity to be either cytotoxic or invasive. Analyses of mutants suggest that PA103 delivers a negative regulator of invasion, or anti-internalization factor, to host cells via a type III secretion system. In this work we show that the type III secreted protein ExoT inhibits the internalization of PA103 by polarized epithelial cells (Madin-Darby canine kidney cells) and J774.1 macrophage-like cells. ExoS, which is closely related to ExoT but has additional ADP-ribosylating activity, can substitute for ExoT as an anti-internalization factor. ExoT contains a signature arginine finger domain found in GTPase-activating proteins. Mutation of the conserved arginine in ExoT diminished its anti-internalization activity and altered its ability to disrupt the actin cytoskeleton. Cell fractionation experiments showed that ExoT is translocated into host cells and that mutation of the arginine finger did not disrupt translocation. In a mouse model of acute pneumonia, PA103ΔUΔT reached the lungs as efficiently as PA103ΔU but showed reduced colonization of the liver. This finding suggests that the ability to resist internalization may be important for virulence in vivo.