A genotypic and phenotypic comparison of type III secretion profiles of Pseudomonas aeruginosa cystic fibrosis and bacteremia isolates

The type III secretion system (TTSS) of Pseudomonas aeruginosa enables delivery of a number of toxins involved in the disruption of eukaryotic epithelial surfaces. Whilst the ability to secrete ExoS facilitates invasion and internalization, the secretion of ExoU mediates acute cytotoxicity. In order to determine any association with the ability to secrete these toxins with the nature and severity of human infection, the TTSS genotypes and phenotypes of 163 clinical isolates were determined by multiplex PCR and Western blotting. An exoS+/exoU- genotype was associated with chronic infection in patients with cystic fibrosis whilst an exoS-/exoU+ genotype was associated with strains isolated from blood. Secretion of the ExoU protein was more commonly seen in isolates obtained from blood, suggesting this ability may be important in the development of acute invasive infection. Detection of TTSS toxins in clinical material may be useful in targeting antimicrobial therapy or identifying individuals infected with aggressive strains of P. aeruginosa.     

Site-directed mutagenesis of the Pseudomonas aeruginosa type III secretion system protein PscJ reveals an essential role for surface-localized residues in needle complex function

The Pseudomonas aeruginosa type III secretion system (TTSS) protein PscJ belongs to the PrgK family of TTSS proteins. These proteins are predicted to form one of the inner membrane localized ring substructures of the TTSS needle complex. To determine which amino acid residues of PscJ are important for its function, the pscJ gene was subjected to site-directed mutagenesis. Fifteen individual PscJ amino acid residues that are located in conserved regions of the PrgK family were targeted for mutagenesis. Eight of these residues could be subjected to non-conservative substitution mutagenesis without affecting the function of the resultant mutant protein. Substitution of the other 7 residues (E26, K52, E105, A107, G126, H133, and V189) resulted in either a non-functional protein or the loss of detectable protein. When the essential residues were mapped on to the crystal structure of the E. coli PrgK homolog EscJ, the majority appeared to localize to surface-exposed regions of the protein suggesting a role for these regions in the assembly of the PscJ ring structure.     

Polarisation of type III translocation by Pseudomonas aeruginosa requires PcrG, PcrV and PopN

Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell. We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PcrV, PopB and PopD. To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants. In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased. When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium. This confirms that ExoS translocation by P. aeruginosa occurs by a polarised mechanism. In contrast, high levels of ExoS were recovered in the tissue culture medium when cells were infected with pcrG, pcrV and popN mutants. Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions. This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells.     

Comparison of type III secretion system virulence among fluoroquinolone-susceptible and -resistant clinical isolates of Pseudomonas aeruginosa.

Fluoroquinolone resistance and type III secretion system (TTSS) virulence are independently associated in Pseudomonas aeruginosa infections with poor patient outcomes. In the present study, the virulence of fluoroquinolone-susceptible and -resistant isolates of P. aeruginosa was compared, focusing on TTSS virulence. Clinical isolates (n = 45) exhibiting a broad range of susceptibilities to fluoroquinolones, with differing mechanisms of resistance and associated with varying disease sites, were selected for the study. PCR, Southern blot and western immunoblot analyses were performed to determine the presence of TTSS-encoding genes and secretion of gene products. The cytotoxicity of the clinical isolates towards human lung epithelial cells was also determined. Clinical isolates encoding only the exoS cytotoxin gene occurred more frequently than those encoding only exoU (62% vs. 27%; p 0.0007). Compared with exoS(+) isolates, exoU(+) isolates were more likely to be fluoroquinolone-resistant (92% vs. 61%, p 0.05) and to exhibit both a gyrA mutation and the efflux pump over-expressed (EPO) phenotype (91% vs. 59%; p 0.06). Almost all exoU(+) strains secreted ExoU and exhibited increased cytotoxicity compared with ExoS-secreting strains (7% vs. 92.5%, relative to a PA103 reference strain control). These data suggest that exoU(+) and fluoroquinolone resistance may be co-selected traits that result in highly virulent and resistant strains. Adverse outcomes associated with infections caused by fluoroquinolone-resistant strains may, in part, be attributable to this co-association, which warrants further clinical investigation.     

Secretion of Pseudomonas aeruginosa type III cytotoxins is dependent on pseudomonas quinolone signal concentration

Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent post-translational control, specifically governing type III cytotoxin secretion, exists in this species.     

Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing

Pseudomonas aeruginosa strains PAO1 and CHA showing type III system-dependent cytotoxicity towards macrophages ex vivo are able to induce rapid death of adult fly Drosophila melanogaster accompanied by bacterial multiplication to high-titers. The role of P. aeruginosa type III secretion system in rapid fly killing was demonstrated here by using several isogenic CHA mutants, selectively affected in this system. The activation of P. aeruginosa pexsCBA, the regulatory operon of the type III system, and the activation of the Drosophila gene diptericin, showed the host-pathogen recognition during infection process.     

Antibiotic and metal resistance among hospital and outdoor strains of Pseudomonas aeruginosa

Phenotypic analyses of antibiotic and metal resistance of a collection of 130 strains of Pseudomonas aeruginosa from various outdoor (i.e. soil, water, animals) and hospital (environment, patients, individuals with cystic fibrosis) settings were performed. Resistance was scored according to the origin of the strains and their likely exposure to antibiotics and chemicals. Most of the 76 outdoor strains showed a wild-type antibiotic resistance phenotype, i.e. resistance to minocycline and trimethoprim–sulfamethoxazole. Sixty percent of hospital strains showed a multiresistance phenotype (from 3 to 16 antibiotics) and confirmed that frequent exposure to antibiotics favored selection and maintenance of antibiotic resistance in P. aeruginosa. Twelve percent of outdoor strains naturally exposed to antiseptics and hydrocarbons showed significant resistance profiles, suggesting that chemical contaminants could contribute to selection of antibiotic resistance. For metal resistance, outdoor strains were more frequently resistant to zinc and cadmium, whereas hospital strains were more frequently resistant to mercury and copper. Differences in metal resistance between the 130 strains investigated were not related to previously characterized processes such as those implicating czcA, involved in cadmium, zinc, and cobalt resistance, or copA and copB, involved in copper resistance. Regulatory or new processes were likely to have contributed to the observed variations. Strains showing strong resistance to antibiotics were the least resistant to metals, and inversely. The lack of significant correlations between antibiotic and metal resistance suggests involvement of distinct processes that are rarely co-selected. The effects of the P. aeruginosa collection size and multi-factorial selective pressure on data sets are discussed.     

Polymorphisms in the Pseudomonas aeruginosa type III secretion protein,PcrV – Implications for anti-PcrV immunotherapy

The type III secretion system of Pseudomonas aeruginosa, responsible for acute infection, is composed of over twenty proteins that facilitate cytotoxin injection directly into host cells. Integral to this process is production and secretion of PcrV. Administration of a recently developed, anti-PcrV immunoglobulin, either as a therapeutic or prophylactic has previously demonstrated efficacy against laboratory strains of P. aeruginosa in a murine model. To determine if this therapy is universally applicable to a variety of P. aeruginosa clinical isolates, genetic heterogeneity of pcrV was analyzed among strains collected from three geographically distinct regions; United States, France and Japan. Sequence analysis of PcrV demonstrated limited variation among the clinical isolates examined. Strains were grouped according to the presence of non-synonymous single nucleotide polymorphisms. Representative isolates from each mutant group were examined for the ability of anti-PcrV to bind the protein secreted by these strains. The protective effect of anti-PcrV IgG against each strain was determined using an epithelial cell line cytotoxicity assay. The majority of strains tested demonstrated reduced cytotoxicity in the presence of anti-PcrV IgG. This study provides insights into the natural sequence variability of PcrV and an initial indication of the amino acid residues that appear to be conserved across strains. It also demonstrates the protective effect of anti-PcrV immunotherapy against a multitude of P. aeruginosa strains from diverse global regions with a variety of mutations in PcrV.     

Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system

We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.     

The Aeromonas dsbA mutation decreased their virulence by triggering type III secretion system but not flagella production

Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Dsb (Disulfide bond) proteins play an important role in catalyzing disulfide bond formation in proteins within the periplasmic space. An A. hydrophila dsbA mutant with attenuated virulence using Dictyostelium amoebae as an alternative host model was identified. The attenuated virulence was tested in other animal models (by intraperitoneal injection in fish and mice) and was correlated with the presence of a defective type III secretion system for the first time in non enteric bacteria. The dsbA mutation was shown in several enteric bacteria to involve the outer membrane secretin. The defect in Aeromonas also seems to involve the outer membrane secretin homologue named AscC. However, unlike what happen in Escherichia coli, no changes in motility or flagella expression were observed for A. hydrophila dsbA mutants. The loss of E. coli motility caused by deletion of dsbA is likely due to defective disulfide bond formation in FlgI, a component of the flagella. No disulfide bond formation in FlgI homologues in Aeromonas flagella biogenesis, either polar or lateral, could be expected according to their amino acid residues sequences.     

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.     

Pseudomonas aeruginosa strains from the chronically infected cystic fibrosis lung display increased invasiveness of A549 epithelial cells over time

The invasive properties of Pseudomonas aeruginosa pose a serious threat to the wellbeing of cystic fibrosis (CF) patients; however the specific factors affecting invasiveness are not well understood, especially in chronic infection. This study characterises the invasive profiles of sequential isolates of the same P. aeruginosa strain collected five to eight years apart from five chronically infected adult CF patients. Strains from three patients were characterised as unique isolates and from two patients as the Australian Epidemic strain (AES-1) by pulsed field gel electrophoresis. The capacity of these strains to invade the human alveolar A549 cell line was examined. Later isolates were significantly more invasive than earlier counterparts from the same patient. Quantitative real-time PCR and Western blotting showed that the increase in invasiveness over time was independent of ExoS expression and secretion. A link between clonality and invasiveness was also identified, with AES-1 isolates more invasive than unique isolates. These results suggest that despite a reduction in some virulence factors such as the Type-3 Secretion System (T3SS) during chronic infection, a particular strain can become more invasive over time. Defining mechanisms behind the increased invasiveness during chronic infection may help identify new therapeutic targets for CF patients.     

Induction of type III secretion by cell-free Chlamydia trachomatis elementary bodies

Chlamydiae secrete type III effector proteins at two distinct stages of their developmental cycle. Elementary bodies (EBs) secrete at least one pre-formed effector protein, Tarp, across the host plasma membrane from an extracellular location. Once internalized, a set of newly transcribed proteins are secreted to modify the inclusion membrane. In an effort to better understand the triggers for chlamydial type III secretion and develop means to identify new effectors, we investigated various inducers of T3SS in other Gram-negative bacterial systems to determine if they were able to activate chlamydial type III secretion from EBs using Tarp secretion as an indicator of activation. Chlamydial EBs are induced to secrete Tarp by exposure to FBS, BSA, or sphingolipid and cholesterol-rich liposomes (SCRLs). The induction by FBS and BSA, but not SCRL, is enhanced in the presence of the calcium-chelator, EGTA. This secretion was temperature dependent and inhibited by paraformaldehyde fixation of the EBs.     

Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila--part I

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.     

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.     

Association of biofilm production with multidrug resistance among clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa from intensive care unit

Background and Aims:

Given choice, bacteria prefer a community-based, surface-bound colony to an individual existence. The inclination for bacteria to become surface bound is so ubiquitous in diverse ecosystems that it suggests a strong survival strategy and selective advantage for surface dwellers over their free-ranging counterparts. Virtually any surface, biotic or abiotic (animal, mineral, or vegetable) is suitable for bacterial colonization and biofilm formation. Thus, a biofilm is “a functional consortium of microorganisms organized within an extensive exopolymeric matrix.”

Materials and Methods:

The present study was undertaken to detect biofilm production from the repertoire stocks of Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa) obtained from clinical specimens. The tube method was performed to qualitatively detect biofilm production.

Results:

A total of 109 isolates of both organisms were included in the study, out of which 42% (46/109) isolates showed biofilm detection. Among the biofilm producers, 57% of P. aeruginosa and 73% of A. baumannii showed multidrug resistance (MDR) pattern which was statistically significant in comparison to nonbiofilm producers (P < 0.001).

Conclusion:

To the best of our knowledge, this is the only study to have tested the biofilm production in both P. aeruginosa and A. baumannii in a single study. Biofilm production and MDR pattern were found to be significantly higher in A. baumannii than P. aeruginosa. Antibiotic resistance was significantly higher among biofilm producing P. aeruginosa than non producers. Similarly, antibiotic resistance was significantly higher among biofilm producing A. baumannii than non producers.     

Prevalence of type III secretion system in effective biocontrol pseudomonads

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN⁺ biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN⁻ biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems.     

Conserved type III secretion system exerts important roles in Chlamydia trachomatis

Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis.     

Identification and specificity of pilus adsorption proteins of filamentous bacteriophages infecting Pseudomonas aeruginosa

Filamentous bacteriophages Pf1 and Pf3 infect Pseudomonas aeruginosa strains K and O, respectively. We show here that the capsids of these bacteriophages each contain a few copies of a minor coat protein (designated g3p) of high molecular mass, which serves as a pilus adsorption protein, much like the protein g3p of the Ff bacteriophages which infect Escherichia coli. Bacteriophage Pf1 was observed to interact with the type IV PAK pilus whereas bacteriophage Pf3 interacted with the conjugative RP4 pilus and not with the type IV PAO pilus. The specificity was found to be mediated by their pilus-binding proteins. This is evidence of a conserved pathway of infection among different classes of filamentous bacteriophage. However, there are likely to be subtle differences yet to be discovered in the way these virions effect entry into their targeted bacterial cells.