Pseudomonas aeruginosa is capable of natural transformation in biofilms

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience

Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites

The malarial carbamoyl phosphate synthetase II gene as a target for DNAzyme therapy

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels.The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present.Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial.Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake intoparasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels

Characterisation of a cysteine protease expressed by Eimeria tenella and identification of its post-traductionnal regulator

Session Poster : Invasion and motilityCysteine proteases of the papain family are major virulent factors expressed by protozoa. They have been involved in many steps of parasites life cycle like cell invasion, intracellular replication, gametocyte formation and parasite differentiation. Their multiple roles in key steps of parasites biology make them attractive new therapeutic targets. Using BlastP, we identified 5 genes encoding for cysteine proteases in the genome of E. tenella. We named them Eimeripain, EtCPL, EtCPC1, EtCPC2 and EtCPC3 encoding respectively for one cathepsin B, one cathepsin L, and three cathepsin C. Complementary approaches of molecular biology and biochemistry revealed that most of these proteases are highly expressed and active in the unsporulated oocysts, suggesting a role in sporulation and/or gametogenesis. Eimeripain is the only activity that persists throughout the life cycle. We show that a specific inhibitor of Human cathepsin B, CA074-ME, inhibits Eimeripain and affects the capacity of sporozoites to invade MDBK cells. These data suggest that Eimeripain plays a central and pleiotropic role in Eimeria life cycle. Cysteine protease inhibitors from the Chagasin family are proteins expressed by protozoa that specifically bind to and inhibit cysteine cathepsins. As such, they participate to parasite pathogenesis. We identified a cysteine protease inhibitor, Eimestatine, expressed by E. tenella, which specifically inhibits the activity of Eimeripain in biochemical assays. Preliminary data suggest that Eimestatine forms a complex at each life stage, which may indicate a tight regulation of Eimeripain throughout the infectious process

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X₇ receptor-deficient mice

The P2X₇ receptor (P2X₇R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X₇R gene knockout (P2X₇R^−/−; on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X₇R^−/− mice lost significantly more weight than C57Bl/6J mice from day 8 p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X₇R^−/− mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X₇R^−/− mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X₇R^−/− mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X₇ receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X₇R^−/− and C57Bl/6J mice produced more IL-1β and TNF than BALB/c mice. There was one important point of differentiation between the P2X₇R^−/− and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X₇R-deficient mice

Biochemical activities of CPs throughout sporulation.

<p>(A) Activities detected on Z-FR-AMC. Lysates of oocysts (1 mg/ml) taken at 0, 6, 12, 24 and 48 h after the beginning of sporulation were incubated with the substrate Z-FR-AMC (10 µM). The morphology of oocysts (under light microscopy) throughout the course of sporulation is shown. The scale bar represents 2 µm. (B) Activities detected on Z-FR-AMC and Z-LR-AMC in presence of the global cysteine protease inhibitor E-64 or the human cathepsin B specific inhibitor CA-074. Lysates (1 mg/ml) of oocysts taken at 0 h (black bars) and 48 h (white bars) after commencement of sporulation were pre-incubated with the inhibitors before adding the substrates. The data represents two independent experiments.</p

Effect of CA-074 Me on MDBK cell invasion by <i>E. tenella</i> sporozoites.

<p>Purified sporozoites were incubated with 10 to 100 µM of CA-074ME, a specific cathepsin B inhibitor permeable to membranes or with 1% DMSO. Sporozoites were then washed and incubated with MDBK cells. Infected cells were fixed and intracellular parasites were detected by IF using specific sera against sporozoites, and counted. The data represent three independent experiments. * denotes significant differences at p<0.05 in the capacity of CA-074 Me- treated parasites to invade cells compared to the control.</p