Periodic Reporting for period 1 - T6SS-PSEUDO-LIP (Type VI-dependent Pseudomonas aeruginosa phospholipases and host manipulation) Período documentado: 2016-02-01 hasta 2018-01-31 Resumen del contexto y de los objetivos generales del proyecto Pseudomonas aeruginosa is an extracellular Gram-negative bacterium able to cause healthcare associated infections, including pneumonia, urinary tract infections, wound infections, acute otitis and septicaemia. It is a major cause of chronic infection in patients with cystic fibrosis and on the World Health Organization list of antibiotic resistant priority pathogens. Like many other Gram-negative bacteria, P. aeruginosa manipulates eukaryotic host cells using secreted effectors and notably the type III secretion system (T3SS)-dependent exoenzymes ExoS, ExoT, ExoY and ExoU. More recently the type VI secretion system (T6SS) was shown to secrete anti-bacterial toxins and thus help outcompeting and manipulating the resident microbiota in the host. However, it has also been demonstrated that T6SS-effectors, such as PldA and PldB phospholipases, are injected into eukaryotic host cells and promote bacterial internalization or activation of several essential eukaryotic processes, such as triggering of the mitogen-activated protein kinase (MAPK) pathway or autophagy. P. aeruginosa T3SS and T6SS could thus be seen as effector-injecting nanomachines manipulating the host for bacterial benefit.The aims of this project were to study the expression of PldA and PldB in clinical isolates of P. aeruginosa (WP1), to decipher the role of PldA and PldB as virulence factor during in vivo infection using a murine model of respiratory tract infection (WP3), to analyze if PldA might play a major role in the H2-T6SS mediated entry of P. aeruginosa in non-phagocytic cells (WP4) and studied downstream signalling events that might occur upon Pld-dependent P. aeruginosa entry in non-phagocytic cells (WP5). Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha WP1: The aim was to study the expression of PldA and PldB in clinical isolates of P. aeruginosa.Task 1: The prevalence of pldA and pldB in clinical isolates representative of chronic respiratory infections in cystic fibrosis patients (n=49) and for other clinically relevant infections (32 urinary tract infections, 46 septicemia and 58 acute respiratory infections in non-CF patients) and for environmental isolates (n=47) was determined by PCR.Task 2:The expression of pldA and pldB was screened by qRT-PCR. Using isolates positive for pldA and/or pldB, qRT-PCR was performed to assess the relative expression of these two genes.Task 3: A collection of P. aeruginosa isolates (n=50) possessing a wide range of antibiotic susceptibility phenotype, including the most relevant worldwide disseminated multidrug resistant clones (was tested for pldA and pldB expression by PCR.Task 4: Since IMP-13-producing P. aeruginosa isolates were demonstrated to be responsible for outbreaks of urinary tract infections, we tested by qRT-PCR the impact of urine on pldA and pldB expression in IMP-13-producing P. aeruginosa isolate.WP2: ΔpldA, ΔpldB, ΔpldAΔpldB, ΔpldA + pldA, ΔpldB + pldB, ΔpldAΔpldB + pldA, ΔpldAΔpldB + pldB isolates were generated in a PAO1 background by allelic replacement.WP3: This WP was not performed since the published data on which the hypothesis was based could not be validated (see below).WP4: The goal was to demonstrate that PldA plays a major role in the H2-T6SS mediated entry of P. aeruginosa in non-phagocytic cells. Invasion assays (=gentamicin protection assay) in 2 types of non-phagocytic cells (HeLa and A459 cells). No difference in term of entry in these two cell lines was observed when comparing WT and mutants.WP5: The goal was to study the upstream (PI3K pathway) and downstream (actin recruitment, host chromatin modifications) signalling events occurring upon Pld-dependent P. aeruginosa entry in non-phagocytic cells.After few weeks, preliminary results obtained from the WP5 Task 3 were the most promising. Accordingly, we investigated in details how Pseudomonas aeruginosa was able to induce histone modification upon infection, with the hypothesis that these epigenetic modifications might result from the type VI secretion system (T6SS).Accordingly, specific new objectives were established :To investigate whether P. aeruginosa (three different stains) is able to induce histone modifications. We looked at phosphorylation of serine 10 of histone H3 (PSer10H), global acetylation of histone H3 (AcH3), trimethylation of lysine 9 of histone H3 (triMetLys9H3), and global acetylation of histone H4 (AcH4). Mutant in T3SS and T6SS were then used to confirm the role of T3SS in the dephosphorylation of H3. All individual exo mutants, i.e. ΔexoS, ΔexoT, ΔexoY, as well as a mutant lacking all three T3SS effector genes, ΔexoSTY were tested for their ability to induce dephosphorylation of H3. We demonstrated that T3SS-dependent Ser10H3 dephosphorylation was not associated with any of the known P. aeruginosa T3SS effectors, we tested confirmed that PopB-PopD translocon, that forms a pore in the host cell membrane, could trigger histone modifications. We demonstrated using flow-cytometry that PopB-PopD-dependent K+ efflux occurs upon infection and is responsible for Ser10H3 dephosphorylationand confirm that PopB-PopD translocon stay anchor in the membrane of infected cells using immunofluorescence microscopy.To further assess the role of PopB-PopD-dependent P Ser10H3 during the infectious process, we performed in vivo infection using Galleria mellonella. Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) Analysis of the prevalence and expression of PldA and PldB phospholipases in relevant clinical isolates of P. aeruginosa, led to establish that pldA is more prevalent in strains responsible for pulmonary infections compared to those responsible for urinary tract infection, septicaemia and environmental isolates (Task 1). In addition, we demonstrated that pldA is more prevalent in multidrug resistant isolates, particularly in IMP-type carbapenemase producing P. aeruginosa. We also demonstrated that in a worldwide disseminated multidrug resistant clones (IMP-13 producing P. aeruginosa of ST-621) pldA and pldB are overexpressed in stationary phase (Task 2) and in presence of biological fluids (here urine) (Task 3). The WP1 was performed by a Master student that I (Laurent Dortet) supervised. Regarding this WP1, a manuscript is under preparation (Thibaud Boulant, Yves-Marie Boudehen, Thierry Naas, Alain Filloux and Laurent Dortet. Increased prevalence of PldA, a trans-kingdom H2-T6SS effector involved in bacterial warfare and host cell interaction, in multidrug resistant Pseudomonas aeruginosa. Int J Antimicrobial Agents, in preparation.)The most promising results were obtained from the objectives presented in WP5 where we check if Pseudomonas aeruginosa was able to induce histone modification upon infection, with the hypothesis that these epigenetic modifications might result from the activity of the type VI secretion system (T6SS). Accordingly, this part of the project was investigated in further depth and resulted in a publication in Nature Microbiology in February 2018 (Dortet. L., Lombardi, C., Cretin, F., Dessen, A. and Filloux, A., 2018. Pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon alters the host epigenome. Nat Microbiol., 3(3):378-386). extended-figure-s11.jpg
