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CRISPR-Cas9 mutagenesis of A549 cells for variants resistant to the ExoU toxin from Pseudomonas aeruginosa

ABSTRACT: Pseudomonas aeruginosa is a leading cause of nosocomial infection and is responsible for severe acute and chronic infections such as pneumonia, urinary tract infections and lung infections in cystic fibrosis patients. The bacterium possesses a type 3 secretion system whose expression is linked with poor outcomes in both animal models and human patients. In particular, this syringe-like apparatus injects ExoU, the most toxic T3SS effector. However, the mechanism of ExoU in P. aeruginosa’s virulence remains elusive compare to the other T3SS toxins. It was previously suggested that the toxin uses a host trafficking system to reach the plasma membrane and to induce cell necrosis. In this work, we demonstrated that once injected into the cell cytoplasm, ExoU binds to DNAJC5+ vesicles to rapidly reach the plasma membrane.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154751 | GEO | 2020/12/01

REPOSITORIES: GEO

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Quantitative proteomic analysis of Burkholderia pseudomallei Bsa Type III secretion system effectors using hyper-secreting mutants

Project description:Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. While the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterised. We constructed a mutant lacking bsaP, a homologue of the T3SS gatekeeper family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hyper-secrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

2015-01-28 | PXD001656 | Pride

A ParDE toxin-antitoxin system is responsible for maintenance of the Yersinia virulence plasmid, but not for type III secretion-associated growth inhibition

Project description:Many Gram-negative pathogens utilize the type III secretion system (T3SS) to translocate virulence-promoting effector proteins into eukaryotic host cells. Activity of this system results in severe reduction of bacterial growth and division, summarized as secretion-associated growth inhibition (SAGI). In Y. enterocolitica, the T3SS and related proteins are encoded on a virulence plasmid. We identified a ParDE-like toxin-antitoxin system on this virulence plasmid in genetic proximity to yopE, encoding a T3SS effector. Effectors are strongly upregulated upon activation of the T3SS, indicating a potential role of the ParDE system in the SAGI or maintenance of the virulence plasmid. Expression of the toxin ParE in trans resulted in reduced growth and elongated bacteria, highly reminiscent of the SAGI. Nevertheless, activity of ParDE is not causal for the SAGI. T3SS activation did not influence ParDE activity; conversely, ParDE had no impact on T3SS assembly or activity itself. However, we found that ParDE ensures presence of the T3SS across bacterial populations by reducing loss of the virulence plasmid, especially under conditions relevant for infection. Despite this effect, a subset of bacteria lost the virulence plasmid and regained the ability to divide under secreting conditions, facilitating the possible emergence of T3SS-negative bacteria in late acute and persistent infections.

2023-06-09 | PXD040518 | Pride

In vivo protein interaction network analysis reveals porin-localized toxin inactivation in antibiotic resistant bacteria

Project description:The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital acquired infections worldwide, and a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. To gain insight on A. baumannii antibiotic resistance mechanisms, we analyzed the protein interaction network of a multidrug-resistant A. baumannii clinical strain Ab5075. Using in vivo chemical cross-linking and mass spectrometry, we identified 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter- molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 were identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO were verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrated changes in meropenem or imipenem sensitivity in Ab5075. These results provide the first view of a porin-localized toxin inactivation model and increase understanding of bacterial antibiotic resistance mechanisms.

2016-12-22 | PXD004236 | Pride

CRISPR-Cas9 mutagenesis of U937 cells for variants resistant to Yersinia pestis intoxication

Project description:The plague agent, Yersinia pestis, employs a type III secretion system (T3SS) to selectively destroy human immune cells, thereby enabling its replication in the bloodstream and transmission to new hosts via fleabite. The host factors responsible for the selective destruction of immune cells by plague bacteria were not known. Here we show that LcrV, the needle cap protein of the Y. pestis T3SS, binds N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors.

2019-07-04 | GSE133826 | GEO

Toxin mediates sepsis caused by methicillin-resistant Staphylococcus epidermidis

Project description:Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species Staphylococcus epidermidis are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused my methicillin-resistant S. epidermidis is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. While toxins have never been clearly indicated in CNS infections, our study shows that an important type of infection caused by the predominant CNS species, S. epidermidis, is mediated to a large extent by a toxin. Of note, these findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches. We used microarrays to detail the global gene expression between S. epidermidis strain Rp62A and S. epidermidis strain Rp62A isogenic Δpsm-mec deletion mutants

2017-01-06 | GSE85265 | GEO

Project description:Nosocomial infections in a veterinary hospital

| PRJNA784350 | ENA

Plasma Membrane Integrity Signaling

Project description:Plasma Membrane Integrity Signaling

| PRJNA701242 | ENA

Lodderomyces elongisporus

Project description:A yeast linked to nosocomial bloodstream infections

| PRJNA12822 | ENA

RNA-seq for PA4595

Project description:Pseudomonas aeruginosa is an important human pathogen which uses the type III secretion system 21 (T3SS) as a primary virulence factor to establish infections in humans. The results presented in this 22 report revealed that the ATP-binding protein PA4595 (named ArtR, a Regulator that is an ATP-activated 23 Repressor of T3SS) represses T3SS expression in P. aeruginosa. The expression of T3SS genes, 24 including exoS, exoY, exoT, exsCEBA and exsD-pscB-L, increased significantly when artR was 25 knockout. The effect of ArtR on ExsA is at the transcriptional level, not at the translational level. The 26 regulatory role and cytoplasm localization of ArtR suggest it belongs to the REG sub-family of 27 ATP-binding cassette (ABC) family. Purified GST-tagged ArtR showed ATPase activity in vitro. The 28 conserved aspartate residues in the dual Walker B motifs prove to be essential for the regulatory 29 function of ArtR. The regulation of T3SS by ArtR is unique, which does not involve the known 30 GacS/A-RsmY/Z-RsmA-ExsA pathway or Vfr. This is the first REG subfamily of ATP-binding 31 cassette that is reported to regulate T3SS genes in bacteria. The results specify a novel player in the 32 regulatory networks of T3SS in P. aeruginosa.

2019-03-04 | GSE127751 | GEO

Increase in membrane cholesterol of neurons in culture recapitulates AlzheimerM-bM-^@M-^Ys disease early phenotypes.

Project description:Levels of membrane-associated cholesterol were shown to be increased in the brain of individuals with sporadic AlzheimerM-bM-^@M-^Ys disease (AD) and correlated with the severity of the disease. We previously found that heavy membrane cholesterol burden promotes amyloid precursor protein (APP) endocytosis and processing, leading to increased amyloid-M-oM-^AM-"M-oM-^@M- M-oM-^@M-(AM-oM-^AM-") secretion. We hypothesized that such an increase of cholesterol could trigger sporadic AD. We thus acutely loaded the plasma membrane of neurons in culture with cholesterol to reach the 30 % increase observed in AD brains. We showed by multiplex electro-chemiluminescence immuno-assay that transient membrane cholesterol loading produced a significant increase of AM-oM-^AM-"42 secretion. We also found that early endosomes were enlarged and more prone to aggregation using confocal and electron microscopy and that APP vesicular transport in neuronal processes was slowed down using fluorescence live-imaging. In addition, treatment of neurons with cholesterol induced changes in gene expression profile that are reminiscent of early AD. This model of membrane cholesterol increase in cultured neurons reproduces most of early AD changes and could thus be relevant for deciphering early mechanisms and design new targets for sporadic AD. In this study, we loaded the plasma membrane of neurons with 30% more cholesterol and observed the effects on gene expression. We compared gene expression of primary hippocampal neurons treated or not with cholesterol using 4 independant replicates in each group.

2015-01-15 | E-GEOD-46221 | biostudies-arrayexpress

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