ClpV3 affects pathogenicity in Pseudomonas aeruginosa
Ontology highlight
ABSTRACT:
ORGANISM(S): Pseudomonas aeruginosa PAO1
PROVIDER: GSE148116 | GEO | 2020/04/20
REPOSITORIES: GEO
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Project description:Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and compete with microbial competitors. These T6SS machines can be loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the three main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control of the regulator RsmA in repressing most known T6SS-related genes. Moreover, it is noticeable that a sigma factor activator (SFA) protein is encoded in each of H2- and H3-T6SS clusters, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins that usually work in concert with the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of all H2-T6SS-linked VgrG and effector arsenal to enable very effective interbacterial killing. Such RpoN-dependent control is exerted both directly and indirectly. The function of the SFA protein is specific since the H3-T6SS associated Sfa3 cannot complement loss of Sfa2. Our study further delineates the multiple regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely to respond and adapt to a range of environmental conditions that a versatile organism like P. aeruginosa would encounter.
2022-02-23 | GSE185398 | GEO
Project description:In order to determine the effect of ALA on geneexpression, P. aeruginosa PAO1 was incubated with and without 30 μM ALA under T3SS-inducing conditions (calcium depletion by addition of NTA to growth medium), followed by RNA extraction and microarray analysis.
2020-08-06 | GSE155769 | GEO
Project description:Melioidosis is a disease caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm), commonly found in soil and water of endemic areas. Naturally acquired human melioidosis infections can result from either exposure through percutaneous inoculation, inhalation, or ingestion of soil-contaminated food or water. Our prior studies recognized Bpm as an effective enteric pathogen, capable of establishing acute or chronic gastrointestinal infections following oral inoculation (Sanchez-Villamil, Tapia et al. 2020). However, the specific mechanisms and virulence factors involved in the pathogenesis of Bpm during intestinal infection are unknown. In our current study, we standardized an in vitro intestinal infection model using mouse primary intestinal epithelial cells (mIECs) and demonstrated that Bpm requires a functional T6SS for full virulence. Further, we performed dual RNA-seq analysis on Bpm-infected mIECs to evaluate differentially expressed host and bacterial genes in the presence or absence of a T6SS. Our results showed a dysregulation in the TNF- signaling via NF-B pathway in the absence of the T6SS, with some of the genes involved in inflammatory processes and cell death also affected. Analysis of the bacterial transcriptome identified virulence factors and regulatory proteins playing a role during infection, with association to the T6SS. By using a Bpm transposon mutant library and isogenic mutants, we showed that deletion of the bicA gene, encoding a putative T3SS/T6SS regulator, ablated intracellular survival and plaque formation by Bpm and impacted survival and virulence when using murine models of acute and chronic gastrointestinal infection. Overall, these results highlight the importance of the type 6 secretion system in the gastrointestinal pathogenesis of Bpm.
2022-08-15 | GSE201263 | GEO
Project description:Secretion systems are used as weapons by a variety of Gram-negative bacteria. Among them the Type VI Secretion System (T6SS) gained more interest throughout the last years. The system functions as a molecular nano-weapon: it is used in inter-kingdom competition by various bacteria to deliver toxic effectors in target cells. Here we describe the role of the T6SS in Photorhabdus laumondii subsp. laumondii strain DJC, an entomopathogenic biocontrol agent able to live in different environmental niches, such as in symbiosis with nematodes and in the rhizosphere on plant roots. Using bioinformatic and protein motif analyses we identified four T6SS gene clusters (T6SS-1, T6SS-2, T6SS-3 and T6SS-4) and multiple orphan T6SS related genes in the genome of P. laumondii. Furthermore, we highlighted 11 T6SS effector-immunity pairs, including three undescribed membrane disrupting effectors, each with putatively different antibacterial activities. By label-free mass spectrometry of P. laumondii wild type cells and respective T6SS-deficient strains, we could point out a cross-link between T6SS and other Photorhabdus’ virulence related mechanisms such as PVCs, T3SS and pyocins. Furthermore, a change in motility as well as in the secondary metabolism was observed upon T6SS-deficiency. Here, we shed light on the T6SS in P. laumondii DJC and suggesting a cross-link of various virulence mechanisms, which could help to gain knowledge on T6SS and better figure out the Photorhabdus ability to live in polymicrobial environments.
2024-04-05 | PXD035720 | Pride
Project description:Regulatory networks including virulence-related transcriptional factors (TFs) determine bacterial pathogenicity in response to different environmental cues. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen of humans, recruits numerous TFs in quorum sensing (QS) system, type III secretion system (T3SS) and Type VI secretion system (T6SS) to mediate the pathogenicity. Although many virulence-related TFs have been illustrated individually, very little is known about their crosstalks and regulatory network. Here, based on chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and transcriptome profiling (RNA-seq), we primarily focused on understanding the crosstalks of 20 virulence-related TFs, which led to construction of a virulence regulatory network named PAGnet (Pseudomonas aeruginosa Genomic integrated regulatory network), including 82 crosstalk targets. The PAGnet uncovered the intricate mechanism of virulence regulation and revealed master regulators in QS, T3SS and T6SS pathways. In particular, GacA and ExsA showed novel functions in QS and nitrogen metabolism. In addition, an online PAGnet platform was provided to analyze these TFs and more virulence factors. Taken together, the present study revealed the function-specific crosstalks of virulence regulatory network, which might provide new strategies for treating infections in P. aeruginosa in the future.
2019-07-16 | GSE128430 | GEO
Project description:PAO1 and FRD1 were cultured planktonically and in biofilms with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined for PAO1 cultured planktonically, for FRD1 cultured planktonically, for PAO1 cutured in biofilms, and for FRD1 cultured in biofilms. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74445 | GEO
Project description:Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates (19 of the 23 detected effectors and accessory harpin proteins), whereas a distinct subgroup of four effectors is exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins with a highly similar secretion pattern was exclusively secreted in medium and high virulence strains. Taken together, our data shows that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicates distinct strategies for establishing virulence of P. syringae pv. aptata in plants.
2023-06-02 | PXD040715 | Pride
Project description:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, most notably those with cystic fibrosis or whose immune systems are compromised. As a global regulator, RpoN has been characterized to control a group of virulence-related factors and quorum sensing (QS) genes in P. aeruginosa, but its detailed molecular regulatory mechanism is largely elusive. To further gain insights into the direct targets of RpoN in vivo, this study focused on identifying the potential targets of RpoN directly regulating QS system and T6SS. We performed a chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) assay that identified 1, 068 binding sites of RpoN, including mostly metabolic genes, a group of genes in QS (lasI, rhlI and pqsR) and type VI secretion system (T6SS) (hcpA and hcpB). The direct regulation of these genes by RpoN has been biochemically and genetically verified. Results revealed that, on one hand, the deletion of rpoN resulted in reduced production of pyocyanin, motility and proteolytic activity. On the other hand, the production of rhamnolipids and biofilm formation was higher in the RpoN mutant than in the wild-type. In sum, the results indicated that RpoN had direct and profound effects on QS and T6SS, which provides new cues to better understand the detailed regulatory networks of virulence.
2018-06-30 | GSE110445 | GEO
Project description:The opportunistic pathogen Pseudomonas aeruginosa controls the production of virulence factors via its quorum sensing (QS) systems. The addition of 100 M 4-GB to succinate-containing medium led to a strong reduction of pyocyanin production in strain PAO1gbuA, whereas pyocyanin production by the wildtype was not affected. To investigate possible transcriptional differences between the two strains RNAseq analysis of strains PAO1 and PAO1gbuA in the presence of 4-GB was performed. RNA was extracted in late exponential phase. For the isolation of RNA for RNAseq analysis, pre-cultures of strains PAO1 and PAO1gbuA were grown in medium B containing 20 mM succinate for 12 h at 37C. Cells were washed and used to inoculate main cultures in medium B containing 20 mM succinate and 100 M 4-GB in triplicates with an OD600 of 0.001. After incubation for 11 h at 37C, the respective triplicates were combined, harvested by centrifugation and resuspended in medium B without complementing solutions. From these cell suspensions, RNA was extracted with the innuPREP RNA Minikit from Analytik Jena (Jena, Germany) according to the manufacturers instructions. Residual DNA was removed by digestion with 10 U RNase-free DNase I (Thermo scientific) for 1 h in the presence of RiboLock RNase inhibitor (Thermo scientific). After DNA digestion, the RNA was again purified with the same kit. RNA quality was checked by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Boblingen, Germany). Ribosomal RNA molecules were removed from total RNA with the Ribo-Zero rRNA Removal Kit (Illumina, San Diega, USA) and removal of rRNA was checked with the Agilent RNA Pico 6000 Kit on an Agilent 2100 Bioanalyzer. cDNA libraries were prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA), and the resulting cDNA was sequenced paired end on an Illumina MiSeq system using 75 bp read length.
2016-06-08 | E-MTAB-4777 | biostudies-arrayexpress
Project description:PAO1 was cultured planktonically to stationary phase with 10 mM calcium and no added calcium. The transcriptional response to calcium addition was determined. Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa strains PAO1 and FRD1 to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 and the inner membrane-anchored five-bladed -propeller protein, PA0327. Mutations in both PA0320 and PA0327 affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in PA0327 had a pleotrophic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin.
2015-12-02 | GSE74444 | GEO