Shift in ribonucleotide reductase gene expression in Pseudomonas aeruginosa during infection.
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ABSTRACT:
SUBMITTER: Sjoberg BM
PROVIDER: S-EPMC3191977 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an H2O2 addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (nrdJ). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. IMPORTANCE The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an H2O2 addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.
| S-EPMC10134789 | biostudies-literature
Project description:Ribonucleotide reductases (RNRs) are a family of sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides (dNTPs), the building blocks for DNA synthesis and repair. Although any living cell must contain one RNR activity to continue living, bacteria have the capacity to encode different RNR classes in the same genome, allowing them to adapt to different environments and growing conditions. Pseudomonas aeruginosa is well known for its adaptability and surprisingly encodes all three known RNR classes (Ia, II and III). There must be a complex transcriptional regulation network behind this RNR activity, dictating which RNR class will be expressed according to specific growing conditions. In this work, we aim to uncover the role of the transcriptional regulator NrdR in P. aeruginosa. We demonstrate that NrdR regulates all three RNR classes, being involved in differential control depending on whether the growth conditions are aerobic or anaerobic. Moreover, we also identify for the first time that NrdR is not only involved in controlling RNR expression but also regulates topoisomerase I (topA) transcription. Finally, to obtain the entire picture of NrdR regulon, we performed a global transcriptomic analysis comparing the transcription profile of wild-type and nrdR mutant strains. The results provide many new data about the regulatory network that controls P. aeruginosa RNR transcription, bringing us a step closer to the understanding of this complex system.
| S-EPMC4409342 | biostudies-literature
Project description:The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related ?-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb)2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth.
| S-EPMC4520616 | biostudies-literature
Project description:Pseudomonas aeruginosa strain PAO1 has become the reference strain in many laboratories. One enzyme that is essential for its cell division is the ribonucleotide reductase (RNR) enzyme that supplies the deoxynucleotides required for DNA synthesis and repair. P. aeruginosa is one of the few microorganisms that encodes three different RNR classes (Ia, II and III) in its genome, enabling it to grow and adapt to diverse environmental conditions, including during infection. In this work, we demonstrate that a lack of RNR activity induces cell elongation in P. aeruginosa PAO1. Moreover, RNR gene expression during anaerobiosis differs among P. aeruginosa strains, with class III highly expressed in P. aeruginosa clinical isolates relative to the laboratory P. aeruginosa PAO1 strain. A single point mutation was identified in the P. aeruginosa PAO1 strain class III RNR promoter region that disrupts its anaerobic transcription by the Dnr regulator. An engineered strain that induces the class III RNR expression allows P. aeruginosa PAO1 anaerobic growth and increases its virulence to resemble that of clinical strains. Our results demonstrate that P. aeruginosa PAO1 is adapted to laboratory conditions and is not the best reference strain for anaerobic or infection studies.
| S-EPMC5645315 | biostudies-literature
Project description:Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium's primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.
| S-EPMC5984494 | biostudies-literature
Project description:Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.
| S-EPMC3174184 | biostudies-literature
Project description:BackgroundThe bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility.ResultsMicroarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14). Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center). Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses.ConclusionsResults reported in this study show that, as opposed to swarm center cells, tendril tip populations of a swarming colony displays general down-regulation of genes associated with virulence and up-regulation of genes involved in energy metabolism. These results allow us to propose a model where tendril tip cells function as «scouts» whose main purpose is to rapidly spread on uncolonized surfaces while swarm center population are in a state allowing a permanent settlement of the colonized area (biofilm-like).
| S-EPMC3091734 | biostudies-literature
Project description:Most infections result from colonization by more than one microbe. Within such polymicrobial infections, microbes often display synergistic interactions that result in increased disease severity. Although many clinical studies have documented the occurrence of synergy in polymicrobial infections, little is known about the underlying molecular mechanisms. A prominent pathogen in many polymicrobial infections is Pseudomonas aeruginosa, a Gram-negative bacterium that displays enhanced virulence during coculture with Gram-positive bacteria. In this study we discovered that during coinfection, P. aeruginosa uses peptidoglycan shed by Gram-positive bacteria as a cue to stimulate production of multiple extracellular factors that possess lytic activity against prokaryotic and eukaryotic cells. Consequently, P. aeruginosa displays enhanced virulence in a Drosophila model of infection when cocultured with Gram-positive bacteria. Inactivation of a gene (PA0601) required for peptidoglycan sensing mitigated this phenotype. Using Drosophila and murine models of infection, we also show that peptidoglycan sensing results in P. aeruginosa-mediated reduction in the Gram-positive flora in the infection site. Our data suggest that P. aeruginosa has evolved a mechanism to survey the microbial community and respond to Gram-positive produced peptidoglycan through production of antimicrobials and toxins that not only modify the composition of the community but also enhance host killing. Additionally, our results suggest that therapeutic strategies targeting Gram-positive bacteria might be a viable approach for reducing the severity of P. aeruginosa polymicrobial infections.
| S-EPMC3549110 | biostudies-literature
Project description:Insights into the host factors and mechanisms mediating the primary host responses after pathogen presentation remain limited, due in part to the complexity and genetic intractability of host systems. Here, we employ the model Drosophila melanogaster to dissect and identify early host responses that function in the initiation and progression of Pseudomonas aeruginosa pathogenesis. First, we use immune potentiation and genetic studies to demonstrate that flies mount a heightened defense against the highly virulent P. aeruginosa strain PA14 when first inoculated with strain CF5, which is avirulent in flies; this effect is mediated via the Imd and Toll signaling pathways. Second, we use whole-genome expression profiling to assess and compare the Drosophila early defense responses triggered by the PA14 vs. CF5 strains to identify genes whose expression patterns are different in susceptible vs. resistant host-pathogen interactions, respectively. Our results identify pathogenesis- and defense-specific genes and uncover a previously undescribed mechanism used by P. aeruginosa in the initial stages of its host interaction: suppression of Drosophila defense responses by limiting antimicrobial peptide gene expression. These results provide insights into the genetic factors that mediate or restrict pathogenesis during the early stages of the bacterial-host interaction to advance our understanding of P. aeruginosa-human infections.
| S-EPMC549001 | biostudies-literature
Project description:Autophagy, a conserved pathway that delivers intracellular materials into lysosomes for degradation, is involved in development, aging, and a variety of diseases. Accumulating evidence demonstrates that autophagy plays a protective role against infectious diseases by diminishing intracellular pathogens, including bacteria, viruses, and parasites. However, the mechanism by which autophagy regulates innate immunity remains largely unknown. Here, we show that autophagy is involved in host defense against a pathogenic bacterium Pseudomonas aeruginosa in the metazoan Caenorhabditis elegans. P. aeruginosa infection induces autophagy via a conserved extracellular signal-regulated kinase (ERK). Intriguingly, impairment of autophagy does not influence the intestinal accumulation of P. aeruginosa, but instead induces intestinal necrosis. Inhibition of necrosis results in the survival of autophagy-deficient worms after P. aeruginosa infection. These findings reveal a previously unidentified role for autophagy in protection against necrosis triggered by pathogenic bacteria in C. elegans and implicate that such a function of autophagy may be conserved through the inflammatory response in diverse organisms.
| S-EPMC4151725 | biostudies-literature