An easy method for quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins.

Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided.

ReViTA: A novel in vitro transcription system to study gene regulation.

ReViTA (Reverse in VitroTranscription Assay) is a novel in vitro transcription-based method to study gene expression under the regulation of specific transcription factors. The ReViTA system uses a plasmid with a control sequence, the promoter region of the studied gene, the transcription factor of interest, and an RNA polymerase saturated with σ70. The main objective of this study was to evaluate the method; thus, as a proof of concept, two different transcription factors were used, a transcriptional inducer, AlgR, and a repressor, LexA, from Pseudomonas aeruginosa. After the promoters were incubated with the transcription factors, the plasmid was transcribed into RNA and reverse transcribed to cDNA. Gene expression was measured using qRTPCR. Using the ReViTA plasmid, transcription induction of 55% was observed when AlgR protein was added and a 27% transcription reduction with the repressor LexA, compared with the samples without transcription factors. The results demonstrated the correct functioning of ReViTA as a novel method to study transcription factors and gene expression. Thus, ReViTA could be a rapid and accessible in vitro method to evaluate genes and regulators of various species.

Pseudomonas aeruginosa Nonphosphorylated AlgR Induces Ribonucleotide Reductase Expression under Oxidative Stress Infectious Conditions.

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.

Novel Alligator Cathelicidin As-CATH8 Demonstrates Anti-Infective Activity against Clinically Relevant and Crocodylian Bacterial Pathogens.

Host defense peptides (HDPs) represent an alternative way to address the emergence of antibiotic resistance. Crocodylians are interesting species for the study of these molecules because of their potent immune system, which confers high resistance to infection. Profile hidden Markov models were used to screen the genomes of four crocodylian species for encoded cathelicidins and eighteen novel sequences were identified. Synthetic cathelicidins showed broad spectrum antimicrobial and antibiofilm activity against several clinically important antibiotic-resistant bacteria. In particular, the As-CATH8 cathelicidin showed potent in vitro activity profiles similar to the last-resort antibiotics vancomycin and polymyxin B. In addition, As-CATH8 demonstrated rapid killing of planktonic and biofilm cells, which correlated with its ability to cause cytoplasmic membrane depolarization and permeabilization as well as binding to DNA. As-CATH8 displayed greater antibiofilm activity than the human cathelicidin LL-37 against methicillin-resistant Staphylococcus aureus in a human organoid model of biofilm skin infection. Furthermore, As-CATH8 demonstrated strong antibacterial effects in a murine abscess model of high-density bacterial infections against clinical isolates of S. aureus and Acinetobacter baumannii, two of the most common bacterial species causing skin infections globally. Overall, this work expands the repertoire of cathelicidin peptides known in crocodylians, including one with considerable therapeutic promise for treating common skin infections.

BosR: A novel biofilm-specific regulator in Pseudomonas aeruginosa.

Biofilms are the most common cause of bacterial infections in humans and notoriously hard to treat due to their ability to withstand antibiotics and host immune defenses. To overcome the current lack of effective antibiofilm therapies and guide future design, the identification of novel biofilm-specific gene targets is crucial. In this regard, transcriptional regulators have been proposed as promising targets for antimicrobial drug design. Therefore, a Transposon insertion sequencing approach was employed to systematically identify regulators phenotypically affecting biofilm growth in Pseudomonas aeruginosa PA14 using the TnSeq analysis tools Bio-TraDIS and TRANSIT. A screen of a pool of 300,000 transposon insertion mutants identified 349 genes involved in biofilm growth on hydroxyapatite, including 47 regulators. Detection of 19 regulatory genes participating in well-established biofilm pathways validated the results. An additional 28 novel prospective biofilm regulators suggested the requirement for multiple one-component transcriptional regulators. Biofilm-defective phenotypes were confirmed for five one-component transcriptional regulators and a protein kinase, which did not affect motility phenotypes. The one-component transcriptional regulator bosR displayed a conserved role in P. aeruginosa biofilm growth since its ortholog in P. aeruginosa strain PAO1 was also required for biofilm growth. Microscopic analysis of a chromosomal deletion mutant of bosR confirmed the role of this regulator in biofilm growth. Overall, our results highlighted that the gene network driving biofilm growth is complex and involves regulators beyond the primarily studied groups of two-component systems and cyclic diguanylate signaling proteins. Furthermore, biofilm-specific regulators, such as bosR, might constitute prospective new drug targets to overcome biofilm infections.

Gradual adaptation of facultative anaerobic pathogens to microaerobic and anaerobic conditions.

Many notable human pathogens are facultative anaerobes. These pathogens exhibit redundant metabolic pathways and a whole array of regulatory systems to adapt to changing oxygen levels. However, our knowledge of facultative anaerobic pathogens is mostly based on fully aerobic or anaerobic cultures, which does not reflect real infection conditions, while the microaerobic range remains understudied. Here, we examine the behavior of pathogenic and nonpathogenic strains of two facultative anaerobes, Escherichia coli and Pseudomonas aeruginosa, during the aerobic-anaerobic transition. To do so, we introduce a new technique named AnaeroTrans, in which we allow self-consumption of oxygen by steady-state cultures and monitor the system by measuring the gas-phase oxygen concentration. We explore the different behavior of the studied species toward oxygen and examine how this behavior is associated with the targeted infection sites. As a model, we characterize the adaptation profile of the ribonucleotide reductase network, a complex oxygen-dependent enzymatic system responsible for the generation of the deoxyribonucleotides. We also explore the actions of the most important anaerobic regulators and how these regulators influence bacterial fitness. Our results allow us to classify the different elements that compose the aerobic-anaerobic transition into reproducible stages, thus showing the different adaptation mechanisms of the studied species.

Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms.

The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.

Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons.

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of ß-amyloid. Their interaction is mandatory for neurotoxic effects of ß-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrPC-overexpressing mice. In addition, α-synuclein binds strongly on PrPC-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.

Regulation of ribonucleotide synthesis by the Pseudomonas aeruginosa two-component system AlgR in response to oxidative stress.

Ribonucleotide reductases (RNR) catalyze the last step of deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. Three forms of RNR exist: classes I, II, and III. While eukaryotic cells use only class Ia RNR, bacteria can harbor any combination of classes, granting them adaptability. The opportunistic pathogen Pseudomonas aeruginosa surprisingly encodes all three classes, allowing it to thrive in different environments. Here we study an aspect of the complex RNR regulation whose molecular mechanism has never been elucidated, the well-described induction through oxidative stress, and link it to the AlgZR two-component system, the primary regulator of the mucoid phenotype. Through bioinformatics, we identify AlgR binding locations in RNR promoters, which we characterize functionally through EMSA and physically through AFM imaging. Gene reporter assays in different growth models are used to study the AlgZR-mediated control on the RNR network under various environmental conditions and physiological states. Thereby, we show that the two-component system AlgZR, which is crucial for bacterial conversion to the mucoid phenotype associated with chronic disease, controls the RNR network and directs how the DNA synthesis pathway is modulated in mucoid and non-mucoid biofilms, allowing it to respond to oxidative stress.

Pseudomonas aeruginosa Exhibits Deficient Biofilm Formation in the Absence of Class II and III Ribonucleotide Reductases Due to Hindered Anaerobic Growth.

Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this complex growth pattern, essential for P. aeruginosa chronic infections.

Function of the Pseudomonas aeruginosa NrdR Transcription Factor: Global Transcriptomic Analysis and Its Role on Ribonucleotide Reductase Gene Expression.

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.