Murepavadin promotes the killing efficacies of aminoglycoside antibiotics against Pseudomonas aeruginosa by enhancing membrane potential.

Murepavadin is a peptidomimetic that specifically targets the lipopolysaccharide transport protein LptD of Pseudomonas aeruginosa. Here, we found that murepavadin enhances the bactericidal efficacies of tobramycin and amikacin. We further demonstrated that murepavadin enhances bacterial respiration activity and subsequent membrane potential, which promotes intracellular uptake of aminoglycoside antibiotics. In addition, the murepavadin-amikacin combination displayed a synergistic bactericidal effect in a murine pneumonia model.

Current and novel therapies for management of Acinetobacter baumannii-associated pneumonia.

Acinetobacter baumannii is a common pathogen associated with hospital-acquired pneumonia showing increased resistance to carbapenem and colistin antibiotics nowadays. Infections with A. baumannii cause high patient fatalities due to their capability to evade current antimicrobial therapies, emphasizing the urgency of developing viable therapeutics to treat A. baumannii-associated pneumonia. In this review, we explore current and novel therapeutic options for overcoming therapeutic failure when dealing with A. baumannii-associated pneumonia. Among them, antibiotic combination therapy administering several drugs simultaneously or alternately, is one promising approach for optimizing therapeutic success. However, it has been associated with inconsistent and inconclusive therapeutic outcomes across different studies. Therefore, it is critical to undertake additional clinical trials to ascertain the clinical effectiveness of different antibiotic combinations. We also discuss the prospective roles of novel antimicrobial therapies including antimicrobial peptides, bacteriophage-based therapy, repurposed drugs, naturally-occurring compounds, nanoparticle-based therapy, anti-virulence strategies, immunotherapy, photodynamic and sonodynamic therapy, for utilizing them as additional alternative therapy while tackling A. baumannii-associated pneumonia. Importantly, these innovative therapies further require pharmacokinetic and pharmacodynamic evaluation for safety, stability, immunogenicity, toxicity, and tolerability before they can be clinically approved as an alternative rescue therapy for A. baumannii-associated pulmonary infections.

Size-Controlled Synthesis of Gold Nanoparticles Tethering Antimicrobial Peptides with Potent Broad-Spectrum Antimicrobial and Antibiofilm Activities.

New antimicrobials are urgently needed to combat the rising global health concern of antibiotic resistance. Antimicrobial peptides (AMPs) are one of the leading candidates as new antimicrobials since they target bacterial membranes and are therefore less prone to bacterial resistance. However, poor enzymatic stability, high production costs, and toxicity are drawbacks that limit their clinical use. Conjugation of AMPs to gold nanoparticles (NPs) may help to improve enzymatic stability and, thus, their overall antimicrobial efficiency. We did a one-pot synthesis of size-controlled (10 nm) gold NPs selectively conjugated to lipopeptides and determined their antibacterial activity. The conjugates exhibited potent (0.13-1.25 µM) antimicrobial activity against clinical isolates, including Gram-positive methicillin-resistant Staphylococcus aureus (S. aureus) ATCC33593, Gram-negative Escherichia coli (E. coli) CTX-M-14, multidrug-resistant Pseudomonas aeruginosa LESB58 and Acinetobacter baumannii ATCC19606, and showed promising activity (90% inhibition of initial biofilms and 80% reduction of preformed biofilms) against S. aureus and E. coli DH5α biofilms at low micromolar concentrations. The conjugates were stable in rat serum and not toxic to representative mammalian cell lines in vitro (≤64 µM) and in vivo (≤100 µM).

Biodistribution of Native and Nanoformulated Innate Defense Regulator Peptide 1002.

Innate defense regulator-1002 (IDR-1002) is a synthetic peptide with promising immunomodulatory and antibiofilm properties. An appreciable body of work exists around its mechanism of action at the cellular and molecular level, along with its efficacy across several infection and inflammation models. However, little is known about its absorption, distribution, and excretion in live organisms. Here, we performed a comprehensive biodistribution assessment with a gallium-67 radiolabeled derivative of IDR-1002 using nuclear tracing techniques. Various dose levels of the radiotracer (2-40 mg/kg) were administered into the blood, peritoneal cavity, and subcutaneous tissue, or instilled into the lungs. The peptide was well tolerated at all subcutaneous and intraperitoneal doses, although higher levels were associated with delayed absorption kinetics and precipitation of the peptide within the tissues. Low intratracheal doses were rapidly absorbed systemically, and small increases in the dose level were lethal. Intravenous doses were rapidly cleared from the blood at lower levels, and upon escalation, were toxic with a high proportion of the dose accumulating within the lung tissue. To improve biocompatibility and prolong its circulation within the blood, IDR-1002 was further formulated onto high molecular weight hyperbranched polyglycerol (HPG) polymers. Constructs prepared at 5:1 and 10:1 peptide-to-polymer ratios were colloidally stable, maintained the biological profile of the peptide payload and helped reduce red blood cell lysis. The 5:1 construct circulated well in the blood, but higher peptide loading was associated with rapid clearance by the reticuloendothelial system. Many peptides face pharmacokinetic and biocompatibility challenges, but formulations such as those with HPG have the potential to overcome these limitations.

From Data to Decisions: Leveraging Artificial Intelligence and Machine Learning in Combating Antimicrobial Resistance - a Comprehensive Review.

The emergence of drug-resistant bacteria poses a significant challenge to modern medicine. In response, Artificial Intelligence (AI) and Machine Learning (ML) algorithms have emerged as powerful tools for combating antimicrobial resistance (AMR). This review aims to explore the role of AI/ML in AMR management, with a focus on identifying pathogens, understanding resistance patterns, predicting treatment outcomes, and discovering new antibiotic agents. Recent advancements in AI/ML have enabled the efficient analysis of large datasets, facilitating the reliable prediction of AMR trends and treatment responses with minimal human intervention. ML algorithms can analyze genomic data to identify genetic markers associated with antibiotic resistance, enabling the development of targeted treatment strategies. Additionally, AI/ML techniques show promise in optimizing drug administration and developing alternatives to traditional antibiotics. By analyzing patient data and clinical outcomes, these technologies can assist healthcare providers in diagnosing infections, evaluating their severity, and selecting appropriate antimicrobial therapies. While integration of AI/ML in clinical settings is still in its infancy, advancements in data quality and algorithm development suggest that widespread clinical adoption is forthcoming. In conclusion, AI/ML holds significant promise for improving AMR management and treatment outcome.

Allenamides as a Powerful Tool to Incorporate Diversity: Thia-Michael Lipidation of Semisynthetic Peptides and Access to ß-Keto Amides.

Lipopeptides are an important class of biomolecules for drug development. Compared with conventional acylation, a chemoselective lipidation strategy offers a more efficient strategy for late-stage structural derivatisation of a peptide scaffold. It provides access to chemically diverse compounds possessing intriguing and non-native moieties. Utilising an allenamide, we report the first semisynthesis of antimicrobial lipopeptides leveraging a highly efficient thia-Michael addition of chemically diverse lipophilic thiols. Using chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an optimised allenamide-mediated thia-Michael addition effected rapid and near quantitative lipidation, affording vinyl sulfide-linked lipopeptide derivatives. Harnessing the utility of this new methodology, 22 lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework. These included alkyl thiols, substituted aromatic thiols, heterocyclic thiols and those bearing additional functional groups (e.g., amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity. Furthermore, we report facile routes to transform the allenamide into a ß-keto amide on unprotected peptides, offering a powerful "jack-of-all-trades" synthetic intermediate to enable further peptide modification.

Identification of novel targets of azithromycin activity against Pseudomonas aeruginosa grown in physiologically relevant media.

Pseudomonas aeruginosa causes severe multidrug-resistant infections that often lead to bacteremia and sepsis. Physiologically relevant conditions can increase the susceptibility of pathogens to antibiotics, such as azithromycin (AZM). When compared to minimal-inhibitory concentrations (MICs) in laboratory media, AZM had a 16-fold lower MIC in tissue culture medium with 5% Mueller Hinton broth (MHB) and a 64-fold lower MIC in this tissue culture medium with 20% human serum. AZM also demonstrated increased synergy in combination with synthetic host-defense peptides DJK-5 and IDR-1018 under host-like conditions and in a murine abscess model. To mechanistically study the altered effects of AZM under physiologically relevant conditions, global transcriptional analysis was performed on P. aeruginosa with and without effective concentrations of AZM. This revealed that the arn operon, mediating arabinosaminylation of lipopolysaccharides and related regulatory systems, was down-regulated in host-like media when compared to MHB. Inactivation of genes within the arn operon led to increased susceptibility of P. aeruginosa to AZM and great increases in synergy between AZM and other antimicrobial agents, indicating that dysregulation of the arn operon might explain increased AZM uptake and synergy in host-like media. Furthermore, genes involved in central and energy metabolism and ribosome biogenesis were dysregulated more in physiologically relevant conditions treated with AZM, likely due to general changes in cell physiology as a result of the increased effectiveness of AZM in these conditions. These data suggest that, in addition to the arn operon, there are multiple factors in host-like environments that are responsible for observed changes in susceptibility.

Friends or enemies? The complicated relationship between Pseudomonas aeruginosa and Staphylococcus aureus.

Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) are opportunistic pathogens that are most commonly co-isolated from chronic wounds and the sputum of cystic fibrosis patients. Over the last few years, there have been plenty of contrasting results from studies involving P. aeruginosa and S. aureus co-cultures. The general concept that P. aeruginosa outcompetes S. aureus has been challenged and there is more evidence now that they can co-exist. Nevertheless, it still remains difficult to mimic polymicrobial infections in vitro and in vivo. In this review, we discuss recent advances in regard to Pa-Sa molecular interactions, their physical responses, and in vitro and in vivo models. We believe it is important to optimize growth conditions in the laboratory, determine appropriate bacterial starting ratios, and consider environmental factors to study the co-existence of these two pathogens. Ideally, optimized growth media should reflect host-mimicking conditions with or without host cells that allow both bacteria to co-exist. To further identify mechanisms that could help to treat these complex infections, we propose to use relevant polymicrobial animal models. Ultimately, we briefly discuss how polymicrobial infections can increase antibiotic tolerance.

Surfing motility is a complex adaptation dependent on the stringent stress response in Pseudomonas aeruginosa LESB58.

Cystic fibrosis (CF) is a genetic disease that affects mucin-producing body organs such as the lungs. Characteristic of CF is the production of thick, viscous mucus, containing the glycoprotein mucin, that can lead to progressive airway obstruction. Recently, we demonstrated that the presence of mucin induced a rapid surface adaptation in motile bacteria termed surfing motility, which data presented here indicates is very different from swarming motility. Pseudomonas aeruginosa, the main colonizing pathogen in CF, employs several stress coping mechanisms to survive the highly viscous environment of the CF lung. We used motility-based assays and RNA-Seq to study the stringent stress response in the hypervirulent CF isolate LESB58 (Liverpool Epidemic Strain). Motility experiments revealed that an LESB58 stringent response mutant (ΔrelAΔspoT) was unable to surf. Transcriptional profiling of ΔrelAΔspoT mutant cells from surfing agar plates, when compared to wild-type cells from the surfing edge, revealed 2,584 dysregulated genes. Gene Ontology and KEGG enrichment analysis revealed effects of the stringent response on amino acid, nucleic acid and fatty acid metabolism, TCA cycle and glycolysis, type VI secretion, as well as chemotaxis, cell communication, iron transport, nitrogen metabolic processes and cyclic-di-GMP signalling. Screening of the ordered PA14 transposon library revealed 224 mutants unable to surf and very limited overlap with genes required for swarming. Mutants affecting surfing included two downstream effector genes of the stringent stress response, the copper regulator cueR and the quinolone synthase pqsH. Both the cueR and pqsH cloned genes complemented the surfing deficiency of ΔrelAΔspoT. Our study revealed insights into stringent stress dependency in LESB58 and showed that surfing motility is stringently-controlled via the expression of cueR and pqsH. Downstream factors of the stringent stress response are important to investigate in order to fully understand its ability to colonize and persist in the CF lung.

Contribution of Swarming Motility to Dissemination in a Pseudomonas aeruginosa Murine Skin Abscess Infection Model.

Swarming motility in Pseudomonas aeruginosa is a multicellular adaptation induced by semisolid medium with amino acids as a nitrogen source. By phenotypic screening, we differentiated swarming from other complex adaptive phenotypes, such as biofilm formation, swimming and twitching, by identifying a swarming-specific mutant in ptsP, a metabolic regulator. This swarming-deficient mutant was tested in an acute murine skin abscess infection model. Bacteria were recovered at significantly lower numbers from organs of mice infected with the ∆ptsP mutant. We also tested the synthetic peptide 1018 for activity against different motilities and efficacy in vivo. Treatment with peptide 1018 mimicked the phenotype of the ∆ptsP mutant in vitro, as swarming was inhibited at low concentrations (<2 µg/mL) but not swimming or twitching, and in vivo, as mice had a reduced bacterial load recovered from organs. Therefore, PtsP functions as a regulator of swarming, which in turn contributes to dissemination and colonization in vivo.

Towards understanding single-channel characteristics of OccK8 purified from Pseudomonas aeruginosa.

Antibiotic resistance in Gram-negative bacteria causes serious health issues worldwide. Bacteria employ several resistance mechanisms to cope with antimicrobials. One of their strategies is to reduce the permeability of antibiotics either through general diffusion porins or substrate-specific channels. In this study, one of the substrate-specific channels from Pseudomonas aeruginosa, OccK8 (also known as OprE), was investigated using single-channel electrophysiology. The study also includes the investigation of permeability properties of several amino acids with different charged groups (i.e. arginine, glycine and glutamic acid) through OccK8. We observed four different conformations of the same OccK8 channel when inserted in lipid bilayers. This is in contrast to previous studies where heterologous expressed OccK8 in E. coli showed only one conformation. We hypothesized that the difference in our study was due to the expression and purification of the native channel from P. aeruginosa. The single-channel uptake characteristics of the porin showed that negatively charged glutamic acid preferentially interacted with the channel while the positively charged arginine molecule showed infrequent interaction with OccK8. The neutral amino acid glycine did not show any interaction at the physiological conditions.

Synergy between conventional antibiotics and anti-biofilm peptides in a murine, sub-cutaneous abscess model caused by recalcitrant ESKAPE pathogens.

With the antibiotic development pipeline running dry, many fear that we might soon run out of treatment options. High-density infections are particularly difficult to treat due to their adaptive multidrug-resistance and currently there are no therapies that adequately address this important issue. Here, a large-scale in vivo study was performed to enhance the activity of antibiotics to treat high-density infections caused by multidrug-resistant Gram-positive and Gram-negative bacteria. It was shown that synthetic peptides can be used in conjunction with the antibiotics ciprofloxacin, meropenem, erythromycin, gentamicin, and vancomycin to improve the treatment outcome of murine cutaneous abscesses caused by clinical hard-to-treat pathogens including all ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae) pathogens and Escherichia coli. Promisingly, combination treatment often showed synergistic effects that significantly reduced abscess sizes and/or improved clearance of bacterial isolates from the infection site, regardless of the antibiotic mode of action. In vitro data suggest that the mechanisms of peptide action in vivo include enhancement of antibiotic penetration and potential disruption of the stringent stress response.

Hyaluronic acid-based nanogels improve in vivo compatibility of the anti-biofilm peptide DJK-5.

Anti-biofilm peptides are a subset of antimicrobial peptides and represent promising broad-spectrum agents for the treatment of bacterial biofilms, though some display host toxicity in vivo. Here we evaluated nanogels composed of modified hyaluronic acid for the encapsulation of the anti-biofilm peptide DJK-5 in vivo. Nanogels of 174 to 194 nm encapsulating 33-60% of peptide were created. Efficacy and toxicity of the nanogels were tested in vivo employing a murine abscess model of a Pseudomonas aeruginosa LESB58 high bacterial density infection. The dose of DJK-5 that could be administered intravenously to mice without inducing toxicity was more than doubled after encapsulation in nanogels. Upon subcutaneous administration, the toxicity of the DJK-5 in nanogels was decreased four-fold compared to non-formulated peptide, without compromising the anti-abscess effect of DJK-5. These findings support the use of nanogels to increase the safety of antimicrobial and anti-biofilm peptides after intravenous and subcutaneous administration.

TonB-Dependent Receptor Repertoire of Pseudomonas aeruginosa for Uptake of Siderophore-Drug Conjugates.

The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

The concept of Helicobacter pylori biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common H. pylori lab strain, G27. The goal of this study was to evaluate biofilm formation by G27 in response to common culture conditions and to explore the biofilm matrix. Our results indicate that while various types of growth media did not dramatically affect biofilm formation, surface selection had a significant effect on the final biofilm mass. Furthermore, enzymatic assays and confocal microscopy revealed that proteins appear to be the primary structural component of the H. pylori extracellular matrix; extracellular DNA (eDNA) and polysaccharides were also present but appear to play a secondary role. Finally, we found that two well-characterized antibiofilm cationic peptides differentially affected early and late-stage biofilms. Together these results provide interesting avenues for future investigations that will seek to understand H. pylori biofilm formation.IMPORTANCE The study of H. pylori biofilm formation is still in its infancy. As such, there is great variability in how biofilm assays are performed across labs. While several groups have begun to investigate factors that influence H. pylori biofilm formation, it is not yet understood how H. pylori biofilm formation may vary based on commonly used conditions. These inconsistencies lead to difficulties in interpretation and comparison between studies. Here, we set out to characterize biofilm formation by a commonly available lab strain, G27. Our findings provide novel insight into optimal biofilm conditions, the biofilm matrix, and possible mechanisms to block or disrupt biofilm formation.

The bacteriophage-derived transcriptional regulator, LscR, activates the expression of levansucrase genes in Pseudomonas syringae.

Synthesis of the exopolysaccharide levan occurs in the bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, when this bacterium encounters moderate to high concentrations of sucrose inside its host plant. The process is mediated by the temperature-dependent expression and secretion of two levansucrases, LscB and LscC. Previous studies showed the importance of a prophage-associated promoter element in driving the expression of levansucrase genes. Herein, heterologous screening for transcriptional activators revealed that the prophage-borne transcriptional regulator, LscR, from P. syringae mediates expression of levansucrase. A lscR-deficient mutant was generated and exhibited a levan-negative phenotype when grown on a sucrose-rich medium. This phenotype was confirmed by zymographic analysis and Western blots which demonstrated absence of levansucrase in the supernatant and total cell lysates. Transcriptional analysis showed a down-regulation of expression levels of levansucrase and glycosyl hydrolase genes in the lscR-deficient mutant. Ultimately, a direct binding of LscR to the promoter region of levansucrase was demonstrated using electrophoretic mobility shift assays allowing to conclude that a bacteriophage-derived regulator dictates expression of bacterial genes involved in in planta fitness.

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii.

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii Here, we investigated the mechanism of action of these molecules in A. baumannii We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.

Antibiofilm Peptides: Potential as Broad-Spectrum Agents.

The treatment of bacterial diseases is facing twin threats, with increasing bacterial antibiotic resistance and relatively few novel compounds or strategies under development or entering the clinic. Bacteria frequently grow on surfaces as biofilm communities encased in a polymeric matrix. The biofilm mode of growth is associated with 65 to 80% of all clinical infections. It causes broad adaptive changes; biofilm bacteria are especially (10- to 1,000-fold) resistant to conventional antibiotics and to date no antimicrobials have been developed specifically to treat biofilms. Small synthetic peptides with broad-spectrum antibiofilm activity represent a novel approach to treat biofilm-related infections. Recent developments have provided evidence that these peptides can inhibit even developed biofilms, kill multiple bacterial species in biofilms (including the ESKAPE [Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species] pathogens), show strong synergy with several antibiotics, and act by targeting a universal stress response in bacteria. Thus, these peptides represent a promising alternative treatment to conventional antibiotics and work effectively in animal models of biofilm-associated infections.

Swarming motility is modulated by expression of the putative xenosiderophore transporter SppR-SppABCD in Pseudomonas aeruginosa PA14.

In the present study, we characterised the putative peptide ABC transporter SppABCD, which is co-transcribed with the TonB-dependent receptor SppR in Pseudomonas aeruginosa PA14. However, our data show that this transporter complex is not involved in the uptake of peptides. The fact that the TonB-dependent receptor SppR is regulated by an iron starvation ECF sigma factor suggested that this transporter is probably involved in the uptake of xenosiderophores. Therefore, we screened culture supernatants of 23 siderophore-producing bacteria for their ability to induce the expression of the SppR-regulating ECF sigma factor. However, none of them had an effect on the expression of this ECF sigma factor. Since the spp operon is not expressed under standard laboratory conditions, we overexpressed it from plasmids in PA14, which led to an impairment of its swarming motility on semisolid agar. Since we excluded the possibility that the uptake of a culture medium component was responsible for the observed phenotype, we hypothesize that the Spp transport system is involved in the uptake of a compound from the periplasmic space or a compound secreted by P. aeruginosa. Furthermore, we found that rhamnolipid synthesis was decreased while biofilm and exopolysaccharide synthesis was slightly increased upon overexpression of the spp operon. Moreover, we observed an impact of spp overexpression on regulation of genes involved in siderophore and phenazine biosynthesis.

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

UNLABELLED: Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE: One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.