A novel colistin adjuvant identified by virtual screening for ArnT inhibitors.

BACKGROUND: Colistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa. OBJECTIVES: Identification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants. METHODS: The available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A. RESULTS: A putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially. CONCLUSIONS: This study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.

Aminoarabinosylation of Lipid A Is Critical for the Development of Colistin Resistance in Pseudomonas aeruginosa.

Lipid A aminoarabinosylation is invariably associated with colistin resistance in Pseudomonas aeruginosa; however, the existence of alternative aminoarabinosylation-independent colistin resistance mechanisms in this bacterium has remained elusive. By combining reverse genetics with experimental evolution assays, we demonstrate that a functional lipid A aminoarabinosylation pathway is critical for the acquisition of colistin resistance in reference and clinical P. aeruginosa isolates. This highlights lipid A aminoarabinosylation as a promising target for the design of colistin adjuvants against P. aeruginosa.

Ferric Uptake Regulator Fur Is Conditionally Essential in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the ferric uptake regulator (Fur) protein controls both metabolism and virulence in response to iron availability. Differently from other bacteria, attempts to obtain fur deletion mutants of P. aeruginosa failed, leading to the assumption that Fur is an essential protein in this bacterium. By investigating a P. aeruginosa conditional fur mutant, we demonstrate that Fur is not essential for P. aeruginosa growth in liquid media, biofilm formation, and pathogenicity in an insect model of infection. Conversely, Fur is essential for growth on solid media since Fur-depleted cells are severely impaired in colony formation. Transposon-mediated random mutagenesis experiments identified pyochelin siderophore biosynthesis as a major cause of the colony growth defect of the conditional fur mutant, and deletion mutagenesis confirmed this evidence. Impaired colony growth of pyochelin-proficient Fur-depleted cells does not depend on oxidative stress, since Fur-depleted cells do not accumulate higher levels of reactive oxygen species (ROS) and are not rescued by antioxidant agents or overexpression of ROS-detoxifying enzymes. Ectopic expression of pch genes revealed that pyochelin production has no inhibitory effects on a fur deletion mutant of Pseudomonas syringae pv. tabaci, suggesting that the toxicity of the pch locus in Fur-depleted cells involves a P. aeruginosa-specific pathway(s).IMPORTANCE Members of the ferric uptake regulator (Fur) protein family are bacterial transcriptional repressors that control iron uptake and storage in response to iron availability, thereby playing a crucial role in the maintenance of iron homeostasis. While fur null mutants of many bacteria have been obtained, Fur appears to be essential in Pseudomonas aeruginosa for still unknown reasons. We obtained Fur-depleted P. aeruginosa cells by conditional mutagenesis and showed that Fur is dispensable for planktonic growth, while it is required for colony formation. This is because Fur protects P. aeruginosa colonies from toxicity exerted by the pyochelin siderophore. This work provides a functional basis to the essentiality of Fur in P. aeruginosa and highlights unique properties of the Fur regulon in this species.

Regulatory Landscape of the Pseudomonas aeruginosa Phosphoethanolamine Transferase Gene eptA in the Context of Colistin Resistance.

Pseudomonas aeruginosa has the genetic potential to acquire colistin resistance through the modification of lipopolysaccharide by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), mediated by the arn operon or the eptA gene, respectively. However, in vitro evolution experiments and genetic analysis of clinical isolates indicate that lipopolysaccharide modification with L-Ara4N is invariably preferred over PEtN addition as the colistin resistance mechanism in this bacterium. Since little is known about eptA regulation in P. aeruginosa, we generated luminescent derivatives of the reference strain P. aeruginosa PAO1 to monitor arn and eptA promoter activity. We performed transposon mutagenesis assays to compare the likelihood of acquiring mutations leading to arn or eptA induction and to identify eptA regulators. The analysis revealed that eptA was slightly induced under certain stress conditions, such as arginine or biotin depletion and accumulation of the signal molecule diadenosine tetraphosphate, but the induction did not confer colistin resistance. Moreover, we demonstrated that spontaneous mutations leading to colistin resistance invariably triggered arn rather than eptA expression, and that eptA was not induced in resistant mutants upon colistin exposure. Overall, these results suggest that the contribution of eptA to colistin resistance in P. aeruginosa may be limited by regulatory restraints.

Generation of Stable and Unmarked Conditional Mutants in Pseudomonas aeruginosa.

The functional and physiological characterization of bacterial genes required for growth and/or cell survival is limited by the inability to generate deletion mutants lacking the specific gene of interest. This limitation can be circumvented by generating conditional mutants in which the loss of the endogenous copy of the gene is compensated by the introduction of the wild-type allele under the control of an inducible promoter, which allows for tightly regulated expression of the gene of interest. Besides the confirmation and/or functional investigation of essential genes, conditional mutants can also be useful to investigate the effect of finely controlled expression of nonessential genes. In this chapter, we describe a method that can be used to generate stable and unmarked conditional mutants in Pseudomonas aeruginosa.

RecA and Specialized Error-Prone DNA Polymerases Are Not Required for Mutagenesis and Antibiotic Resistance Induced by Fluoroquinolones in Pseudomonas aeruginosa.

To cope with stressful conditions, including antibiotic exposure, bacteria activate the SOS response, a pathway that induces error-prone DNA repair and mutagenesis mechanisms. In most bacteria, the SOS response relies on the transcriptional repressor LexA and the co-protease RecA, the latter being also involved in homologous recombination. The role of the SOS response in stress- and antibiotic-induced mutagenesis has been characterized in detail in the model organism Escherichia coli. However, its effect on antibiotic resistance in the human pathogen Pseudomonas aeruginosa is less clear. Here, we analyzed a recA deletion mutant and confirmed, by conjugation and gene expression assays, that RecA is required for homologous recombination and SOS response induction in P. aeruginosa. MIC assays demonstrated that RecA affects P. aeruginosa resistance only towards fluoroquinolones and genotoxic agents. The comparison of antibiotic-resistant mutant frequency between treated and untreated cultures revealed that, among the antibiotics tested, only fluoroquinolones induced mutagenesis in P. aeruginosa. Notably, both RecA and error-prone DNA polymerases were found to be dispensable for this process. These data demonstrate that the SOS response is not required for antibiotic-induced mutagenesis in P. aeruginosa, suggesting that RecA inhibition is not a suitable strategy to target antibiotic-induced emergence of resistance in this pathogen.

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa.

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the ß clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa.

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

Effect of lipid A aminoarabinosylation on Pseudomonas aeruginosa colistin resistance and fitness.

Colistin represents the last-line treatment option against many multidrug-resistant Gram-negative pathogens. Several lines of evidence indicate that aminoarabinosylation of the lipid A moiety of lipopolysaccharide (LPS) is an essential step for the development of colistin resistance in Pseudomonas aeruginosa. However, whether it is sufficient to confer resistance in this bacterium remains unclear. The aim of this work was to investigate the specific contribution of lipid A aminoarabinosylation to colistin resistance in P. aeruginosa and evaluate the effect of this resistance mechanism on bacterial fitness. Recombinant strains constitutively expressing the enzymes for lipid A aminoarabinosylation were generated in a small collection of reference and clinical isolates and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR), lipid A extraction and mass spectrometry. The effect of aminoarabinosylated lipid A on colistin resistance was found to be strain- and culture condition-dependent. Higher levels of resistance were generally obtained in the presence of divalent cations, which appear to be important for aminoarabinosylation-mediated colistin resistance. High colistin resistance was also observed for most strains in human serum and in artificial sputum medium, which should partly mimic growth conditions during infection. The results of growth, biofilm, cell envelope integrity and Galleria mellonella infection assays indicate that lipid A aminoarabinosylation does not cause relevant fitness costs in P. aeruginosa.

Mutational analysis of the essential lipopolysaccharide-transport protein LptH of Pseudomonas aeruginosa to uncover critical oligomerization sites.

Lipopolysaccharide (LPS) is a critical component of the outer membrane (OM) of many Gram-negative bacteria. LPS is translocated to the OM by the LPS transport (Lpt) system. In the human pathogen Pseudomonas aeruginosa, the periplasmic Lpt component, LptH, is essential for LPS transport, planktonic and biofilm growth, OM stability and infectivity. LptH has been proposed to oligomerize and form a protein bridge that accommodates LPS during transport. Based on the known LptH crystal structure, here we predicted by in silico modeling five different sites likely involved in LptH oligomerization. The relevance of these sites for LptH activity was verified through plasmid-mediated expression of site-specific mutant proteins in a P. aeruginosa lptH conditional mutant. Complementation and protein expression analyses provided evidence that all mutated sites are important for LptH activity in vivo. It was observed that the lptH conditional mutant overcomes the lethality of nonfunctional lptH variants through RecA-mediated homologous recombination between the wild-type lptH gene in the genome and mutated copies in the plasmid. Finally, biochemical assays on purified recombinant proteins showed that some LptH variants are indeed specifically impaired in oligomerization, while others appear to have defects in protein folding and/or stability.

Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1.

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for resistance to cationic antimicrobial peptides (e.g., polymyxins), survival in human blood, and pathogenicity in animal models. The lipid A of the human pathogen Pseudomonas aeruginosa can be hydroxylated in both secondary acyl chains, but the genetic basis and physiological role of these hydroxylations are still unknown. Through the generation of single and double deletion mutants in the lpxO1 and lpxO2 homologs of P. aeruginosa PAO1 and lipid A analysis by mass spectrometry, we demonstrate that both LpxO1 and LpxO2 are responsible for lipid A hydroxylation, likely acting on different secondary acyl chains. Lipid A hydroxylation does not appear to affect in vitro growth, cell wall stability, and resistance to human blood or antibiotics in P. aeruginosa. In contrast, it is required for infectivity in the Galleria mellonella infection model, without relevantly affecting in vivo persistence. Overall, these findings suggest a role for lipid A hydroxylation in P. aeruginosa virulence that could not be directly related to outer membrane integrity.

Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence.

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P. aeruginosa LptA homologue, confirmed the crucial role of the Lpt system and, thus, of LPS in OM biogenesis in this species. Surprisingly, independent high-throughput transposon mutagenesis studies identified viable P. aeruginosa insertion mutants in the lptE gene, suggesting that it might be dispensable for bacterial growth. To test this hypothesis, we generated an lptE conditional mutant in P. aeruginosa PAO1. LptE depletion only slightly impairs P. aeruginosa growth in vitro. Conversely, LptE is important for cell envelope stability, antibiotic resistance and virulence in an insect model. Interestingly, the maturation and OM localization of LPS is only marginally affected in LptE-depleted cells, while the levels of the OM component LptD are strongly reduced. This suggests that P. aeruginosa LptE might not be directly involved in LPS transport, although it is clearly essential for the maturation and/or stability of LptD. While poor functionality of LptD caused by LptE depletion is somehow tolerated by P. aeruginosa, this has a high cost in terms of cell integrity, drug resistance and virulence, highlighting LptE function(s) as an interesting target to weaken P. aeruginosa defenses and reduce its infectivity.

In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis.

The periplasmic protein TolB as a potential drug target in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant "superbug" for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.