Hydroxyl Radical Overproduction in the Envelope: an Achilles' Heel in Peptidoglycan Synthesis.

While many mechanisms governing bacterial envelope homeostasis have been identified, others remain poorly understood. To decipher these processes, we previously developed an assay in the Gram-negative model Escherichia coli to identify genes involved in maintenance of envelope integrity. One such gene was ElyC, which was shown to be required for envelope integrity and peptidoglycan synthesis at room temperature. ElyC is predicted to be an integral inner membrane protein with a highly conserved domain of unknown function (DUF218). In this study, and stemming from a further characterization of the role of ElyC in maintaining cell envelope integrity, we serendipitously discovered an unappreciated form of oxidative stress in the bacterial envelope. We found that cells lacking ElyC overproduce hydroxyl radicals (HO•) in their envelope compartment and that HO• overproduction is directly or indirectly responsible for the peptidoglycan synthesis arrest, cell envelope integrity defects, and cell lysis of the ΔelyC mutant. Consistent with these observations, we show that the ΔelyC mutant defect is suppressed during anaerobiosis. HO• is known to cause DNA damage but to our knowledge has not been shown to interfere with peptidoglycan synthesis. Thus, our work implicates oxidative stress as an important stressor in the bacterial cell envelope and opens the door to future studies deciphering the mechanisms that render peptidoglycan synthesis sensitive to oxidative stress. IMPORTANCE Oxidative stress is caused by the production and excessive accumulation of oxygen reactive species. In bacterial cells, oxidative stress mediated by hydroxyl radicals is typically associated with DNA damage in the cytoplasm. Here, we reveal the existence of a pathway for oxidative stress in the envelope of Gram-negative bacteria. Stemming from the characterization of a poorly characterized gene, we found that HO• overproduction specifically in the envelope compartment causes inhibition of peptidoglycan synthesis and eventually bacterial cell lysis.

The Chaperone Activities of DsbG and Spy Restore Peptidoglycan Biosynthesis in the elyC Mutant by Preventing Envelope Protein Aggregation.

Peptidoglycan (PG) is the main structural component of bacterial envelopes. It protects bacterial cells against variations in osmotic pressure and cell lysis. The newly discovered Escherichia coli factor ElyC has been shown to be important for peptidoglycan biosynthesis at low temperatures. PG production in ΔelyC mutant cells is totally blocked after a few hours of growth at 21°C, triggering cell lysis. In this study, we took a candidate approach to identify genetic suppressors of the ΔelyC mutant cell lysis phenotype. We identified the periplasmic proteins DsbG and Spy as multicopy suppressors and showed that their overproduction restores PG biosynthesis in the ΔelyC mutant. Interestingly, we found that DsbG acts by a novel mechanism, which is independent of its known reductase activity and substrates. DsbG, like Spy, acts as a chaperone to reduce the amounts of protein aggregates in the envelopes of ΔelyC cells. In fact, we found that the amount of protein aggregates was greater in the ΔelyC mutant than in the wild type. Taken together, our results show a protein-folding defect in the envelope compartments of ΔelyC cells that blocks PG production, and they reveal a new physiological activity of DsbG.IMPORTANCE Peptidoglycan biosynthesis is a dynamic and well-controlled pathway. The molecular assembly of PG and the regulatory pathways ensuring its maintenance are still not well understood. Here we studied the newly discovered Escherichia coli factor ElyC, which is important for PG biosynthesis at low temperatures. We revealed an important protein-folding defect in the ΔelyC mutant and showed that overproduction of the periplasmic chaperone DsbG or Spy was sufficient to correct the protein-folding defect and restore PG biosynthesis. These results show that the PG defect in the absence of ElyC is caused, at least in part, by a protein-folding problem in the cell envelope. Furthermore, we showed, for the first time, that the periplasmic protein DsbG has chaperone activity in vivo.

Adaptation of a bacterial membrane permeabilization assay for quantitative evaluation of benzalkonium chloride as a membrane-disrupting agent.

We describe the use of the ortho-nitrophenyl-ß-galactoside (ONPG) assay developed by Lehrer et al. to which a new mathematical data treatment was applied. In this simplified assay, only one enzymatic assay is needed to provide direct evidence of the kinetics of Escherichia coli membrane permeabilization induced by different concentrations of benzalkonium chloride (BAC). Analysis of the data obtained from the revised ONPG assay with our adapted mathematical formula indicates that BAC induces permeabilization of the bacterial outer and inner membranes in a two-step process. The two effective concentration (EC50) values obtained in this study, combined with the results from an outer membrane permeabilization assay, suggest that the two steps observed in the permeabilization process are related to the two different bacterial membranes. We show that membrane permeabilization occurs very fast upon the addition of bacterial cells to the BAC solutions and demonstrate that sub-lethal concentrations of BAC disturb the integrity of the Gram-negative bacterial membranes. Overall, our work broadens our knowledge on the mode of action of BAC on bacterial cells and emphasizes that BAC, and quaternary ammonium compounds in general, should not be used at sub-lethal concentrations in order to lower the risk of bacterial tolerance and resistance to antibiotics.

Biology and Assembly of the Bacterial Envelope.

All free-living bacterial cells are delimited and protected by an envelope of high complexity. This physiological barrier is essential for bacterial survival and assures multiple functions. The molecular assembly of the different envelope components into a functional structure represents a tremendous biological challenge and is of high interest for fundamental sciences. The study of bacterial envelope assembly has also been fostered by the need for novel classes of antibacterial agents to fight the problematic of bacterial resistance to antibiotics. This chapter focuses on the two most intensively studied classes of bacterial envelopes that belong to the phyla Firmicutes and Proteobacteria. The envelope of Firmicutes typically has one membrane and is defined as being monoderm whereas the envelope of Proteobacteria contains two distinct membranes and is referred to as being diderm. In this chapter, we will first discuss the multiple roles of the bacterial envelope and clarify the nomenclature used to describe the different types of envelopes. We will then define the architecture and composition of the envelopes of Firmicutes and Proteobacteria while outlining their similarities and differences. We will further cover the extensive progress made in the field of bacterial envelope assembly over the last decades, using Bacillus subtilis and Escherichia coli as model systems for the study of the monoderm and diderm bacterial envelopes, respectively. We will detail our current understanding of how molecular machines assure the secretion, insertion and folding of the envelope proteins as well as the assembly of the glycosidic components of the envelope. Finally, we will highlight the topics that are still under investigation, and that will surely lead to important discoveries in the near future.

The biology of Mur ligases as an antibacterial target.

With antibiotic resistance mechanisms increasing in diversity and spreading among bacterial pathogens, the development of new classes of antibacterial agents against judiciously chosen targets is a high-priority task. The biochemical pathway for peptidoglycan biosynthesis is one of the best sources of antibacterial targets. Within this pathway are the Mur ligases, described in this review as highly suitable targets for the development of new classes of antibacterial agents. The amide ligases MurC, MurD, MurE and MurF function with the same catalytic mechanism and share conserved amino acid regions and structural features that can conceivably be exploited for the design of inhibitors that simultaneously target more than one enzyme. This would provide multi-target antibacterial weapons with minimized likelihood of target-mediated resistance development.

A genome-wide screen for bacterial envelope biogenesis mutants identifies a novel factor involved in cell wall precursor metabolism.

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.

Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases.

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.

Caveolin-1 modifies the immunity to Pseudomonas aeruginosa.

The inflammatory response to Pseudomonas aeruginosa is not properly regulated in the lungs of patients with cystic fibrosis (CF). In the lung epithelium of individuals with wild-type CF transmembrane conductance regulator, lipid rafts containing CF transmembrane conductance regulator are rapidly formed in response to P. aeruginosa infection, and this response is closely linked to resistance to infection and disease. We found these rafts also contained high levels of caveolin-1 and thus examined the sensitivity of cav1 knockout (KO) mice to P. aeruginosa challenge in both acute and chronic P. aeruginosa infection models. We found that cav1 KO mice had increased sensitivity to P. aeruginosa infection, as represented by an increased mortality rate, elevated bacterial burdens recovered from lungs and spleens, and elevated inflammatory responses. These findings correlated with the decreased ability of cav1-deficient neutrophils to phagocytose P. aeruginosa. In addition, P. aeruginosa colonized cav1 KO mice much better compared with the wild-type controls in a model of chronic infection, indicting an important contribution of Cav-1 to innate host immunity to P. aeruginosa infection in the setting of both acute pneumonia and chronic infection typical of CF.

Biophysical studies of the interactions between the phage varphiKZ gp144 lytic transglycosylase and model membranes.

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.

Pseudomonas aeruginosa MurE amide ligase: enzyme kinetics and peptide inhibitor.

The enzyme kinetics of the amide ligase MurE, a cell wall biosynthesis enzyme, from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu (uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamate). When coupled to a competitive bio-panning technique using a M13 phage display library encoding approximately 2.7 x 10(9) random peptide permutations and the specific substrates meso-A2pm (meso-diaminopimelic acid) and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with an IC(50) value of 500 microM. The inhibition was shown to be time-dependent and was reversed by the addition of meso-A2pm or UDP-MurNAc-Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp1 as a mixed inhibitor against both substrates with K(i) values of 160 and 80 microM respectively. MurEp1 was found to interfere in meso-A2pm and UDP-MurNAc-Ala-Glu binding necessary for amide bond formation. Modelling of Ps. aeruginosa MurE and docking of MurEp1 on the Ps. aeruginosa MurE surface indicated that MurEp1 binds at the juxtaposition of both meso-A2pm- and UDP-MurNAc-Ala-Glu-binding sites in the closed conformational state of the enzyme. Identification of the MurEp1 residues involved in MurE binding and inhibition will allow the development of a novel class of inhibitors having a novel mode of action against MurE.

Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF.

BACKGROUND: To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. RESULTS: Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 microM, and the Ki was established at 420 microM with respect to the mixed type of inhibition against D-Ala-D-Ala. CONCLUSION: MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development.

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis.

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) underlies the hypersusceptibility of cystic fibrosis (CF) patients to chronic airway infections, particularly with Pseudomonas aeruginosa. CFTR is involved in the specific recognition of P. aeruginosa, thereby contributing to effective innate immunity and proper hydration of the airway surface layer (ASL). In CF, the airway epithelium fails to initiate an appropriate innate immune response, allowing the microbe to bind to mucus plugs that are then not properly cleared because of the dehydrated ASL. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and that are needed for bacterial clearance, a process that is defective in CF patients hypersusceptible to infection with this organism.

Discovery of new MurF inhibitors via pharmacophore modeling and QSAR analysis followed by in-silico screening.

The pharmacophoric space of streptococcal MurF was explored using a set of 39 known inhibitors. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and physicochemical descriptors that access self-consistent quantitative structure-activity relationship (QSAR) (r(2)=0.93,F=56.9,r(LOO)(2)=0.91,r(PRESS)(2) against eight external test inhibitors=0.75). Two orthogonal pharmacophores (of cross-correlation r(2)=0.26) emerged in the QSAR equation suggesting the existence of at least two distinct binding modes accessible to ligands within MurF binding pocket. The validity of the QSAR equation and the associated pharmacophore models was experimentally established by the identification of three promising new MurF inhibitors retrieved from the NCI database. Docking studies conducted on active hits supported the binding modes suggested by the pharmacophore/QSAR analysis.

Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase.

The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.

Parallel solid synthesis of inhibitors of the essential cell division FtsZ enzyme as a new potential class of antibacterials.

As a model system for designing new inhibitors of bacterial cell division, we studied the essential and highly conserved FtsZ GTPase from Pseudomonas aeruginosa. A collection of GTP analogues were prepared using the solid-phase parallel synthesis approach. The synthesized GTP analogues inhibited the GTPase activity of FtsZ with IC(50) values between 450microM and 2.6mM, and 5 compounds inhibited Staphylococcus aureus growth in a biological assay. The FtsZ spectrophotometric assay developed for screening of synthesized compounds is the first step in identification of antibacterials targeting the bacterial cell division essential proteins.

Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme.

The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.

Peptide inhibitors of the essential cell division protein FtsA.

The revolutionary era of antibiotics has been overwhelmed by the evolutionary capacity of microorganisms such as Pseudomonas aeruginosa to develop resistance to all classes of antibiotics. In the perspective of identifying new antimicrobials using novel strategies, we targeted the essential and highly conserved FtsA protein from the bacterial cell division machinery of P.aeruginosa. In a series of experiments we cloned, overproduced and purified the FtsA and FtsZ proteins. Expression of FtsA into Escherichia coli cells led to its accumulation in inclusion bodies. We developed a protocol permitting the purification and refolding of enzymatically active FtsA hydrolysing ATP. The purified enzyme was used to screen for peptide inhibitors of ATPase activity using phage display. Selective biopanning assays were done and phages were eluted using ATP, a non-hydrolysable ATP analogue and the protein FtsZ known to interact with FtsA in the divisome during the process of bacterial cell division. We identified two consensus peptide sequences interacting with FtsA and a competitive ELISA was used to identify peptides having high affinity for the target protein. Five of the six peptides synthesized showed specific inhibition of ATPase activity of FtsA with IC50 values between 0.7 and 35 mM. Discovery of peptides inhibiting the essential cell division machinery in bacteria is the first step for the future development of antimicrobial agents via peptidomimetism.

Ultrastructural and cytochemical study of cell wall modification by lactoferrin, lactoferricin and penicillin G against Staphylococcus aureus.

To investigate the effect of lactoferrin or lactoferricin with or without penicillin G, light and transmission electron microscopy were performed on thin sections of two Staphylococcus aureus strains. Lactoferrin affected the ultrastructure of S. aureus and groups of undivided cells were observed after lactoferrin treatment with or without penicillin G. These results suggest that lactoferrin can affect staphylococcal cell separation and therefore prevent dissemination of daughter cells from spreading infection. After treatment with lactoferrin, S. aureus cells were less covered (P<0.05) with wheatgerm agglutinin labelled with gold, thus suggesting that lactoferrin affected the synthesis of peptidoglycan and/or the binding to N-acetyl-beta-D-glucosamine. Lactoferricin with or without penicillin G induced the lysis of many bacteria, formation of mesosomal structures and modifications of cell wall. Lactating female CD-1 mice were infected by intramammary delivery of a penicillin-resistant S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. Electron microscopy examination showed that 2 days of systemic treatments with lactoferrin affected the morphology and aggregation of S. aureus. In conclusion, lactoferrin and lactoferricin affect S. aureus morphology which may facilitate its killing by penicillin G.