Solute channels of the outer membrane: from bacteria to chloroplasts

Chloroplasts, unique organelles of plants, originated from endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. It is assumed that the outer envelope membrane, which delimits the chloroplast from the surrounding cytosol, was thus inherited from its Gram-negative bacterial ancestor. This plastid-specific membrane is thus equipped with elements of prokaryotic and eukaryotic origin. In particular, the membrane-intrinsic outer envelope proteins (OEPs) form solute channels with properties reminiscent of porins and channels in the bacterial outer membrane. OEP channels are characterised by distinct specificities for metabolites and a quite peculiar expression pattern in specialised plant organs and plastids, thus disproving the assumption that the outer envelope is a non-specific molecular sieve. The same is true for the outer membrane of Gram-negative bacteria, which functions as a permeability barrier in addition to the cytoplasmic membrane, and embeds different classes of channel pores. The channels of these prokaryotic prototype proteins, ranging from unspecific porins to specific channels to ligand-gated receptors, are exclusively built of P-barrels. Although most of the OEP channels are formed by P-strands as well, phylogeny based on sequence homology alone is not feasible. Thus, the comparison of structural and functional properties of chloroplast outer envelope and bacterial outer membrane channels is required to pinpoint the ancestral OEP `portrait gallery'

Characterization of modular bacteriophage endolysins from giant phiKZ related myoviridae phages OBP, 201phi2-1 and PVP-SEl

Peptidoglycan lytic enzymes (endolysins) of bacteriophages have a major role in bacterial lysis at the end of the phage replication cycle. These endolysins turned out to be potential antibacterial compounds to combat a broad range of Gram-positive pathogens, yet Gram-negative bacteria remain unharmed due to their impermeable outer membrane. With this background, we recently characterized three new endolysins from Gramnegative origin: OBPgp279 (Pseudomonas fluorescens phage OBP), PVP-SElgpl46 (Salmonella Enteritidis phage PVP-SEl) and 20lphi2-lgp229 (Pseudomonas chlororaphis phage 201phi2-1). These endolysins share a modular structure with anNterminal cell wall binding domain and a C-terrninal catalytic domain, a unique property of endolysins belonging to giant phiKZ related phages and some other giant, non-related myoviruses. All three endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, a feature linked with their modular composition. In case of OBPgp279, the presence of the cell wall binding domain is responsible for 38 % of the total muralytic activity. Moreover, the binding domain of PVP-SE1gp146 has a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules. Remarkably, PVP-SElgp146 shows thermoresistant properties up to temperatures of 90°C, making it a potential candidate as antibacterial in hurdle technology for food preservation. OBPgp279, on the other hand. is able to pass the outer membrane of P. aeruginosa PAO 1 using an unknown mechanism, thereby gaining access to its peptidoglycan and reduce the bacterium with !logarithmic unit. Addition of the outer membrane permeabilizer EDTA significantly increased the antibacterial activity of the three endolysins up to 2-3 logarithmic units. This research offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties ofOBPgp279, PVP-SE1gp146 and 201phi2- lgp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used for treatment of Gram-negative bacterial infections

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI-Translocation consortium. The synergistic combination of structural data, in vitro assays and computer simulations has proven to give new insights towards the identification and description of physico–chemical properties modulating permeation. Once similar general rules are identified, we believe that the use of virtual screening techniques will be very helpful in searching for new molecular scaffolds with enhanced permeation, and that molecular modeling will be of fundamental assistance to the optimization stage

Investigation of tetrasubstituted heterocycles reveals hydantoins as a promising scaffold for development of novel antimicrobials with membranolytic properties

Mimics of antimicrobial peptides (AMPs) have been proposed as a promising class of antimicrobial agents. We report the analysis of five tetrasubstituted, cationic, amphipathic heterocycles as potential AMP mimics. The analysis showed that the heterocyclic scaffold had a strong influence on the haemolytic activity of the compounds, and the hydantoin scaffold was identified as a promising template for drug lead development. Subsequently, a total of 20 hydantoin derivatives were studied for their antimicrobial potency and haemolytic activity. We found 19 of these derivatives to have very low haemolytic toxicity and identified three lead structures, 2dA, 6cG, and 6dG with very promising broad-spectrum antimicrobial activity. Lead structure 6dG displayed minimum inhibitory concentration (MIC) values as low as 1 μg/mL against Gram-positive bacteria and 4–16 μg/mL against Gram-negative bacteria. Initial mode of action (MoA) studies performed on the amine derivative 6cG, utilizing a luciferase-based biosensor assay, suggested a strong membrane disrupting effect on the outer and inner membrane of Escherichia coli. Our findings show that the physical properties and structural arrangement induced by the heterocyclic scaffolds are important factors in the design of AMP mimics

The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

Predicting the outer membrane proteome of Pasteurella multocida based on consensus prediction enhanced by results integration and manual confirmation

Background Outer membrane proteins (OMPs) of Pasteurella multocida have various functions related to virulence and pathogenesis and represent important targets for vaccine development. Various bioinformatic algorithms can predict outer membrane localization and discriminate OMPs by structure or function. The designation of a confident prediction framework by integrating different predictors followed by consensus prediction, results integration and manual confirmation will improve the prediction of the outer membrane proteome. Results In the present study, we used 10 different predictors classified into three groups (subcellular localization, transmembrane β-barrel protein and lipoprotein predictors) to identify putative OMPs from two available P. multocida genomes: those of avian strain Pm70 and porcine non-toxigenic strain 3480. Predicted proteins in each group were filtered by optimized criteria for consensus prediction: at least two positive predictions for the subcellular localization predictors, three for the transmembrane β-barrel protein predictors and one for the lipoprotein predictors. The consensus predicted proteins were integrated from each group into a single list of proteins. We further incorporated a manual confirmation step including a public database search against PubMed and sequence analyses, e.g. sequence and structural homology, conserved motifs/domains, functional prediction, and protein-protein interactions to enhance the confidence of prediction. As a result, we were able to confidently predict 98 putative OMPs from the avian strain genome and 107 OMPs from the porcine strain genome with 83% overlap between the two genomes. Conclusions The bioinformatic framework developed in this study has increased the number of putative OMPs identified in P. multocida and allowed these OMPs to be identified with a higher degree of confidence. Our approach can be applied to investigate the outer membrane proteomes of other Gram-negative bacteria

Mechanisms of Bacterial Extracellular Electron Exchange.

The biochemical mechanisms by which microbes interact with extracellular soluble metal ions and insoluble redox-active minerals have been the focus of intense research over the last three decades. The process presents two challenges to the microorganism; firstly electrons have to be transported at the cell surface, which in Gram negative bacteria presents an additional problem of electron transfer across the ~ 6 nm of the outer membrane. Secondly the electrons must be transferred to or from the terminal electron acceptors or donors. This review covers the known mechanisms that bacteria use to transport electrons across the cell envelope to external electron donors/acceptors. In Gram negative bacteria electron transfer across the outer membrane involves the use of an outer membrane β-barrel and cytochrome. These can be in the form of a porin-cytochrome protein, such as Cyc2 of Acidothiobacillus ferrioxydans, or a multiprotein porin-cytochrome complex like MtrCAB of Shewanella oneidensis MR-1. For mineral respiring organisms there is the additional challenge of transferring the electrons from the cell to mineral surface. For the strict anaerobe Geobacter sulfurreducens this requires electron transfer through conductive pili to associated cytochrome OmcS that directly reduces Fe(III)oxides, while the facultative anaerobe S. oneidensis MR-1 accomplishes mineral reduction through direct membrane contact, contact through filamentous extentions and soluble flavin shuttles, all of which require the outer membrane cytochromes MtrC and OmcA in addition to secreted flavin

Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs

There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus; Photorhabdus; , the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class; Gammaproteobacteria; . Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified; in silico; . Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates.; IMPORTANCE; Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens