antibacTR : dynamic antibacterial-drug-target ranking integrating comparative genomics, structural analysis and experimental annotation

Background : development of novel antibacterial drugs is both an urgent healthcare necessity and a partially neglected field. The last decades have seen a substantial decrease in the discovery of novel antibiotics, which combined with the recent thrive of multi-drug-resistant pathogens have generated a scenario of general concern. The procedures involved in the discovery and development of novel antibiotics are economically challenging, time consuming and lack any warranty of success. Furthermore, the return-on-investment for an antibacterial drug is usually marginal when compared to other therapeutics, which in part explains the decrease of private investment. - Results : in this work we present antibacTR, a computational pipeline designed to aid researchers in the selection of potential drug targets, one of the initial steps in antibacterial-drug discovery. The approach was designed and implemented as part of two publicly funded initiatives aimed at discovering novel antibacterial targets, mechanisms and drugs for a priority list of Gram-negative pathogens: Acinetobacter baumannii, Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. However, at present this list has been extended to cover a total of 74 fully sequenced Gram-negative pathogens. antibacTR is based on sequence comparisons and queries to multiple databases (e.g. gene essentiality, virulence factors) to rank proteins according to their potential as antibacterial targets. The dynamic ranking of potential drug targets can easily be executed, customized and accessed by the user through a web interface which also integrates computational analyses performed in-house and visualizable on-site. These include three-dimensional modeling of protein structures and prediction of active sites among other functionally relevant ligand-binding sites. - Conclusions : given its versatility and ease-of-use at integrating both experimental annotation and computational analyses, antibacTR may effectively assist microbiologists, medicinal-chemists and other researchers working in the field of antibacterial drug-discovery. The public web-interface for antibacTR is available at 'http://bioinf.uab.cat/antibactr'

Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1

During their life cycle, proteins are subject to different modifications involving reactive oxygen species. Such oxidative damage to proteins may lead to the formation of insoluble aggregates and cytotoxicity and is associated with age-related disorders including neurodegenerative diseases, cancer, and diabetes. Superoxide dismutase 1 (SOD1), a key antioxidant enzyme in human cells, is particularly susceptible to such modifications. Moreover, this homodimeric metalloenzyme has been directly linked to both familial and sporadic amyotrophic lateral sclerosis (ALS), a devastating, late-onset motor neuronal disease, with more than 150 ALS-related mutations in the SOD1 gene. Importantly, oxidatively damaged SOD1 aggregates have been observed in both familial and sporadic forms of the disease. However, the molecular mechanisms as well as potential implications of oxidative stress in SOD1-induced cytotoxicity remain elusive. In this study, we examine the effects of oxidative modification on SOD1 monomer and homodimer stability, the key molecular properties related to SOD1 aggregation. We use molecular dynamics simulations in combination with thermodynamic integration to study microscopic-level site-specific effects of oxidative "mutations" at the dimer interface, including lysine, arginine, proline and threonine carbonylation, and cysteine oxidation. Our results show that oxidative damage of even single residues at the interface may drastically destabilize the SOD1 homodimer, with several modifications exhibiting a comparable effect to that of the most drastic ALS-causing mutations known. Additionally, we show that the SOD1 monomer stability decreases upon oxidative stress, which may lead to partial local unfolding and consequently to increased aggregation propensity. Importantly, these results suggest that oxidative stress may play a key role in development of ALS, with the mutations in the SOD1 gene being an additional factor

Interaction of camel Lactoferrin derived peptides with DNA : a molecular dynamics study

Background: lactoferrampin (LFampin), Lactoferricin (LFcin), and LFchimera are three well-known antimicrobial peptides derived from Lactoferrin and proposed as alternatives for antibiotics. Although the intracellular activity of these peptides has been previously demonstrated, their mode of action is not yet fully understood. Here, we performed a molecular dynamics simulation study to understand the molecular interactions between camel Lactoferrin derived peptides, including CLFampin, CLFcin, and CLFchimera, and DNA as an important intracellular target. - Results: our results indicate that all three peptides bind to DNA, albeit with different propensities, with CLFchimera showing the highest binding affinity. The secondary structures of the peptides, modeled on Lactoferrin, did not undergo significant changes during simulation, supporting their functional relevance. Main residues involved in the peptide-DNA interaction were identified based on binding free energy estimates calculated over 200 ns, which, as expected, confirmed strong electrostatic interactions between DNA phosphate groups and positively charged peptide side chains. Interaction between the different concentrations of CLFchimera and DNA revealed that after binding of four copies of CLFchimera to DNA, hydrogen bonds between the two strands of DNA start to break from one of the termini. - Conclusions: importantly, our results revealed that there is no DNA-sequence preference for peptide binding, in line with a broad antimicrobial activity. Moreover, the results showed that the strength of the interaction between DNA and CLFchimera is concentration dependent. The insight provided by these results can be used for the rational redesign of natural antimicrobial peptides targeting the bacterial DN

Quorum sensing signaling and quenching in the multidrug-resistant pathogen stenotrophomonas maltophilia

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf -1 and rpf -2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies

Draft genome sequence of Stenotrophomonas maltophilia strain UV74 reveals extensive variability within its genomic group

We report the draft genome sequence of Stenotrophomonas maltophilia UV74, isolated from a vascular ulcer. This draft genome sequence shall contribute to the understanding of the evolution and pathogenicity of this species, particularly regarding isolates of clinical origin

Draft genome sequence of Stenotrophomonas maltophilia strain M30, isolated from a chronic pressure ulcer in an elderly patient

Stenotrophomonas maltophilia is an emerging opportunistic pathogen with an increasing prevalence of multidrug-resistant strains. Here, we report the draft genome sequence of S. maltophilia strain M30, isolated from a pressure ulcer in an elderly patient

Decoding the genetic and functional diversity of the DSF Quorum-Sensing system in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate

Synthesis and evaluation of aromatic BDSF bioisosteres on biofilm formation and colistin sensitivity in pathogenic bacteria

The diffusible signal factor family (DSF) of molecules play an important role in regulating intercellular communication, or quorum sensing, in several disease-causing bacteria. These messenger molecules, which are comprised of cis-unsaturated fatty acids, are involved in the regulation of biofilm formation, antibiotic tolerance, virulence and the control of bacterial resistance. We have previously demonstrated how olefinic N-acyl sulfonamide bioisosteric analogues of diffusible signal factor can reduce biofilm formation or enhance antibiotic sensitivity in a number of bacterial strains. This work describes the design and synthesis of a second generation of aromatic N-acyl sulfonamide bioisosteres. The impact of these compounds on biofilm production in Acinetobacter baumannii, Escherichia coli, Burkholderia multivorans, Burkholderia cepacia, Burkholderia cenocepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia is evaluated, in addition to their effects on antibiotic tolerance. The ability of these molecules to increase survival rates on co-administration with colistin is also investigated using the Galleria infection model

Stenotrophomonas maltophilia responds to exogenous AHL signals through the LuxR solo SmoR (Smlt1839)

Quorum Sensing (QS) mediated by Acyl Homoserine Lactone (AHL) molecules are probably the most widespread and studied among Gram-negative bacteria. Canonical AHL systems are composed by a synthase (LuxI family) and a regulator element (LuxR family), whose genes are usually adjacent in the genome. However, incomplete AHL-QS machinery lacking the synthase LuxI is frequently observed in Proteobacteria, and the regulator element is then referred as LuxR solo. It has been shown that certain LuxR solos participate in interspecific communication by detecting signals produced by different organisms. In the case of Stenotrophomonas maltophilia, a preliminary genome sequence analysis revealed numerous putative luxR genes, none of them associated to a luxI gene. From these, the hypothetical LuxR solo Smlt1839, here designated SmoR, presents a conserved AHL binding domain and a helix-turn-helix DNA binding motif. Its genomic organization-adjacent to hchA gene-indicate that SmoR belongs to the new family "LuxR regulator chaperone HchA-associated." AHL-binding assays revealed that SmoR binds to AHLs in-vitro, at least to oxo-C8-homoserine lactone, and it regulates operon transcription, likely by recognizing a conserved palindromic regulatory box in the hchA upstream region. Supplementation with concentrated supernatants from Pseudomonas aeruginosa, which contain significant amounts of AHLs, promoted swarming motility in S. maltophilia. Contrarily, no swarming stimulation was observed when the P. aeruginosa supernatant was treated with the lactonase AiiA from Bacillus subtilis, confirming that AHL contributes to enhance the swarming ability of S. maltophilia. Finally, mutation of smoR resulted in a swarming alteration and an apparent insensitivity to the exogenous AHLs provided by P. aeruginosa. In conclusion, our results demonstrate that S. maltophilia senses AHLs produced by neighboring bacteria through the LuxR solo SmoR, regulating population behaviors such as swarming motility

The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm

In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. Here we use the crystallographic structure of Ttg2D at 2.5 Å resolution to reveal that this protein can accommodate four acyl chains. Analysis of the available structures of Ttg2D orthologs shows that they conform a new substrate-binding-protein structural cluster. Native and denaturing mass spectrometry experiments confirm that Ttg2D, produced both heterologously and homologously and isolated from the periplasm, can carry two diacyl glycerophospholipids as well as one cardiolipin. Binding is notably promiscuous, allowing the transport of various molecular species. In vitro binding assays coupled to native mass spectrometry show that binding of cardiolipin is spontaneous. Gene knockout experiments in P. aeruginosa multidrug-resistant strains reveal that the Ttg2 system is involved in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes