Cyclic Antibiotic Research Articles

Streptomyces wadayamensis MppP: A Novel PLP‐Dependent L‐Arginine Hydroxylase in L‐Enduracididine Biosynthesis

The nonproteinogenic amino acid L‐enduracididine (L‐End) is a critical component of the mannopeptimycins, a family of cyclic glycopeptide antibiotics with potent activities against drug‐resistant pathogens like MRSA. Creating derivatives of mannopeptimycin and other L‐End‐containing natural products is hampered by the limited availability of L‐End. We are investigating the L‐End biosynthetic pathway in order to develop an efficient enzymatic or chemo‐enzymatic route to produce this unusual amino acid. Recently, we found that MppP is a PLP‐dependent L‐Arg hydroxylase in L‐End biosynthesis in Streptomyces wadayamensis. It reacts with L‐Arg and dioxygen to yield two products, 2‐ketoarginine and 4‐hydroxy‐2‐ketoarginine in a ratio of ~1.7:1. Surprisingly, 1 equivalent of H2O2 is produced for each equivalent of O2 consumed. In addition, the ratio of O2 consumption to L‐Arg consumption was ~1.4:1, suggesting that one product requires 2 equivalents of O2, and the other only 1 equivalent. We have determined the structures of MppP in four states: the internal aldimine, the external aldimine with the substrate, L‐Arg, the product complex with 4‐hydroxy‐2‐ketoarginine, and the product complex with 2‐ketoarginine. According to the structures, the N‐terminal helix is disordered in the internal aldimine and covers the active site only when the substrate is bound. A glutamate residue in the N‐terminal helix, E15, makes a hydrogen bonding interaction with the carboxylate of the substrate, L‐Arg. This observation prompted us to make three N‐terminal variants of SwMppP: E15A, E15Q, and the truncation mutant, SwMppP23‐276. Steady state kinetics showed that the two point mutants had no effect on kcat or KM,L‐Arg. The truncation mutant, however, showed an approximately 10‐fold increase in KM and a 3‐fold decrease in kcat, which reduced the pseudo‐second order rate constant by almost 25‐fold. Interestingly, although the steady state kinetics (as measured by dioxygen consumption) are indistinguishable for the E15A and E15Q variants, the E15A variant does not produce 4‐hydroxy‐2‐ketoarginine, only the abortive product 2‐ketoarginine. Likewise, the truncation mutant also produced only 2‐ketoarginine. Our structural and kinetic characterization of the wild‐type and variant forms of SwMppP have allowed us to propose a revised model where the oxygen incorporated in the hydroxy‐arginine product is derived from water rather than from dioxygen.Support or Funding InformationNational Science Foundation CHE‐1606842

Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics.

Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo-LipD is very similar to other four-helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo-LipD are more restricted than in apo-LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo-LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg2+ or Ca2+ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo-LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS-ACP from A. friuliensis that would allow the acyl-CoA ligase to interact preferentially with the LipD instead of binding to the FAS-ACP.

Patients hospitalized with an infective exacerbation of bronchiectasis unrelated to cystic fibrosis: Clinical, physiological and sputum characteristics.

ABSTRACTBackground and objectiveBronchiectasis is a growing health burden both globally and in Australasia. Associated with repeated respiratory infections, the disease often results in hospital admission, impaired quality of life, reduced lung function and shortened life expectancy. We describe the local clinical, physiological and sputum characteristics in patients hospitalized with an infective exacerbation of bronchiectasis.MethodsThis study examined the medical records of all 61 adults admitted to a metropolitan Australian hospital with an infective exacerbation of bronchiectasis in a calendar year.ResultsBaseline characteristics include: mean (SD) age of participants was 66 (14) years; 56% were women and 42% were current or ex‐smokers. The majority had other coexisting medical conditions, with asthma in 44%, COPD in 59% and both asthma and COPD in 31%. Seventy‐two percent were on regular inhaled medication, 23% on cyclical antibiotics and 26% undertook regular respiratory physiotherapy. Bronchodilator reversibility was present in 17% and small airway reversibility in 41%. Sputum demonstrated normal flora in 17%, Pseudomonas aeruginosa in 32%, Haemophilus influenzae in 15% and both organisms in 17%. Mean numbers of exacerbations per year requiring hospitalization was 2.3. Sixty‐two percent of subjects had an Index of Relative Socio‐Economic Disadvantage in deciles 1–5. Risk factors for exacerbations included a history of asthma or COPD, documented small airway reversibility and presence of P. aeruginosa.ConclusionPatients hospitalized with an infective exacerbation of bronchiectasis are predominantly older with co‐morbidities and of lower socio‐economic status. Presence of P. aeruginosa was a risk factor for repeated exacerbations, as was a history of asthma, COPD or small airway reversibility.

ITRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus

LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD(+)H, NADP(+)H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold <0.5)). Based on proteome analysis, the putative pathways of AMP-jsa9 action against B. cereus can be summarized as: (i) inhibition of bacterial sporulation, thiamine biosynthesis, energy metabolism, DNA transcription and translation, and cell wall biosynthesis, through direct regulation of protein levels; and (ii) indirect effects on the same pathways through the accumulation of ROS and the consequent impairment of cellular functions, resulting from downregulation of antioxidant proteins, especially CAT and SOD. Biological significanceThe mode of action of LI-F type antimicrobial peptides (AMP-jsa9) against B. cereus was elucidated at the proteomic level. Two pathways of AMP-jsa9 action upon B. cereus cells were identified and the mechanism of bleb formation on the surfaces of bacterial cells was predicted based on the results of ultrastructural observation and proteomic analysis. These results are helpful in understanding the mechanism of LI-F type peptides and in providing the theoretical base for applying AMP-jsa9 or its analogs to combat Gram-positive pathogenic bacteria in the food and feed industries.

Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications

The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies.

PWE-047 Metronidazole is A Less Effective Therapy for Small Intestinal Bacterial Overgrowth

Introduction Choice of antibiotic treatment in patients who test positively for Small Intestinal Bacterial Overgrowth (SIBO) can be difficult. No controlled trial has examined the clinical efficacy of antibiotics in SIBO. This study aimed to identify which antibiotics are the most effective in treating SIBO in a real world retrospective cohort. Methods 520 adult patients (360 female, 160 male, mean age 49) who underwent hydrogen and glycocholate-methane breath tests for a suspicion of SIBO between the dates 01/01/2010 and 31/12/2013 were evaluated. The clinical information of patients who tested positively in both tests was recorded. Information was also gathered as to the antibiotic they were prescribed and whether they received symptomatic benefit from their treatment. Results A total of 90 patients who tested positive were prescribed antibiotics (59 female, 31 male, mean age 56). 69 (76.7%) of these patients benefitted from their management. 32 patients who tested positive were given cyclical antibiotics. The efficacy of cyclical antibiotics compared with the combined efficacy rates of other antibiotic regimes in treating patients with a positive breath test result was 93.8%. (93.8% vs 67.2%, Chi-square = 8.1, p = 0.004). 15 patients who tested positive were given rifaximin. The efficacy of rifaximin in treating patients with a positive breath test result compared with the combined efficacy of other antibiotic regimes was 80%. (80% vs 76% Chi-square = 0.112, p = 0.738). 18 patients who tested positive were given metronidazole. The efficacy of metronidazole in treating patients who tested positive in the breath tests compared with combined efficacy rates of other antibiotics was much lower at 50% (50% vs 83%, Chi-square = 8.9441, p = 0.0028). Conclusion Metronidazole appeared inferior to rifaximin monotherapy for treating SIBO. Cyclical antibiotics appeared most effective. These findings should be explored further in randomised controlled prospective trials to determine the optimal treatment regime. Disclosure of Interest None Declared

Chitosan-Based Film of Tyrothricin for Enhanced Antimicrobial Activity against Common Skin Pathogens Including Staphylococcus aureus.

Chitosan-based film-forming gel is regarded as a promising vehicle for topical delivery of antimicrobial agents to skin wounds, since it protects from microbial infection and the cationic polymer itself possesses antibacterial activity. In this study, possible synergistic interaction against common skin pathogens between the cationic polymer and tyrothricin (TRC), a cyclic polypeptide antibiotic, was investigated, by determining the concentration to inhibit 90% of bacterial isolates (MIC). The addition of the polysaccharide to TRC dramatically reduced the MIC values of TRC by 1/33 and 1/4 against both methicillin-resistant and methicillinsusceptible Staphylococcus aureus, respectively. The synergism of TRC and chitosan combination against both strains was demonstrated by the checkerboard method, with a fractional inhibitory concentration index below 0.5. Moreover, co-treatment of TRC and chitosan exhibited antibacterial activity against Pseudomonas aeruginosa, due to the antibacterial activity of chitosan, whereas TRC itself did not inhibit the gram-negative bacterial growth. These findings suggested that the use of chitosan-based film for topical delivery of TRC could be an alternative to improve TRC antimicrobial activity against strains that are abundant in skin wounds.

How to Test and Treat Small Intestinal Bacterial Overgrowth: an Evidence-Based Approach.

Small intestinal bacterial overgrowth (SIBO) is characterized by an excessive amount of bacteria in the small intestine and a constellation of symptoms that include bloating, pain, gas, and diarrhea. Although known for many decades, there is a lack of consensus and clarity regarding the natural history and methods for its diagnosis. Several tests have been proposed, including the glucose breath test, lactulose breath test, small intestinal aspiration and culture, and others. However, there is a lack of standardization of these tests and their interpretation. Treatment of SIBO remains empirical; generally, broad spectrum antibiotics are recommended for 2 weeks (amoxicillin, rifaximin, ciprofloxacin, etc.) but evidence for their use is fair. Clearly, there is a strong need to develop a systematic approach for the management of SIBO and to perform multicenter clinical trials for the treatment of SIBO. In this review, we will discuss the current evidence for the diagnosis and treatment of SIBO, which includes (1) elimination/modification of the underlying causes, (2) induction of remission (antibiotics and elemental diet), and (3) maintenance of remission (promotility drugs, dietary modifications, repeat or cyclical antibiotics).

Solid-phase synthesis and in vitro biological activity of a Thr4→Ser4 analog of daptomycin.

Daptomycin is a Ca(+2)-dependent cyclic lipodepsipeptide antibiotic used clinically to treat serious infections caused by Gram-positive bacteria. The recent appearance of daptomycin-resistant strains, daptomycin's lack of activity in the presence of lung surfactant, and its incompletely understood mechanism of action underscores the need for establishing detailed structure-activity relationships. Here we report a solid-phase synthesis of a daptomycin analog in which Thr4, 3-MeGlu12 and Kyn13 in daptomycin were replaced with Ser, Glu and Trp residues, respectively (Dap-S4-E12-W13). The Thr4 to Ser4 substitution was detrimental to activity, as Dap-S4-E12-W13 was at least 20-fold less active at physiological Ca(+2) concentration than Dap-E12-W13. Much of its activity could be recovered at high (100 mM) Ca(+2) concentration, suggesting that the residue at position 4 affects Ca(+2) binding and, consequently, biological activity.

Structure and Antibacterial Activity of Ambobactin, a New Telomycin-Like Cyclic Depsipeptide Antibiotic Produced by Streptomyces ambofaciens F3.

A new telomycin-like cyclic depsipeptide, ambobactin (1), was isolated from the metabolites of Streptomyces ambofaciens F3, an endophyte of Platycladus orientalis. Its structure was elucidated on the basis of extensive spectroscopic analysis and advanced Marfey’s method. Ambobactin is structurally related with telomycin, except that the configuration of the 3-methyltryptophanes in their structures is different. It exhibited strong antibacterial activity against both Gram-positive and Gram-negative bacteria. Furthermore, this investigation revealed that S. ambofaciens F3 is a new producer of telomycin-like antibiotics.

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus.

The rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MS(n) spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.

Examination of a Structural Model of Peptidomimicry by Cyclic Acyldepsipeptide Antibiotics in Their Interaction with the ClpP Peptidase.

The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N-acylphenylalanine is thought to structurally mimic the ClpP-binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N-acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N-capped with methyl-branched acyl groups-like the side chains of residues in the (I/L)GF motifs-were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N-acylphenylalanine moiety is not simply a stand-in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl-branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.

Intérêt de l’antibioprophylaxie et manifestations urologiques des femmes blessées médullaires au cours de la grossesse

More and more women with spinal cord injury (SCI) can have a pregnancy. Few data have been published on these pregnancies and their urological impact. We report a series of these cases illustrating the urological experience of specialized centers. This is a retrospective study that identified pregnancies in women with SCI followed-up between 2000 and 2014. Data covering all urological and obstetric events during pregnancies, before and after implementation of weekly oral cyclic antibiotics (WOCA) program, were collected from medical records. Fifteen women with SCI who gave birth to 20 children were included. Three of them were quadriplegics and 12 were paraplegics. All of them performed themselves intermittent catheterization: 11 by urethral way and 4 by high way because carrying a continent cystostomy. Mean follow-up period before pregnancy was 14.6 years [3-27 years IC 95%] and the mean follow-up for the study was 8.5 months [6.5-9 IC 95%]. We observed a reduction of symptomatic urinary tract infections after WOCA onset (13/13 before vs. 0/7 after, P=0.001), a reduction of recurrent urinary infections (6/13 vs. 0/7, P=0.03), a reduction of threats to premature births (8/13 vs. 0/7, P=0.001). Multi-resistant bacteria appeared to increase (0/13 vs. 3/7, P=0.01). We also observed a trend to significant reduction of premature births number (4/13 vs. 0/13, P=0.1) and that of low birth weight (3/13 versus 0/7, P=0.1). The overall neonatal survival rate was 100%. Our study before/after introduction of a weekly oral cycle antibiotic prophylaxis during pregnancy for women with spinal cord injury showed a significant reduction in symptomatic urinary tract infections and a tendency to improve pregnancy outcomes. 4.

Autophagy regulates colistin-induced apoptosis in PC-12 cells.

Colistin is a cyclic cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. Our recent study demonstrated that colistin induces apoptosis in primary chick cortex neurons and PC-12 cells. Although apoptosis and autophagy have different impacts on cell fate, there is a complex interaction between them. Autophagy plays an important role as a homeostasis regulator by removing excessive or unnecessary proteins and damaged organelles. The aim of the present study was to investigate the modulation of autophagy and apoptosis regulation in PC-12 cells in response to colistin treatment. PC-12 cells were exposed to colistin (125 to 250 μg/ml), and autophagy was detected by visualization of monodansylcadaverine (MDC)-labeled vacuoles, LC3 (microtubule-associated protein 1 light chain 3) immunofluorescence microscopic examination, and Western blotting. Apoptosis was measured by flow cytometry, Hoechst 33258 staining, and Western blotting. Autophagosomes were observed after treatment with colistin for 12 h, and the levels of LC3-II gene expression were determined; observation and protein levels both indicated that colistin induced a high level of autophagy. Colistin treatment also led to apoptosis in PC-12 cells, and the level of caspase-3 expression increased over the 24-h period. Pretreatment of cells with 3-methyladenine (3-MA) increased colistin toxicity in PC-12 cells remarkably. However, rapamycin treatment significantly increased the expression levels of LC3-II and beclin 1 and decreased the rate of apoptosis of PC-12 cells. Our results demonstrate that colistin induced autophagy and apoptosis in PC-12 cells and that the latter was affected by the regulation of autophagy. It is very likely that autophagy plays a protective role in the reduction of colistin-induced cytotoxicity in neurons.

Solid-Phase Total Synthesis of Daptomycin and Analogs

An entirely solid-phase synthesis of daptomycin, a cyclic lipodepsipeptide antibiotic currently in clinical use, was achieved using a combination of α-azido and Fmoc amino acids. This methodology was applied to the synthesis of several daptomycin analogs, one of which did not contain kynurenine or the synthetically challenging amino acid (2S,3R)-methylglutamate yet exhibited an MIC approaching that of daptomycin.

New antibiotics produced by Bacillus subtilis strains

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mes6nteroides VKPM B-4177 resistant to glycopep-> tide antibiotics, as well as antifungal activity. The strains were identified as belonging to the "B. subtilis" com- plex based on their morphological and physiological characteristics, as well as by sequencing of the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaen and pentaen, respectively). Strain INA 01086 produced also a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.

In Vitro Antibacterial Screening of Six Proline-Based Cyclic Dipeptides in Combination with β-Lactam Antibiotics Against Medically Important Bacteria

The in vitro synergistic antibacterial activity of six proline-based cyclic dipeptides [cyclo(D-Pro-L-Leu), cyclo(L-Pro-L-Met), cyclo(D-Pro-L-Phe), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-D-Tyr)] in combination imipenem and ceftazidime was investigated in the present manuscript. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the cyclic dipeptides were compared with those of the standard antibiotics (imipenem and ceftazidime). The synergistic antibacterial activities of cyclic dipeptides with imipenem or ceftazidime were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effect of six cyclic dipeptides with imipenem predominantly recorded synergistic interaction (FIC index <0.5), whereas combination of certain cyclic dipeptides with ceftazidime recorded additive. The time-kill curve showed that the growth of the test bacteria was completely attenuated after 12-24h of treatment with a 50:50 ratio of proline-based cyclic dipeptides and antibiotics. These synergistic effects have a potential role in delaying the development of resistance as the antibacterial activity is achieved with the very low concentrations of cyclic dipeptides and antibiotics. The cytotoxicity of cyclic dipeptides was tested against VERO cell line (African green monkey kidney cell line), and no cytotoxicity was recorded for cyclic dipeptides up to 100μg/mL. These findings suggest that combination of cyclic dipeptides and antibiotics might be a good strategy for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for antimicrobial chemotherapy. Moreover, these combinations may lead to the development of a new and vital antimicrobial combination against the infections caused by pathogenic bacteria. The in vitro synergistic activity of cyclic dipeptides with antibiotics against medically important bacteria is reported here for the first time.

Colistin-induced apoptosis in PC12 cells: Involvement of the mitochondrial apoptotic and death receptor pathways

Colistin, a cyclic cationic polypeptide antibiotic that is used to treat infections, may cause neurotoxicity. However, whether colistin can induce apoptosis and the precise mechanism of apoptosis involved in PC12 cells remains to be determined. The aim of the present study was to determine reactive oxygen species (ROS) level and DNA damage, as well as apoptotic factors such as p53, cytochrome c, Bax, Bcl-2, Fas, Fas-L and caspase family via western blotting in PC12 cells treated with colistin sulfate. The results showed that colistin sulfate increased ROS levels significantly. An increase of ROS levels induces the release of cytochrome c and DNA damage. DNA damage can activate p53, which leads to the upregulation of Bax and downregulation of Bcl-2. The imbalance of Bax/Bcl-2 promotes additional release of cytochrome c. The release of cytochrome c contributes to the activation of caspase-9 and the subsequent activation of caspase-3. An increase of Fas and Fas-L induced the activation of caspase-8 leading to the activation of caspases-3, the latter induces apoptosis. Therefore, these results demonstrate that the apoptotic pathway of colistin-induced apoptosis in PC12 cells is involved in both the mitochondrial and death receptor pathway.

Restriction of the conformational dynamics of the cyclic acyldepsipeptide antibiotics improves their antibacterial activity.

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP–ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.

The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.