Inhibitors Of Cell Wall Biosynthesis Research Articles

Molecular Dynamics Simulation of Atomic Interactions in the Vancomycin Binding Site.

Vancomycin is a glycopeptide antibiotic produced by Amycolaptopsis orientalis used to treat serious infections by Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. Vancomycin inhibits cell wall biosynthesis by targeting lipid II, which is the membrane-bound peptidoglycan precursor. The heptapeptide aglycon structure of vancomycin binds to the d-Ala-d-Ala of the pentapeptide stem structure in lipid II. The third residue of vancomycin aglycon is asparagine, which is not directly involved in the dipeptide binding. Nonetheless, asparagine plays a crucial role in substrate recognition, as the vancomycin analogue with asparagine substituted by aspartic acid (VD) shows a reduction in antibacterial activities. To characterize the function of asparagine, binding of vancomycin and its aspartic-acid-substituted analogue VD to l-Lys-d-Ala-d-Ala and l-Lys-d-Ala-d-Lac was investigated using molecular dynamic simulations. Binding interactions were analyzed using root-mean-square deviation (RMSD), two-dimensional (2D) contour plots, hydrogen bond analysis, and free energy calculations of the complexes. The analysis shows that the aspartate substitution introduced a negative charge to the binding cleft of VD, which altered the aglycon conformation that minimized the repulsive lone pair interaction in the binding of a depsipeptide. Our findings provide new insight for the development of novel glycopeptide antibiotics against the emerging vancomycin-resistant pathogens by chemical modification at the third residue in vancomycin to improve its binding affinity to the d-Ala-d-Lac-terminated peptidoglycan in lipid II found in vancomycin-resistant enterococci and vancomycin-resistant S. aureus.

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis.

Resistance against antimicrobial peptides (AMPs) is often mediated by detoxification modules that rely on sensing the AMP through a BceAB-like ATP-binding cassette (ABC) transporter that subsequently activates a cognate two-component system (TCS) to mount the cell response. Here, the Lactococcus lactis ABC transporter YsaDCB is shown to constitute, together with TCS-G, a detoxification module that protects L. lactis against bacitracin and the bacteriocin Lcn972, both AMPs that inhibit cell wall biosynthesis. Initially, increased expression of ysaDCB was detected by RT-qPCR in three L. lactis resistant to Lcn972, two of which were also resistant to bacitracin. These mutants shared, among others, single-point mutations in ysaB coding for the putative Bce-like permease. These results led us to investigate the function of YsaDCB ABC-transporter and study the impact of these mutations. Expression in trans of ysaDCB in L. lactis NZ9000, a strain that lacks a functional detoxification module, enhanced resistance to both AMPs, demonstrating its role as a resistance factor in L. lactis. When the three different ysaB alleles from the mutants were expressed, all of them outperformed the wild-type transporter in resistance against Lcn972 but not against bacitracin, suggesting a distinct mode of protection against each AMP. Moreover, PysaD promoter fusions, designed to measure the activation of the detoxification module, revealed that the ysaB mutations unlock transcriptional control by TCS-G, resulting in constitutive expression of the ysaDCB operon. Finally, deletion of ysaD was also performed to get an insight into the function of this gene. ysaD encodes a secreted peptide and is part of the ysaDCB operon. YsaD appears to modulate signal relay between the ABC transporter and TCS-G, based on the different response of the PysaD promoter fusions when it is not present. Altogether, the results underscore the unique features of this lactococcal detoxification module that warrant further research to advance in our overall understanding of these important resistance factors in bacteria.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Synthetic-Bioinformatic Natural Product Antibiotics with Diverse Modes of Action.

Bacterial natural products have inspired the development of numerous antibiotics in use today. As resistance to existing antibiotics has become more prevalent, new antibiotic lead structures and activities are desperately needed. An increasing number of natural product biosynthetic gene clusters, to which no known molecules can be assigned, are found in genome and metagenome sequencing data. Here we access structural information encoded in this underexploited resource using a synthetic-bioinformatic natural product (syn-BNP) approach, which relies on bioinformatic algorithms followed by chemical synthesis to predict and then produce small molecules inspired by biosynthetic gene clusters. In total, 157 syn-BNP cyclic peptides inspired by 96 nonribosomal peptide synthetase gene clusters were synthesized and screened for antibacterial activity. This yielded nine antibiotics with activities against ESKAPE pathogens as well as Mycobacterium tuberculosis. Not only are antibiotic-resistant pathogens susceptible to many of these syn-BNP antibiotics, but they were also unable to develop resistance to these antibiotics in laboratory experiments. Characterized modes of action for these antibiotics include cell lysis, membrane depolarization, inhibition of cell wall biosynthesis, and ClpP protease dysregulation. Increasingly refined syn-BNP-based explorations of biosynthetic gene clusters should allow for more rapid identification of evolutionarily inspired bioactive small molecules, in particular antibiotics with diverse mechanism of actions that could help confront the imminent crisis of antimicrobial resistance.

Tuberculosis and beta-lactam antibiotics.

Tuberculosis (TB) is one of the oldest health problems in the world and it remains unresolved. Multidrug-resistant-TB and extensively resistant-TB are a serious problem for control programs. The evaluation of available antibiotics has gained importance in recent years for the treatment of resistant TB. Beta-lactam antibiotics inhibit cell wall biosynthesis in the bacteria; the presence of beta-lactamase enzyme in TB bacilli raises the question of whether this group of antibiotics can be used in treatment. As a result, it has been reported that the combination of beta-lactam antibiotics with beta-lactamase is effective against Mycobacterium tuberculosis both in vitro and in vivo. The aim of this article is to review and discuss up-to-date knowledge and future perspective on beta-lactam antibiotics and TB.

Vancomycin C-Terminus Guanidine Modifications and Further Insights into an Added Mechanism of Action Imparted by a Peripheral Structural Modification.

A series of vancomycin C-terminus guanidine modifications is disclosed that improves antimicrobial activity, enhances the durability of antimicrobial action against selection or induction of resistance, and introduces a synergistic mechanism of action independent of d-Ala-d-Ala binding and inhibition of cell wall biosynthesis. The added mechanism of action results in induced bacterial cell permeability, which we show may involve interaction with cell envelope teichoic acid. Significantly, the compounds examined that contain two combined peripheral modifications, a (4-chlorobiphenyl)methyl (CBP) and C-terminus guanidinium modification, offer opportunities for new treatments against not only vancomycin-sensitive but especially vancomycin-resistant bacteria where they act by two synergistic and now durable mechanisms of action independent of d-Ala-d-Ala/d-Lac binding and display superb antimicrobial potencies (MIC 0.6-0.15 μg/mL, VanA VRE). For the first time, we demonstrate that the synergistic behavior of the peripheral modifications examined requires the presence of both the CBP and guanidine modifications in a single molecule versus their combined use as an equimolar mixture of singly modified compounds. Finally, we show that a prototypical member of the series, G3-CBP-vancomycin (15), exhibits no hemolytic activity, displays no mammalian cell growth inhibition, possesses improved and especially attractive in vivo pharmacokinetic (PK) properties, and displays excellent in vivo efficacy and potency against an especially challenging multidrug-resistant (MRSA) and VanA vancomycin-resistant (VRSA) Staphylococcus aureus bacterial strain.

Bacterial cell wall modification by glycolipid probes

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. The structures of mammalian glycans can be perturbed through metabolic incorporation, in which non‐natural sugars are taken up by cells, converted to nucleotide‐sugar intermediates, and incorporated via biosynthetic pathways. Applications of metabolic incorporation in bacteria are complicated by the diversity of pathways bacteria use to acquire, metabolize, and catabolize monosaccharide intermediates. We developed a strategy to alter glycan structure that avoids intracellular processing using lipid‐linked glycans, intermediates directly used in glycan biosynthesis. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified glycolipid donors whose addition restores cell wall arabinan, demonstrating that non‐natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. Our lipid‐based probe could rescue wild type cells treated with an inhibitor of cell wall biosynthesis. We then focused on incorporating derivatives with chemical handles for labeling such that bacterial glycans can be captured and visualized. To this end, we have generated the first probe to label arabinose‐containing structures in mycobacteria and corynebacteria.Support or Funding InformationNIH: RO1‐AI063596, NSERC PGS‐D

From Modules to Networks: a Systems-Level Analysis of the Bacitracin Stress Response in Bacillus subtilis.

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor.IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

Cellular Noise and Response to Antibiotics

Genetically identical cells express different phenotypes, even when they experience identical environmental conditions. This form of cellular noise includes differences in the rates of growth, as well as the probability of survival against antibiotic pressure. To better understand the relationship between cellular growth and death by antibiotics, we explore how wild type and antibiotic resistant Escherichia coli bacteria survive drug pressure. Specifically, we investigate the effects of ampicillin, a cell wall biosynthesis inhibitor, on single cells using microfluidics and bioimaging. We will present our experimental approach, as well as our findings relating cellular noise during growth to various survival strategies against antibiotic pressure.

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Comparative Metabolomics Reveals Key Pathways Associated With the Synergistic Killing of Colistin and Sulbactam Combination Against Multidrug-Resistant Acinetobacter baumannii.

Background: Polymyxins are a last-line class of antibiotics against multidrug-resistant Acinetobacter baumannii. However, polymyxin resistance can emerge with monotherapy, highlighting the need for synergistic combination therapies. Polymyxins in combination with β-lactams have shown remarkable synergy against multidrug-resistant A. baumannii. Methods: Liquid chromatography–mass spectrometry-based metabolomics was conducted to investigate the metabolic perturbations in an A. baumannii clinical isolate, AB090342, in response to colistin (1 mg/L), sulbactam (128 mg/L), and their combination at 1, 4, and 24 h. Metabolomics data were analyzed using univariate and multivariate statistics, and metabolites showing ≥2-fold changes were subjected to pathway analysis. Results: The synergistic activity of colistin–sulbactam combination was initially driven by colistin through perturbation of fatty acid and phospholipid levels at 1 h. Cell wall biosynthesis was perturbed by sulbactam alone and the combination over 24 h; this was demonstrated by the decreased levels of two important precursors, uridine diphosphate-N-acetylglucosamine and uridine diphosphate-N-acetylmuramate, together with perturbed lysine and amino sugar metabolism. Moreover, sulbactam alone and the combination significantly depleted nucleotide metabolism and the associated arginine biosynthesis, glutamate metabolism, and pentose phosphate pathway. Notably, the colistin–sulbactam combination decreased amino acid and nucleotide levels more dramatically at 4 h compared with both monotherapies. Conclusions: This is the first metabolomics study revealing the time-dependent synergistic activity of colistin and sulbactam against A. baumannii, which was largely driven by sulbactam through the inhibition of cell wall biosynthesis. Our mechanistic findings may help optimizing synergistic colistin combinations in patients.

Bacterial Cell Wall Modification with a Glycolipid Substrate.

Despite the ubiquity and importance of glycans in biology, methods to probe their structures in cells are limited. Mammalian glycans can be modulated using metabolic incorporation, a process in which non-natural sugars are taken up by cells, converted to nucleotide-sugar intermediates, and incorporated into glycans via biosynthetic pathways. These studies have revealed that glycan intermediates can be shunted through multiple pathways, and this complexity can be heightened in bacteria, as they can catabolize diverse glycans. We sought to develop a strategy that probes structures recalcitrant to metabolic incorporation and that complements approaches focused on nucleotide sugars. We reasoned that lipid-linked glycans, which are intermediates directly used in glycan biosynthesis, would offer an alternative. We generated synthetic arabinofuranosyl phospholipids to test this strategy in Corynebacterium glutamicum and Mycobacterium smegmatis, organisms that serve as models of Mycobacterium tuberculosis. Using a C. glutamicum mutant that lacks arabinan, we identified synthetic glycosyl donors whose addition restores cell wall arabinan, demonstrating that non-natural glycolipids can serve as biosynthetic intermediates and function in chemical complementation. The addition of an isotopically labeled glycan substrate facilitated cell wall characterization by NMR. Structural analysis revealed that all five known arabinofuranosyl transferases could process the exogenous lipid-linked sugar donor, allowing for the full recovery of the cell envelope. The lipid-based probe could also rescue wild-type cells treated with an inhibitor of cell wall biosynthesis. Our data indicate that surrogates of natural lipid-linked glycans can intervene in the cell's traditional workflow, indicating that biosynthetic incorporation is a powerful strategy for probing glycan structure and function.

The Lasso Peptide Siamycin-I Targets Lipid II at the Gram-Positive Cell Surface.

Ribosomally synthesized post-translationally modified peptides (RiPPs) are a diverse class of biologically active molecules produced by many environmental bacteria. While thousands of these compounds have been identified, mostly through genome mining, a relatively small number has been investigated at the molecular level. One less understood class of RiPPs is the lasso peptides. These are 20-25 amino acid residue compounds bearing an N-terminal macrocyclic ring and a C-terminal tail that is threaded through the ring. We have carried out a detailed investigation on the mechanism of action of the siamycin-I lasso peptide. We demonstrate that siamycin-I interacts with lipid II, the central building block of the major cell wall component peptidoglycan, which is readily accessible on the outside of the cell. This interaction compromises cell wall biosynthesis in a manner that activates the liaI stress response. Additionally, resistance to siamycin-I can be brought about by mutations in the essential WalKR two-component system that causes thickening of the cell wall. Siamycin-I is the first lasso peptide that has been shown to inhibit cell wall biosynthesis.

Transcriptional profiling and localization of GUL-1, a COT-1 pathway component, in Neurospora crassa

Impairment of theNeurospora crassaCOT-1 kinase results in defects in hyphal polarity. Some of these effects are partially suppressed by inactivation of gul-1 (encoding an mRNA-binding protein involved in translational regulation). Here, we report on the transcriptional profiling of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism. The GUL-1 protein itself was found to be distributed within the entire hyphal cell, along with a clear presence of aggregates that traffic within the cytoplasm. Live imaging of GUL-1-GFP demonstrated that GUL-1 transport is microtubule-dependent. Cellular stress, as imposed by the presence of the cell wall biosynthesis inhibitor Nikkomycin Z or by nitrogen limitation, resulted in a 2–3-fold increase of GUL-1 aggregate association with nuclei. Taken together, this study demonstrates that GUL-1 affects multiple processes, its function is stress-related and linked with cellular traffic and nuclear association.

The Role of Greasy Residues in Teixobactin Derivatives

Teixobactin is a novel antibiotic that inhibits cell wall biosynthesis by binding to cell wall precursors, such as lipid II. Previously, we characterized multiple conformations of the teixobactin-lipid II complex in membrane using modeling and molecular dynamics simulations (Chem. Sci., 2018, 9:6997-7008). Common structural features shared by these conformations include pyrophosphate coordination by the C-terminal cyclodepsipeptide ring and membrane anchoring by the mid-chain hydrophobic residues. Since the cell wall precursors exist in low concentrations at bacterial surfaces, it is likely that teixobactin binds and inserts into the membrane before reaching its targets. Here, we performed microsecond-scale molecular dynamics simulations to investigate the membrane binding and insertion of teixobactin and a highly potent derivative (D-Arg4/Ile10-teixobactin) in the absence of their target molecules. Multiple membrane models were employed in our simulations, including the Highly Mobile Membrane Mimetic model and full-length phospholipid bilayers, consisting of either zwitterionic phosphatidylethanolamines or anionic phosphatidylglycerols. We found that teixobactin derivatives always anchor to the membrane using residues N-Me-D-Phe1, Ile2, D-allo-Ile5, Ile6, and Ile11. In particular, the insertion depths of the N-terminal hydrophobic residues are consistently comparable to those of the mid-chain hydrophobic anchors, implying they also play a significant role in membrane insertion. Additionally, the membrane insertion of Ile11 restricts the orientation of the cyclodepsipeptide ring at the membrane surface, suggesting that one of the lipid II-bound forms characterized in our earlier work would be more prominent due to a compatible cyclodepsipeptide ring orientation. In summary, almost half of the side chains in teixobactin participate in membrane-anchoring and not direct lipid II-binding. These hydrophobic residues appear invariable in several studies of the structure-activity relationship of teixobactin derivatives, suggesting that membrane binding and insertion act as an essential step in its antibacterial mechanism.

Identification of potential inhibitors for Penicillinbinding protein (PBP) from Staphylococcus aureus.

Staphylococcus aureus is an infectious agent that causes severe skin and soft tissue infection in hospitalized patients. Therefore, it is of interest to develop potent inhibitors for S. aureus. Penicillin Binding protein (PBP) is a known drug target for inhibition of cell wall biosynthesis in S. aureus. Hence, PBP was screened with compounds from six databases using virtual screening approaches. Results shows that the screened lead compound produced higher docking score (-9.87 kcal/mol) compared to resistant drugs. Antimicrobial activity using screened lead compounds and resistant drugs showed maximum activity in potential screened compounds compared to resistant compounds.

Synergistic Lethality of a Binary Inhibitor of Mycobacterium tuberculosis KasA.

We report GSK3011724A (DG167) as a binary inhibitor of β-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production

Methanol is a promising feedstock for bioproduction of fuels and chemicals, thus massive efforts have been devoted to engineering non-native methylotrophic platform microorganisms to utilize methanol. Herein, we rationally designed and experimentally engineered the industrial workhorse Corynebacterium glutamicum to serve as a methanol-dependent synthetic methylotroph. The cell growth of the methanol-dependent strain relies on co-utilization of methanol and xylose, and most notably methanol is an indispensable carbon source. Due to the methanol-dependent characteristic, adaptive laboratory evolution was successfully applied to improving methanol utilization. The evolved mutant showed a 20-fold increase in cell growth on methanol-xylose minimal medium and utilized methanol and xylose with a high mole ratio of 3.83:1. 13C-labeling experiments demonstrated that the carbon derived from methanol was assimilated into intracellular building blocks, high-energy carriers, cofactors, and biomass (up to 63% 13C-labeling). By inhibiting cell wall biosynthesis, methanol-dependent glutamate production was also achieved, demonstrating the potential application in bioconversion of methanol into useful chemicals. Genetic mutations detected in the evolved strains indicate the importance of intracellular NAD+/NADH ratio, substrate uptake, and methanol tolerance on methanol utilization. This study reports significant improvement in the area of developing fully synthetic methylotrophs.

Increasing the Antimicrobial Activity of Nisin-Based Lantibiotics against Gram-Negative Pathogens.

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial compounds containing lanthionine and methyl-lanthionine residues. Nisin, one of the most extensively studied and used lantibiotics, has been shown to display very potent activity against Gram-positive bacteria, and stable resistance is rarely observed. By binding to lipid II and forming pores in the membrane, nisin can cause the efflux of cellular constituents and inhibit cell wall biosynthesis. However, the activity of nisin against Gram-negative bacteria is much lower than that against Gram-positive bacteria, mainly because lipid II is located at the inner membrane, and the rather impermeable outer membrane in Gram-negative bacteria prevents nisin from reaching lipid II. Thus, if the outer membrane-traversing efficiency of nisin could be increased, the activity against Gram-negative bacteria could, in principle, be enhanced. In this work, several relatively short peptides with activity against Gram-negative bacteria were selected from literature data to be fused as tails to the C terminus of either full or truncated nisin species. Among these, we found that one of three tails (tail 2 [T2; DKYLPRPRPV], T6 [NGVQPKY], and T8 [KIAKVALKAL]) attached to a part of nisin displayed improved activity against Gram-negative microorganisms. Next, we rationally designed and reengineered the most promising fusion peptides. Several mutants whose activity significantly outperformed that of nisin against Gram-negative pathogens were obtained. The activity of the tail 16 mutant 2 (T16m2) construct against several important Gram-negative pathogens (i.e., Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes) was increased 4- to 12-fold compared to that of nisin. This study indicates that the rational design of nisin can selectively and significantly improve its outer membrane-permeating capacity as well as its activity against Gram-negative pathogens.IMPORTANCE Lantibiotics are antimicrobial peptides that are highly active against Gram-positive bacteria but that have relatively poor activity against most Gram-negative bacteria. Here, we modified the model lantibiotic nisin by fusing parts of it to antimicrobial peptides with known activity against Gram-negative bacteria. The appropriate selection of peptidic moieties that could be attached to (parts of) nisin could lead to a significant increase in its inhibitory activity against Gram-negative bacteria. Using this strategy, hybrids that outperformed nisin by displaying 4- to 12-fold higher levels of activity against relevant Gram-negative bacterial species were produced. This study shows the power of modified peptide engineering to alter target specificity in a desired direction.

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in Aspergillus fumigatus.

RasA is a major regulator of fungal morphogenesis and virulence in Aspergillus fumigatus. The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order. These PTMs include: (1) CAAX motif farnesylation by the farnesyltransferase complex composed of Ram1p and Ram2p; (2) proteolysis of the -AAX residues by Rce1p or Ste24p; (3) methylation of the remaining prenylated cysteine residue by Ste14p, and; (4) palmitoylation at a single conserved cysteine residue mediated by the Erf2p/Erf4p palmitoyltransferase. We previously reported that homologs of each RasA PTM enzyme are conserved in A. fumigatus. Additionally, we delineated a major role for protein farnesylation in A. fumigatus growth and virulence. In this work, we characterize the post-prenylation processing enzymes of RasA in A. fumigatus. The genes encoding the RasA post-prenylation enzymes were first deleted and examined for their roles in growth and regulation of RasA. Only when strains lacked cppB, the A. fumigatus homologue of yeast RCE1, there was a significant reduction in fungal growth and conidial germination. In addition, cppB-deletion mutants displayed hypersensitivity to the cell wall-perturbing agents Calcofluor White and Congo Red and the cell wall biosynthesis inhibitor Caspofungin. In contrast to the previously published data in yeast, the deletion of post-prenylation modifying enzymes did not alter the plasma membrane localization or activation of RasA. To delineate the molecular mechanisms underlying these differences, we investigated the interplay between dual-palmitoylation of the RasA hypervariable region and CAAX proteolysis for stabilization of RasA at the plasma membrane. Our data indicate that, in the absence of proper CAAX proteolysis, RasA accumulation at the plasma membrane is stabilized by dual palmitoyl groups on the dual cysteine residues. Therefore, we conclude CAAX proteolysis and dual-palmitoylation of the hypervariable region is important for maintaining a stable attachment association of RasA with the plasma membrane to support optimal fungal growth and development.