Rationalizing generation of broad spectrum antibiotics with the addition of a positive charge

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that...

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Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.10.3133-3141.2002 ◽

2002 ◽

Vol 46 (10) ◽

pp. 3133-3141 ◽

Cited By ~ 294

Author(s):

George Tegos ◽

Frank R. Stermitz ◽

Olga Lomovskaya ◽

Kim Lewis

Keyword(s):

Broad Spectrum ◽

Plant Pathogens ◽

Bacterial Species ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Human Pathogens ◽

Gram Negative ◽

Low Level ◽

Broad Spectrum Antibiotics ◽

Level Of Activity

ABSTRACT Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 μg/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100- to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These results suggest that plants might have developed means of delivering their antimicrobials into bacterial cells. These findings also suggest that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs.

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The essential inner membrane protein YejM is a metalloenzyme

Scientific Reports ◽

10.1038/s41598-020-73660-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Uma Gabale ◽

Perla Arianna Peña Palomino ◽

HyunAh Kim ◽

Wenya Chen ◽

Susanne Ressl

Keyword(s):

Antibiotic Resistance ◽

Membrane Protein ◽

Outer Membrane ◽

Critical Role ◽

Membrane Properties ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Periplasmic Domain ◽

Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

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An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli

mBio ◽

10.1128/mbio.00939-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 15

Author(s):

Elayne M. Fivenson ◽

Thomas G. Bernhardt

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Outer Membrane ◽

Unknown Function ◽

Barrier Function ◽

Major Determinant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Essential Protein

ABSTRACT Gram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share an intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adapter protein LapB (YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM (PbgA) participates in this regulatory pathway. Suppressors of YejM essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of ΔyejM mutants. Furthermore, the stability of LpxC was shown to be reduced in cells lacking YejM, and genetic and physical interactions between LapB and YejM were detected. Taken together, our results are consistent with a model in which YejM directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis. IMPORTANCE The outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this study, we identified an essential protein of unknown function as a key new factor in modulating LPS synthesis in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

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Thrombin-Derived Peptides Potentiate the Activity of Gram-Positive-Specific Antibiotics against Gram-Negative Bacteria

Molecules ◽

10.3390/molecules26071954 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1954

Author(s):

Charlotte M. J. Wesseling ◽

Thomas M. Wood ◽

Kristine Bertheussen ◽

Samantha Lok ◽

Nathaniel I. Martin

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

Therapeutic Range ◽

Structural Features ◽

Gram Negative Bacteria ◽

Binding Properties ◽

Gram Positive ◽

Gram Negative ◽

Recent Attention ◽

Lps Binding

The continued rise of antibiotic resistance threatens to undermine the utility of the world’s current antibiotic arsenal. This problem is particularly troubling when it comes to Gram-negative pathogens for which there are inherently fewer antibiotics available. To address this challenge, recent attention has been focused on finding compounds capable of disrupting the Gram-negative outer membrane as a means of potentiating otherwise Gram-positive-specific antibiotics. In this regard, agents capable of binding to the lipopolysaccharide (LPS) present in the Gram-negative outer membrane are of particular interest as synergists. Recently, thrombin-derived C-terminal peptides (TCPs) were reported to exhibit unique LPS-binding properties. We here describe investigations establishing the capacity of TCPs to act as synergists with the antibiotics erythromycin, rifampicin, novobiocin, and vancomycin against multiple Gram-negative strains including polymyxin-resistant clinical isolates. We further assessed the structural features most important for the observed synergy and characterized the outer membrane permeabilizing activity of the most potent synergists. Our investigations highlight the potential for such peptides in expanding the therapeutic range of antibiotics typically only used to treat Gram-positive infections.

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An essential membrane protein modulates the proteolysis of LpxC to control lipopolysaccharide synthesis in Escherichia coli

10.1101/2020.02.10.942425 ◽

2020 ◽

Author(s):

Elayne M. Fivenson ◽

Thomas G. Bernhardt

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Outer Membrane ◽

Unknown Function ◽

Barrier Function ◽

Adaptor Protein ◽

Major Determinant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Essential Protein

ABSTRACTGram-negative bacteria are surrounded by a complex cell envelope that includes two membranes. The outer membrane prevents many drugs from entering these cells and is thus a major determinant of their intrinsic antibiotic resistance. This barrier function is imparted by the asymmetric architecture of the membrane with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The LPS and phospholipid synthesis pathways share a common intermediate. Proper membrane biogenesis therefore requires that the flux through each pathway be balanced. In Escherichia coli, a major control point in establishing this balance is the committed step of LPS synthesis mediated by LpxC. Levels of this enzyme are controlled through its degradation by the inner membrane protease FtsH and its presumed adaptor protein LapB(YciM). How turnover of LpxC is controlled has remained unclear for many years. Here, we demonstrate that the essential protein of unknown function YejM(PbgA), which we have renamed ClxD (control of LpxC degradation), participates in this regulatory pathway. Suppressors of ClxD essentiality were identified in lpxC and lapB, and LpxC overproduction was shown to be sufficient to allow survival of ΔclxD mutants. Furthermore, the half-life of LpxC was shown to be reduced in cells lacking ClxD, and genetic and physical interactions between LapB and ClxD were detected. Taken together, our results are consistent with a model in which ClxD directly modulates LpxC turnover by FtsH-LapB to regulate LPS synthesis and maintain membrane homeostasis.SIGNIFICANCEThe outer membrane is a major determinant of the intrinsic antibiotic resistance of Gram-negative bacteria. It is composed of both lipopolysaccharide (LPS) and phospholipid, and the synthesis of these lipid species must be balanced for the membrane to maintain its barrier function in blocking drug entry. In this report, we identify an essential protein of unknown function as a key new factor in maintaining LPS/phospholipid balance in the model bacterium Escherichia coli. Our results provide novel insight into how this organism and most likely other Gram-negative bacteria maintain membrane homeostasis and their intrinsic resistance to antibiotics.

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Exploring Nature’s Treasure to Inhibit β-Barrel Assembly Machinery of Antibiotic Resistant Bacteria: An In-Silico Approach

Letters in Drug Design & Discovery ◽

10.2174/1570180818999201224121342 ◽

2020 ◽

Vol 18 ◽

Author(s):

Shalja Verma ◽

Anand Kumar Pandey

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

In Silico ◽

Membrane Integrity ◽

Natural Compounds ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Bam Complex ◽

In Silico Admet

Background: Development of antibiotic resistance in bacteria is a matter of global concern due to the exceptionally high morbidity and mortality rates. Outer membrane of most Gram-negative bacteria act as a highly efficient barrier and blocks the entry of the majority of antibiotics, making them ineffective. Bam complex, β-barrel assembly machinery complex, contains five subunits (BamA,B,C,D,E) which plays a vital role in folding and insertion of essential outer membrane proteins into membrane thus maintains outer membrane integrity. Bam A and Bam D are essential subunits to fulfil this purpose. Thus, targeting this complex to treat antibiotic resistance can be an incredibly effective approach. Natural bacterial pigment like violacein, phytochemicals like withanone, semasin and several polyphenols have often been reported for their effective antibiotic, antioxidant, anti-inflammatory, antiviral and anti-carcinogenic properties. Objective: Structural inhibition of Bam complex by natural compounds can provide safe and effective treatment to antibiotic resistance by targeting outer membrane integrity. Methods: In-silico ADMET and Molecular docking analysis was performed with 10 natural compounds namely violacein, withanone, sesamin, resveratrol, naringenin, quercetin, epicatechin, gallic acid, ellagic acid and galangin to analyse their inhibitory potential against Bam complex. Results: Docking complexes of Violacein gave high binding energies of -10.385 and -9.46 Kcal/mol at C and D subunits interface, and at A subunits of the Bam complex respectively. Conclusion: Henceforth, violacein can be an effective antibiotic against till date reported resistant Gram-negative bacteria by inhibiting the Bam complex of their outer membrane, therefore urgent need for exhaustive research in this concern is highly demanded.

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The membrane-proximal domain of the periplasmic adapter protein plays a role in vetting substrates utilising channels 1 and 2 of RND efflux transporters

10.1101/2021.10.05.463233 ◽

2021 ◽

Author(s):

Ilyas Alav ◽

Vassiliy N. Bavro ◽

Jessica M. A. Blair

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

Salmonella Enterica ◽

Adaptor Protein ◽

Efflux Pumps ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Serovar Typhimurium ◽

Rnd Transporter ◽

Critical Residues

AbstractActive efflux by resistance-nodulation-division (RND) efflux pumps is a major contributor to antibiotic resistance in clinically relevant Gram-negative bacteria. Tripartite RND pumps, such as AcrAB-TolC of Salmonella enterica serovar Typhimurium, comprise of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. Previously, we elucidated binding sites within the PAP AcrA (termed binding boxes) that were important for AcrB-transporter recognition. Here, we have refined the binding box model by identifying the most critical residues involved in PAP-RND binding and show that the corresponding RND-binding residues in the closely related PAP AcrE are also important for AcrB interactions. In addition, our analysis identified a membrane-proximal domain (MPD)-residue in AcrA (K366), that when mutated, differentially affects transport of substrates utilising different AcrB efflux-channels, namely channels 1 and 2, supporting a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket (PBP) of the transporter and substrate vetting. Our model predicts that there is a close interplay between the MPD of the PAP and the RND transporter in the productive export of substrates utilising the PBP.ImportanceAntibiotic resistance greatly threatens our ability to treat infectious diseases. In Gram-negative bacteria, overexpression of tripartite efflux pumps, such as AcrAB-TolC, contributes to multidrug resistance because they export many different classes of antibiotics. The AcrAB-TolC pump is made up of three components: the periplasmic adaptor protein (PAP) AcrA, the RND-transporter AcrB, and the outer-membrane factor TolC. Here, we identified critical residues of AcrA that are important for its function with AcrB in Salmonella enterica serovar Typhimurium. Also, we show that AcrA shares these critical residues with AcrE, a closely related PAP, explaining their interoperability with AcrB. Importantly, we identified a residue in the membrane-proximal domain of AcrA that when mutated affected how different substrates access AcrB and impacted downstream efflux via TolC channel. Understanding the role that PAPs play in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.

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Investigation of gating in outer membrane porins provides new perspectives on antibiotic resistance mechanisms

10.1101/2021.09.09.459668 ◽

2021 ◽

Author(s):

Archit Kumar Vasan ◽

Nandan Haloi ◽

Rebecca Joy Ulrich ◽

Mary Elizabeth Metcalf ◽

Po-Chao Wen ◽

...

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

Large Scale ◽

Dynamic Equilibrium ◽

Resistance Mechanisms ◽

Health Concern ◽

Gram Negative Bacteria ◽

Bacterial Strains ◽

Gram Negative ◽

Scale Motion

AbstractGram-negative bacteria pose a serious public health concern, primarily due to a higher frequency of antibiotic resistance conferred to them as a result of low permeability of their outer membrane (OM). Antibiotics capable of traversing the OM typically permeate through OM porins; thus, understanding the permeation properties of these porins is instrumental to the development of new antibiotics. A common macroscopic feature of many OM porins is their ability to transition between functionally distinct open and closed states that regulate transport properties and rate. To obtain a molecular basis for these processes, we performed tens of microseconds of molecular dynamics simulations of E. coli OM porin, OmpF. We observed that large-scale motion of the internal loop, L3, leads to widening and narrowing of the pore, suggesting its potential role in gating. Furthermore, Markov state analysis revealed multiple energetically stable conformations of L3 corresponding to open and closed states of the porin. Dynamics between these functional states occurs on the time scale of tens of microseconds and are mediated by the movement of highly conserved acidic residues of L3 to form H-bonds with opposing sides of the barrel wall of the pore. To validate our mechanism, we mutated key residues involved in the gating process that alter the H-bond pattern in the open/closed states and performed additional simulations. These mutations shifted the dynamic equilibrium of the pore towards open or closed states. Complementarily, the mutations favoring the open/closed states lead to increased/decreased accumulation of multiple antibiotics in our whole-cell accumulation assays. Notably, porins containing one of the mutations favoring the closed state has previously been found in antibiotic resistant bacterial strains. Overall, our 180 µs of simulation data (wild type and mutants) with concerted experiments suggests that regulation of the dynamic equilibrium between open and closed states of OM porins could be a mechanism by which Gram-negative bacteria acquire antibiotic resistance.

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Outer Membrane Disruption Overcomes Intrinsic, Acquired, and Spontaneous Antibiotic Resistance

mBio ◽

10.1128/mbio.01615-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Craig R. MacNair ◽

Eric D. Brown

Keyword(s):

Antibiotic Resistance ◽

Outer Membrane ◽

Biofilm Formation ◽

Antibiotic Use ◽

Membrane Disruption ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

Outer Membrane Permeability

ABSTRACT Disruption of the outer membrane (OM) barrier allows for the entry of otherwise inactive antimicrobials into Gram-negative pathogens. Numerous efforts to implement this approach have identified a large number of OM perturbants that sensitize Gram-negative bacteria to many clinically available Gram-positive active antibiotics. However, there is a dearth of investigation into the strengths and limitations of this therapeutic strategy, with an overwhelming focus on characterization of individual potentiator molecules. Herein, we look to explore the utility of exploiting OM perturbation to sensitize Gram-negative pathogens to otherwise inactive antimicrobials. We identify the ability of OM disruption to change the rules of Gram-negative entry, overcome preexisting and spontaneous resistance, and impact biofilm formation. Disruption of the OM expands the threshold of hydrophobicity compatible with Gram-negative activity to include hydrophobic molecules. We demonstrate that while resistance to Gram-positive active antibiotics is surprisingly common in Gram-negative pathogens, OM perturbation overcomes many antibiotic inactivation determinants. Further, we find that OM perturbation reduces the rate of spontaneous resistance to rifampicin and impairs biofilm formation. Together, these data suggest that OM disruption overcomes many of the traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. IMPORTANCE The spread of antibiotic resistance is an urgent threat to global health that necessitates new therapeutics. Treatments for Gram-negative pathogens are particularly challenging to identify due to the robust outer membrane permeability barrier in these organisms. Recent discovery efforts have attempted to overcome this hurdle by disrupting the outer membrane using chemical perturbants and have yielded several new peptides and small molecules that allow the entry of otherwise inactive antimicrobials. However, a comprehensive investigation into the strengths and limitations of outer membrane perturbants as antibiotic partners is currently lacking. Herein, we interrogate the interaction between outer membrane perturbation and several common impediments to effective antibiotic use. Interestingly, we discover that outer membrane disruption is able to overcome intrinsic, spontaneous, and acquired antibiotic resistance in Gram-negative bacteria, meriting increased attention toward this approach.

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New functional identity of the essential inner membrane protein YejM: the cardiolipin translocator is also a metalloenzyme

10.1101/2020.02.13.947838 ◽

2020 ◽

Cited By ~ 1

Author(s):

Uma Gabale ◽

Perla A. Peña Palomino ◽

HyunAh Kim ◽

Wenya Chen ◽

Susanne Ressl

Keyword(s):

Antibiotic Resistance ◽

Membrane Protein ◽

Outer Membrane ◽

Essential Gene ◽

Critical Role ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Periplasmic Domain ◽

Inner Membrane Protein

AbstractRecent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability by translocating and increasing the cardiolipin lipid concentration. How the inner membrane protein YejM with its periplasmic domain acts as a cardiolipin translocator remains unknown. Despite overwhelming structural similarity of the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its cardiolipin translocation properties. Understanding the molecular mechanism by which YejM is involved in OM remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

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