Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.

/pdf/daptomycin-inhibits-cell-envelope-synthesis-by-interfering-110tgoz0nr.pdf

Daptomycin inhibits cell envelope synthesis by interfering with fluid membrane microdomains

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call 'oligotrophic growth state', provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.

https://pure.uva.nl/ws/files/33518215/s41467_019_08719_8.pdf

Extreme slow growth as alternative strategy to survive deep starvation in bacteria

To deal with the contamination of the water environment, which represents an arena for microorganisms and antibiotics, a photocatalytic process has been proposed due to its high efficiency and non-toxicity. This review discusses and investigates antibiotics and antibiotic-resistant bacteria (ARB) removal and inactivation with their associated genes through photocatalysis technique as an efficient and ecofriendly advanced oxidation process (AOP) photocatalysis. The paper summarizes recent studies that dealt with both pollutants and their process parameters, optimal operating conditions, used semiconductor photocatalysts and their corresponding removal efficiency. Thus, it suggests that photocatalysis can offer impressive treatment efficiencies for both organic and microbial pollutants simultaneously. The review also states that both antibiotics and bacteria have a destroying effect against each other, where antibiotics have antimicrobial activity and bacteria have microbial degradation. This phenomenon creates a hybrid system for each pollutant (photocatalysis-bacteria, photocatalysis-antibiotic, bacteria-antibiotic), which accelerates the purification and disinfection of the polluted water caused by pathogens and hazardous pollutants using green and safe technology. Moreover, the reaction mechanism was detailed in order to define the role of reactive oxygen species (ROS). Likewise, photocatalysis coupling with other AOP techniques in this context was discussed to achieve more promising results. The main contribution of this review is to explain the relationship between antibiotics, microbial contaminants and photocatalysis. It also introduces a new area of study and concludes with an outlook on future research topics, such as viral disinfection.

Simultaneous removal of antibiotics and inactivation of antibiotic-resistant bacteria by photocatalysis: A review

Subsequent to binding pocket modifications designed to provide dual d-Ala-d-Ala/d-Ala-d-Lac binding that directly overcome the molecular basis of vancomycin resistance, peripheral structural changes have been explored to improve antimicrobial potency and provide additional synergistic mechanisms of action. A C-terminal peripheral modification, introducing a quaternary ammonium salt, is reported and was found to provide a binding pocket-modified vancomycin analog with a second mechanism of action that is independent of d-Ala-d-Ala/d-Ala-d-Lac binding. This modification, which induces cell wall permeability and is complementary to the glycopeptide inhibition of cell wall synthesis, was found to provide improvements in antimicrobial potency (200-fold) against vancomycin-resistant Enterococci (VRE). Furthermore, it is shown that this type of C-terminal modification may be combined with a second peripheral (4-chlorobiphenyl)methyl (CBP) addition to the vancomycin disaccharide to provide even more potent antimicrobial agents [VRE minimum inhibitory concentration (MIC) = 0.01–0.005 μg/mL] with activity that can be attributed to three independent and synergistic mechanisms of action, only one of which requires d-Ala-d-Ala/d-Ala-d-Lac binding. Finally, it is shown that such peripherally and binding pocket-modified vancomycin analogs display little propensity for acquired resistance by VRE and that their durability against such challenges as well as their antimicrobial potency follow now predictable trends (three > two > one mechanisms of action). Such antibiotics are expected to display durable antimicrobial activity not prone to rapidly acquired clinical resistance.

https://www.pnas.org/content/pnas/early/2017/05/23/1704125114.full.pdf

Peripheral modifications of [Ψ[CH2NH]Tpg4]vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics.

Interactions between bacterial and fungal cells shape many polymicrobial communities. Bacteria elaborate diverse strategies to interact and compete with other organisms, including the deployment of protein secretion systems. The type VI secretion system (T6SS) delivers toxic effector proteins into host eukaryotic cells and competitor bacterial cells, but, surprisingly, T6SS-delivered effectors targeting fungal cells have not been reported. Here we show that the ‘antibacterial’ T6SS of Serratia marcescens can act against fungal cells, including pathogenic Candida species, and identify the previously undescribed effector proteins responsible. These antifungal effectors, Tfe1 and Tfe2, have distinct impacts on the target cell, but both can ultimately cause fungal cell death. ‘In competition’ proteomics analysis revealed that T6SS-mediated delivery of Tfe2 disrupts nutrient uptake and amino acid metabolism in fungal cells, and leads to the induction of autophagy. Intoxication by Tfe1, in contrast, causes a loss of plasma membrane potential. Our findings extend the repertoire of the T6SS and suggest that antifungal T6SSs represent widespread and important determinants of the outcome of bacterial–fungal interactions. The type VI secretion system of Serratia marcescens can deliver effector proteins that target fungi, including pathogenic Candida species, via disruption of nutrient uptake and metabolism or loss of plasma membrane potential.

/pdf/the-type-vi-secretion-system-deploys-antifungal-effectors-2ga1ef6wi5.pdf

The type VI secretion system deploys antifungal effectors against microbial competitors.

The bacterial cytoplasmic membrane is a major inhibitory target for antimicrobial compounds. Commonly, although not exclusively, these compounds unfold their antimicrobial activity by disrupting the essential barrier function of the cell membrane. As a consequence, membrane permeability assays are central for mode of action studies analysing membrane-targeting antimicrobial compounds. The most frequently used in vivo methods detect changes in membrane permeability by following internalization of normally membrane impermeable and relatively large fluorescent dyes. Unfortunately, these assays are not sensitive to changes in membrane ion permeability which are sufficient to inhibit and kill bacteria by membrane depolarization. In this manuscript, we provide experimental advice how membrane potential, and its changes triggered by membrane-targeting antimicrobials can be accurately assessed in vivo. Optimized protocols are provided for both qualitative and quantitative kinetic measurements of membrane potential. At last, single cell analyses using voltage-sensitive dyes in combination with fluorescence microscopy are introduced and discussed.

/pdf/analysis-of-antimicrobial-triggered-membrane-depolarization-1s97cbobla.pdf

Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Despite efforts to develop new antibiotics, antibacterial resistance still develops too fast for drug discovery to keep pace. Often, resistance against a new drug develops even before it reaches the market. This continued resistance crisis has demonstrated that resistance to antibiotics with single protein targets develops too rapidly to be sustainable. Most successful long-established antibiotics target more than one molecule or possess targets, which are encoded by multiple genes. This realization has motivated a change in antibiotic development toward drug candidates with multiple targets. Some mechanisms of action presuppose multiple targets or at least multiple effects, such as targeting the cytoplasmic membrane or the carrier molecule bactoprenol phosphate and are therefore particularly promising. Moreover, combination therapy approaches are being developed to break antibiotic resistance or to sensitize bacteria to antibiotic action. In this Review, we provide an overview of antibacterial multitarget approaches and the mechanisms behind them.

/pdf/multitarget-approaches-against-multiresistant-superbugs-1sadicc3es.pdf

Multitarget Approaches against Multiresistant Superbugs

Daptomycin is a cyclic lipopeptide antibiotic, which was discovered in 1987 and entered the market in 2003. To date, it serves as last resort antibiotic to treat complicated skin infections, bacteremia, and right-sided endocarditis caused by Gram-positive pathogens, most prominently methicillin-resistant Staphylococcus aureus. Daptomycin was the last representative of a novel antibiotic class that was introduced to the clinic. It is also one of the few membrane-active compounds that can be applied systemically. While membrane-active antibiotics have long been limited to topical applications and were generally excluded from systemic drug development, they promise slower resistance development than many classical drugs that target single proteins. The success of daptomycin together with the emergence of more and more multi-resistant superbugs attracted renewed interest in this compound class. Studying daptomycin as a pioneering systemic membrane-active compound might help to pave the way for future membrane-targeting antibiotics. However, more than 30 years after its discovery, the exact mechanism of action of daptomycin is still debated. In particular, there is a prominent discrepancy between in vivo and in vitro studies. In this review, we discuss the current knowledge on the mechanism of daptomycin against Gram-positive bacteria and try to offer explanations for these conflicting observations.

/pdf/more-than-a-pore-a-current-perspective-on-the-in-vivo-mode-6b3eodbp2u.pdf

More Than a Pore: A Current Perspective on the In Vivo Mode of Action of the Lipopeptide Antibiotic Daptomycin.

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.

/pdf/insights-into-suscd-mediated-glycan-import-by-a-prominent-52uixvf7m1.pdf

Declan A. Gray

Papers

Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes.

Extreme slow growth as alternative strategy to survive deep starvation in bacteria

Multitarget Approaches against Multiresistant Superbugs

More Than a Pore: A Current Perspective on the In Vivo Mode of Action of the Lipopeptide Antibiotic Daptomycin.

Insights into SusCD-mediated glycan import by a prominent gut symbiont.