The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species

A significant challenge today within protein lipidology is to understand the relationship between cell structure, lipid membrane integrity, ion homeostasis and the embedded membrane proteins. The bacterial magnesium transporter A (MgtA) is a specialized P-type ATPase important for Mg2+ import into the cytoplasm; disrupted magnesium homeostasis is linked to intrinsic ribosome instability and nitro-oxidative stress in Salmonella strains. MgtAs function is highly dependent on anionic lipids, particularly cardiolipin, and further co-localization of cardiolipin with MgtA at the E. coli cell poles has been revealed. Here, we show that MgtA has functional specificity for cardiolipin 18:1, but it reaches maximum activity only in combination with cardiolipin 16:0, equivalent to the major components of native cardiolipin found in E. coli membranes. This is the first time it has been shown experimentally that two different lipid species from the same class, individually promoting low activity, can enhance activity in combination. Native mass spectrometry verifies the presence of two binding sites for cardiolipin and kinetic studies reveal that a cooperative relationship likely exists between the two different cardiolipin variants (cardiolipin 16:0 and cardiolipin 18:1). This is the first experimental evidence of cooperative effects between lipids of the same class, with only minor variations in their acyl chain composition, acting on a membrane protein. In summary, our results reveal that MgtA exhibits a highly complex interaction with one cardiolipin 18:1 and one cardiolipin 16:0, affecting protein activity and stability, and contributing to our understanding of the particular interactions between lipid environment and membrane proteins. Further, a better understanding of Mg2+ homeostasis in bacteria, due to its role as a virulence regulator, will provide further insights into the regulation and mechanism of bacterial infections.

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Plantaricin NC8 αβ exerts potent antimicrobial activity against Staphylococcus spp. and enhances the effects of antibiotics

Scientific Reports ◽

10.1038/s41598-020-60570-w ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Torbjörn Bengtsson ◽

Robert Selegård ◽

Amani Musa ◽

Kjell Hultenby ◽

Johanna Utterström ◽

...

Keyword(s):

Amino Acids ◽

Bacterial Infections ◽

Antimicrobial Agents ◽

Lipid Membrane ◽

Membrane Integrity ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

Promising Alternative ◽

Conventional Antibiotics ◽

Plantaricin Nc8

AbstractThe use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αβ were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and β were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 αβ caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, α1–22, β7–34 and β1–20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αβ substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αβ is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.

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Microstructural morphology of sphingolipid dispersions as investigated by freeze-fracture EM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141093 ◽

1995 ◽

Vol 53 ◽

pp. 944-945

Author(s):

Vitthal S. Kulkarni ◽

Wayne H. Anderson ◽

Rhoderick E. Brown

Keyword(s):

Acyl Chain ◽

Biological Significance ◽

Freeze Fracture ◽

Fatty Acyl ◽

Fatty Acyl Moiety ◽

Aqueous Dispersions ◽

Head Groups ◽

Lipid Species ◽

Microstructural Morphology ◽

Layer Chromatography

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.

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Bacterial detection and differentiation via direct volatile organic compound sensing with surface enhanced Raman spectroscopy

10.26434/chemrxiv.5393281 ◽

2017 ◽

Author(s):

Caitlin S. DeJong ◽

David I. Wang ◽

Aleksandr Polyakov ◽

Anita Rogacs ◽

Steven J. Simske ◽

...

Keyword(s):

Raman Spectroscopy ◽

Bacterial Infections ◽

Direct Detection ◽

Point Of Care ◽

Surface Enhanced Raman Spectroscopy ◽

E Coli ◽

Recent Emergence ◽

Surface Enhanced ◽

Volatile Organic ◽

Surface Enhanced Raman

Through the direct detection of bacterial volatile organic compounds (VOCs), via surface enhanced Raman spectroscopy (SERS), we report here a reconfigurable assay for the identification and monitoring of bacteria. We demonstrate differentiation between highly clinically relevant organisms: Escherichia coli, Enterobacter cloacae, and Serratia marcescens. This is the first differentiation of bacteria via SERS of bacterial VOC signatures. The assay also detected as few as 10 CFU/ml of E. coli in under 12 hrs, and detected E. coli from whole human blood and human urine in 16 hrs at clinically relevant concentrations of 103 CFU/ml and 104 CFU/ml, respectively. In addition, the recent emergence of portable Raman spectrometers uniquely allows SERS to bring VOC detection to point-of-care settings for diagnosing bacterial infections.

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Probiotics Slow the Growth of Pathogenic Bacteria

International Journal of Probiotics and Prebiotics ◽

10.37290/ijpp2641-7197.14:28-31 ◽

2019 ◽

Vol 14 (1) ◽

pp. 28-31 ◽

Cited By ~ 1

Author(s):

Rowles H. L.

Keyword(s):

Growth Rate ◽

Bacterial Infections ◽

Pathogenic Bacteria ◽

Diarrheal Disease ◽

Growth Curves ◽

Mortality Rates ◽

Controlled Delivery ◽

E Coli ◽

Multiple Strains ◽

Commercial Probiotics

Probiotics are live microorganisms, which when ingested in sufficient amounts, confer health benefits to the host by improving the gut microflora balance. The purpose of this research was to determine whether commercial probiotic products containing multitude of commensal bacteria would reduce the growth rate of pathogenic bacteria, specifically Escherichia coli and Salmonella typhimurium. Growth curves were established, and the growth rates were compared for samples of E. coli, S. typhimurium, Nature’s Bounty Controlled Delivery probiotic, Sundown Naturals Probiotic Balance probiotic, and cocultures of the pathogenic bacteria mixed with the probiotics. The findings of this research were that the commercial probiotics significantly reduced the growth rate of E. coli and S. typhimurium when combined in cocultures. Probiotics containing multiple strains may be taken prophylactically to reduce the risk of bacterial infections caused by E. coli and S. typhimurium. Probiotics could be used to reduce the high global morbidity and mortality rates of diarrheal disease.

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Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽

10.3390/cells8111325 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1325 ◽

Cited By ~ 1

Author(s):

Ke Yue ◽

Tran Nam Trung ◽

Yiyong Zhu ◽

Ralf Kaldenhoff ◽

Lei Kai

Keyword(s):

Functional Analysis ◽

Membrane Proteins ◽

Fluorescent Protein ◽

Water Channel ◽

New Approach ◽

E Coli ◽

Straightforward Method ◽

Lipid Environment ◽

Green Fluorescent ◽

Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

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Evaluation of commercially available aroA delated gene E. coli O78 vaccine in commercial broiler chickens under Middle East simulating field conditions

Scientific Reports ◽

10.1038/s41598-021-81523-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hussein M. Galal ◽

M. I. Abdrabou ◽

Ahmed H. I. Faraag ◽

C. K. Mah ◽

Azza M. Tawfek

Keyword(s):

Middle East ◽

Broiler Chickens ◽

Bacterial Infections ◽

Body Weight Gain ◽

Broiler Chicks ◽

E Coli ◽

Reduced Body ◽

Antibiotics Use ◽

Commercial Broiler ◽

And Performance

AbstractThe broiler industry in the Middle East (ME) faces many challenges related to bacterial infections, including M. gallisepticum, M. synoviae, E. coli, and other gram-negative bacteria, exacerbated by various errors in the brooding process. Antibiotics use in the first three days of life, such as Linco-Spectin 100 SP, tilmicosin, enrofloxacin, tylosin, colistin, and doxycycline, is the trend in the market to control such challenges. This study aimed to evaluate the efficacy of the newly introduced aroA E. coli vaccine (Poulvac E. coli) and its ability to reduce over-reliance on the heavy use of antibiotics in the ME. The study was conducted on 160 broiler chicks, divided into eight even groups. Each group was treated differently in terms of antibiotic therapy and ages at the time of Poulvac E. coli administration and the challenge of virulent avian pathogenic E. coli (APEC), serotype O78. Spray application of Poulvac E. coli at seven days of age plus Linco-Spectin 100 SP during the first three days provided the best results for zero mortality after challenge with APEC, while Poulvac E. coli at seven days with enrofloxacin during the early three days resulted in 10% mortality. Poulvac E. coli hatchery vaccination protected birds against mortality but reduced body weight gain compared to the 7-day group vaccinated with Linco-Spectin 100 SP during the first three days. Poulvac E. coli given on day one or day seven did not affect the immune response to concurrent respiratory viral vaccines and, in some cases, improved response. This study shows that Poulvac E. coli at seven days of age, together with Linco-Spectin 100 during the first three days, has produced the best results in terms of protection and performance in the ME high presence of avian pathogenic E. coli field challenge.

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Three-Year Trend in Escherichia coli Antimicrobial Resistance among Children’s Urine Cultures in an Italian Metropolitan Area

Children ◽

10.3390/children8070597 ◽

2021 ◽

Vol 8 (7) ◽

pp. 597

Author(s):

Luca Pierantoni ◽

Laura Andreozzi ◽

Simone Ambretti ◽

Arianna Dondi ◽

Carlotta Biagi ◽

...

Keyword(s):

Escherichia Coli ◽

Metropolitan Area ◽

Bacterial Infections ◽

Asymptomatic Bacteriuria ◽

Multidrug Resistant ◽

Resistance Rate ◽

First Line ◽

E Coli ◽

First Line Therapy ◽

Tract Infections

Urinary tract infections (UTIs) are among the most common bacterial infections in children, and Escherichia coli is the main pathogen responsible. Several guidelines, including the recently updated Italian guidelines, recommend amoxicillin-clavulanic acid (AMC) as a first-line antibiotic therapy in children with febrile UTIs. Given the current increasing rates of antibiotic resistance worldwide, this study aimed to investigate the three-year trend in the resistance rate of E. coli isolated from pediatric urine cultures (UCs) in a metropolitan area of northern Italy. We conducted a retrospective review of E. coli-positive, non-repetitive UCs collected in children aged from 1 month to 14 years, regardless of a diagnosis of UTI, catheter colonization, urine contamination, or asymptomatic bacteriuria. During the study period, the rate of resistance to AMC significantly increased from 17.6% to 40.2% (p < 0.001). Ciprofloxacin doubled its resistance rate from 9.1% to 16.3% (p = 0.007). The prevalence of multidrug-resistant E. coli rose from 3.9% to 9.2% (p = 0.015). The rate of resistance to other considered antibiotics remained stable, as did the prevalence of extended spectrum beta-lactamases and extensively resistant E. coli among isolates. These findings call into question the use of AMC as a first-line therapy for pediatric UTIs in our population, despite the indications of recent Italian guidelines.

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Interaction of Temporin-L Analogues with the E. coli FtsZ Protein

Antibiotics ◽

10.3390/antibiotics10060704 ◽

2021 ◽

Vol 10 (6) ◽

pp. 704

Author(s):

Angela Di Somma ◽

Carolina Canè ◽

Antonio Moretta ◽

Angela Duilio

Keyword(s):

Protein Interactions ◽

Bacterial Infections ◽

Antibacterial Properties ◽

Bacterial Cell Division ◽

Structural Determinants ◽

E Coli ◽

Functional Studies ◽

Division Process ◽

Z Ring ◽

Peptide Complexes

The research of new therapeutic agents to fight bacterial infections has recently focused on the investigation of antimicrobial peptides (AMPs), the most common weapon that all organisms produce to prevent invasion by external pathogens. Among AMPs, the amphibian Temporins constitute a well-known family with high antibacterial properties against Gram-positive and Gram-negative bacteria. In particular, Temporin-L was shown to affect bacterial cell division by inhibiting FtsZ, a tubulin-like protein involved in the crucial step of Z-ring formation at the beginning of the division process. As FtsZ represents a leading target for new antibacterial compounds, in this paper we investigated in detail the interaction of Temporin L with Escherichia coli FtsZ and designed two TL analogues in an attempt to increase peptide-protein interactions and to better understand the structural determinants leading to FtsZ inhibition. The results demonstrated that the TL analogues improved their binding to FtsZ, originating stable protein-peptide complexes. Functional studies showed that both peptides were endowed with a high capability of inhibiting both the enzymatic and polymerization activities of the protein. Moreover, the TL analogues were able to inhibit bacterial growth at low micromolar concentrations. These observations may open up the way to the development of novel peptide or peptidomimetic drugs tailored to bind FtsZ, hampering a crucial process of bacterial life that might be proposed for future pharmaceutical applications.

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In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

Scientific Reports ◽

10.1038/s41598-021-81140-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kaitlin S. Witherell ◽

Jason Price ◽

Ashok D. Bandaranayake ◽

James Olson ◽

Douglas R. Call

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptide ◽

Membrane Integrity ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Multidrug Resistant Bacteria ◽

E Coli ◽

Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

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Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽

10.3390/membranes11020155 ◽

2021 ◽

Vol 11 (2) ◽

pp. 155

Author(s):

Ekaitz Errasti-Murugarren ◽

Paola Bartoccioni ◽

Manuel Palacín

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Lipid Membrane ◽

Protein Stabilization ◽

New Drugs ◽

Cell Physiology ◽

Protein Preparation ◽

Functional Studies ◽

Membrane Protein Stability ◽

Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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