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

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.

scholarly journals A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

scholarly journals Antibiotic Resistance of Gram-Negative Bacteria from Wild Captured Loggerhead Sea Turtles

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 162 ◽  
Author(s):  
Monica Francesca Blasi ◽  
Luciana Migliore ◽  
Daniela Mattei ◽  
Alice Rotini ◽  
Maria Cristina Thaller ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Sea Turtles ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Marine Animals ◽  
Loggerhead Sea Turtles ◽  
Antibiotic Resistant ◽  
Gram Negative ◽  
Marine Habitats ◽  
Resistant Strains

Sea turtles have been proposed as health indicators of marine habitats and carriers of antibiotic-resistant bacterial strains, for their longevity and migratory lifestyle. Up to now, a few studies evaluated the antibacterial resistant flora of Mediterranean loggerhead sea turtles (Caretta caretta) and most of them were carried out on stranded or recovered animals. In this study, the isolation and the antibiotic resistance profile of 90 Gram negative bacteria from cloacal swabs of 33 Mediterranean wild captured loggerhead sea turtles are described. Among sea turtles found in their foraging sites, 23 were in good health and 10 needed recovery for different health problems (hereafter named weak). Isolated cloacal bacteria belonged mainly to Enterobacteriaceae (59%), Shewanellaceae (31%) and Vibrionaceae families (5%). Although slight differences in the bacterial composition, healthy and weak sea turtles shared antibiotic-resistant strains. In total, 74 strains were endowed with one or multi resistance (up to five different drugs) phenotypes, mainly towards ampicillin (~70%) or sulfamethoxazole/trimethoprim (more than 30%). Hence, our results confirmed the presence of antibiotic-resistant strains also in healthy marine animals and the role of the loggerhead sea turtles in spreading antibiotic-resistant bacteria.

scholarly journals Antibiotic-Resistant Gram-Negative Bacteria in Birds From the Brazilian Atlantic Forest

The Condor ◽  
2003 ◽  
Vol 105 (2) ◽  
pp. 358-361 ◽  
Author(s):  
Andréa M. A. Nascimento ◽  
Luciana Cursino ◽  
Higgor Gonçalves-Dornelas ◽  
Andrea Reis ◽  
Edmar Chartone-Souza ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Kanamycin Resistance ◽  
Minas Gerais ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Brazilian Atlantic Forest ◽  
Antibiotic Resistant ◽  
Gram Negative ◽  
Resistant Phenotype ◽  
Animal Farms

Abstract We evaluated the antibiotic resistance of bacteria isolated from cloacal swabs of wild birds collected with mist nets in the Jequitinhonha river valley, in the state of Minas Gerais, Brazil. A total of 191 isolates from 19 individuals of 16 species was obtained and tested for resistance to five antibiotics. At Salto da Divisa 97% of the isolates exhibited a resistant phenotype, and resistance to more than one antibiotic was frequent (71%). At Jequitinhonha 36% of isolates were resistant, but 94% showed resistance to only one antibiotic. Of the five antibiotics tested, resistance to ampicillin was most frequent (in both areas), whereas kanamycin resistance was found in only one isolate. The data here obtained and other data reported in the literature show that the general premise that antibiotic-resistant bacteria arise primarily in hospitals or animal farms should be reconsidered. Bactérias Gram-Negativas Resistentes a Antibióticos em Aves da Mata Atlântica Brasileira Resumo. Avaliamos a resistência a antibióticos de bactérias isoladas por swab cloacal em aves selvagens capturadas com redes de neblina em duas regiões do Vale do Rio Jequitinhonha, Minas Gerais, Brasil. Foram obtidos 191 isolados de 19 indivíduos de 16 espécies e foi testada a resistência desses isolados a cinco antibióticos. Em Salto da Divisa, 97% dos isolados exibiram fenótipo resistente e foi freqüente (71%) a resistência a mais de um antibiótico. Em Jequitinhonha, 36% dos isolados exibiram fenótipo resistente, dos quais 94% apresentaram resistência a apenas um antibiótico. Em ambas as áreas, a maioria dos isolados apresentou resistência à ampicilina, enquanto somente um único isolado foi resistente à canamicina. Os dados aqui obtidos e outros relatados na literatura mostram que a premissa geral de que bactérias resistentes a antibióticos surgem principalmente em hospitais ou fazendas de animais deve ser reconsiderada.

scholarly journals Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Elizabeth M. Hart ◽  
Thomas J. Silhavy
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Bam Complex ◽  
Essential Function ◽  
The Individual

ABSTRACT The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K), the A-to-P change at position 496 (bamAA496P), and the A-to-S change at position 499 (bamAA499S), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

scholarly journals Crystal structure of calcium bound outer membrane phospholipase A (OmpLA) from Salmonella typhi and in silico anti-microbial screening

2020 ◽  
Author(s):  
Perumal Perumal ◽  
Rahul Raina ◽  
Sundara Baalaji Narayanan ◽  
Arulandu Arockiasamy
Keyword(s):  
Crystal Structure ◽  
Outer Membrane ◽  
In Silico ◽  
Membrane Integrity ◽  
Acid Tolerance ◽  
Salmonella Typhi ◽  
Phospholipase A ◽  
Gram Negative ◽  
Activated State ◽  
Outer Membrane Phospholipase A

AbstractAntimicrobial resistance is widespread in Salmonella infections that affect millions worldwide. Salmonella typhi and other Gram-negative bacterial pathogens encode an outer membrane phospholipase A (OmpLA), crucial for their membrane integrity. Further, OmpLA is implicated in pathogen internalization, haemolysis, acid tolerance, virulence and sustained infection in human hosts. OmpLA is an attractive drug target for developing novel anti-microbials that attenuate virulence, as the abrogation of OmpLA encoding pldA gene causes loss of virulence. Here, we present the crystal structure of Salmonella typhi OmpLA in dimeric calcium bound activated state at 2.95 Å. Structure analysis suggests that OmpLA is a potential druggable target. Further, we have identified and shortlisted small molecules that bind at the dimer interface using structure based in silico screening, docking and molecular dynamics. While it requires further experimental validation, anti-microbial discovery targeting OmpLA from gram-negative pathogens offers an advantage as OmpLA is required for virulence.

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

2021 ◽  
Author(s):  
nandan haloi ◽  
Archit Kumar Vasan ◽  
Emily Jane Geddes ◽  
Arjun Prasanna ◽  
Po-Chao Wen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Broad Spectrum ◽  
Positive Charge ◽  
Low Permeability ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Broad Spectrum Antibiotics

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...

scholarly journals Innolysins: A novel approach to engineer endolysins to kill Gram-negative bacteria

2018 ◽  
Author(s):  
Athina Zampara ◽  
Martine C. Holst Sørensen ◽  
Dennis Grimon ◽  
Fabio Antenucci ◽  
Yves Briers ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Receptor Binding ◽  
Binding Proteins ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Proof Of Concept ◽  
Gram Negative ◽  
E Coli ◽  
Phage Receptor ◽  
Alternative Antimicrobials

ABSTRACTBacteriophage-encoded endolysins degrading the essential peptidoglycan of bacteria are promising alternative antimicrobials to handle the global threat of antibiotic resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since their outer membrane prevents access to the peptidoglycan. Here we present Innolysins, a novel concept for engineering endolysins that allows the enzymes to pass through the outer membrane, hydrolyse the peptidoglycan and kill the target bacterium. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As our proof of concept, we used phage T5 endolysin and receptor binding protein Pb5, which binds irreversibly to the phage receptor FhuA involved in ferrichrome transport inEscherichia coli. In total, we constructed twelve Innolysins fusing endolysin with Pb5 or the binding domain of Pb5 with or without flexible linkers in between. While the majority of the Innolysins maintained their muralytic activity, Innolysin#6 also showed bactericidal activity againstE. colireducing the number of bacteria by 1 log, thus overcoming the outer membrane barrier. Using anE. coli fhuAdeletion mutant, we demonstrated that FhuA is required for bactericidal activity, supporting that the specific binding of Pb5 to its receptor onE. coliis needed for the endolysin to access the peptidoglycan. Accordingly, Innolysin#6 was able to kill other bacterial species that carry conserved FhuA homologs such asShigella sonneiandPseudomonas aeruginosa. In summary, the Innolysin approach expands recent protein engineering strategies allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.IMPORTANCEThe extensive use of antibiotics has led to the emergence of antimicrobial resistant bacteria responsible for infections causing more than 50,000 deaths per year across Europe and the US. In response, the World Health Organization has stressed an urgent need to discover new antimicrobials to control in particular Gram-negative bacterial pathogens, due to their extensive multi-drug resistance. However, the outer membrane of Gram-negative bacteria limits the access of many antibacterial agents to their targets. Here, we developed a new approach, Innolysins that enable endolysins to overcome the outer membrane by exploiting the binding specificity of phage receptor binding proteins. As proof of concept, we constructed Innolysins againstE. coliusing the endolysin and the receptor binding protein of phage T5. Given the rich diversity of phage receptor binding proteins and their different binding specificities, our proof of concept paves the route for creating an arsenal of pathogen specific alternative antimicrobials.

scholarly journals Detection of Multidrug Resistant Gram Negative Bacteria in Healthy Cattle from Maiduguri Metropolitan, Nigeria

2020 ◽  
pp. 68-76
Author(s):  
Adam Mustapha ◽  
Mustafa Alhaji Isa ◽  
Ibrahim Yusuf Ngoshe ◽  
Hashidu Bala
Keyword(s):  
Antibiotic Resistance ◽  
Clinical Laboratory ◽  
Resistance Index ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Salmonella Spp ◽  
Gram Negative ◽  
Healthy Cattle

Aim: Prevalence of multidrug resistant bacteria on apparently health animals has turned antibiotic resistance to multifaceted process and threatens global food security and public health. The aim of the present study was to investigate the resistance profile of isolates from apparently healthy cattle in Maiduguri, Nigeria. Methodology: A total of 120 nasal swab samples were collected from cattle. Colony identification was according to the guidelines of Bergey’s Manual of Determinative Bacteriology. The susceptibility pattern of the isolates was conducted on the identified isolates according to the Modified Kirby-Baur disc diffusion method on Muller-Hilton agar and interpreted according to the procedures of Clinical Laboratory Standards Institute (CLSI, 2018) guidelines. Multiple Antibiotic Resistance Index (MARI) was calculated using the formula, MARI=a/b where “a” is the number of antibiotic resisted and “b” is the total number of antibiotic used in the study. Results: Of the total samples (120) from cattle 96 (80%) detected the following isolates; E. coli was the most commonly recovered isolates (33, 34.4%), followed by Klebsiella spp (28, 29.2%), Salmonella spp (21, 21.9%) and Pseudomonas aeruginosa (14, 14.5%). In this study, all the recovered isolates were found to be multidrug resistant gram negative bacteria, with highest resistance was shown by Salmonella spp. The high MARI observed in all the isolates in this study ranging from 0.7 to 0.9. MARI value of 0.2 > is suggests multiple antibiotic resistant bacteria and indicate presence of highly resistant bacteria. Conclusion: The study indicates highly resistant bacteria are carried by healthy food animals. Thus, there is need for continued monitoring of antibiotics use in animal husbandry to prevent further spread of resistance in Maiduguri, Nigeria.

scholarly journals Formation of a β-barrel membrane protein is catalyzed by the interior surface of the assembly machine protein BamA

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
James Lee ◽  
David Tomasek ◽  
Thiago MA Santos ◽  
Mary D May ◽  
Ina Meuskens ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Molecular Mechanisms ◽  
Gram Negative Bacteria ◽  
Site Specific ◽  
Gram Negative ◽  
Interior Surface ◽  
Essential Component ◽  
Bam Complex ◽  
Β Sheet

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

scholarly journals The essential inner membrane protein YejM is a metalloenzyme

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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