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

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.

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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 outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri

Open Biology ◽

10.1098/rsob.160073 ◽

2016 ◽

Vol 6 (9) ◽

pp. 160073 ◽

Cited By ~ 6

Author(s):

Xia Wang ◽

Feng Jiang ◽

Jianhua Zheng ◽

Lihong Chen ◽

Jie Dong ◽

...

Keyword(s):

Bacterial Infection ◽

Outer Membrane ◽

Mutant Strain ◽

Type Iii Secretion ◽

Shigella Flexneri ◽

Phospholipase A ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Type Iii ◽

Outer Membrane Phospholipase A

Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri , termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

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In silico evolutionary analysis of Helicobacter pylori outer membrane phospholipase A (OMPLA)

BMC Microbiology ◽

10.1186/1471-2180-12-206 ◽

2012 ◽

Vol 12 (1) ◽

pp. 206 ◽

Cited By ~ 3

Author(s):

Hilde S Vollan ◽

Tone Tannæs ◽

Yoshio Yamaoka ◽

Geir Bukholm

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane ◽

In Silico ◽

Phospholipase A ◽

Evolutionary Analysis ◽

Outer Membrane Phospholipase A

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Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

Journal of Bacteriology ◽

10.1128/jb.183.18.5230-5238.2001 ◽

2001 ◽

Vol 183 (18) ◽

pp. 5230-5238 ◽

Cited By ~ 17

Author(s):

Geoffrey R. Langen ◽

Jill R. Harper ◽

Thomas J. Silhavy ◽

S. Peter Howard

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Stress Responses ◽

Elevated Temperatures ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Phospholipase A ◽

Temperature Sensitive ◽

Outer Membrane Phospholipase A ◽

Temperature Sensitive Phenotype

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

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