Undergraduate Research Forum Poster

Introduction

Infections caused by antibiotic-resistant bacteria kill > 1 million people, including 200,000 newborn babies annually worldwide.
These infections are projected to cause 10 million fatalities annually by 2050.
Escherichia coli is a Gram-negative bacterium that inhabit and contaminate many places such as the environment, food, water, and the digestive tracts of humans and animals.
Pathogenic E. coli strains are dangerous to humans and can affect different regions of the body causing ailments like food poisoning, diarrhea, pneumonia, meningitis, and sepsis. The impact from such strains calls for the development of novel antibiotic candidates, especially when considering rising rates of antibiotic resistance.
The lipopolysaccharide (LPS) transport system is crucial for providing protection against external threats and maintaining the membrane integrity of the outer membrane. This includes creating a barrier that selectively restricts access of molecules allowed to pass in and out of the cell.
LPS is vital for cell survival of many Gram-negative bacterial species. It acts as a chaperone of LPS molecules across the periplasmic space.
In inhibition of LPS activity will weaken the membrane’s integrity leading to cell death from antibiotics and the host’s innate immunity.

Project Goal

I aim to identify compounds able to bind LptA and inhibit the transport of LPS to the outer membrane. Such compounds may function as effective antibiotic candidates for Gram-negative bacteria containing LptA or structurally similar proteins.

mage showing the lipopolysaccharide transport system spanning the inner and outer membranes. On the left image, the transport system is effectively moving LPS from the cytosol to the outer leaflet of the outer membrane. On the right image the transport of LPS has been blocked at the periplasm where LptA functions, diminishing the amount of LPS on the outer membrane.

Methods and Results

Construction of an LptA expression strain

E.coli genomic DNA was isolated using the NEB Monarch Genomic Isolation Kit.
Designed primers annealing to the 5’ and 3’-ends were used to amplify the LptA gene using a polymerase chain reaction (PCR).
PCR product ligated into pJET, a blunt-end ligation vector, to develop plasmid pJET-L1.
pJET-L1 was transformed into chemically competent DH5𝛼 using the heat shock technique.
The pJET-L1 and pET-28a (expression cloning vector) were double-digested with XhoI and NcoI restriction enzymes. Digestion products were analyzed by agarose gel electrophoresis.
The LptA gene insert and linearized pET-28a vector were then ligated using T4 Ligase to construct plasmid pET-28a-L1.
pET28a-L1 was transformed into BL21 (DE3) for protein overexpression & purification.

Overexpression and purification of LptA from E. coli cells

Image is a schematic detailing the overexpression and purification of LptA methodology. 1:Tranformation of BL21 (DE3) with pET-28a-L1. 2:Protein expression in transformed cells. 3: cell lysis (burst bacterial cells). 4: Protein purification by Ni-NTA Affinity Chromatography. 5: sample concentration. 6: Buffer exchange. 7: protein quantification. 8: Red-Tris-NTA labeling.

Assessment of the binding of E. coli LPS extracts to purified LptA in vitro

Microscale thermophoresis binding assay estimates the LptA-LPS dissociation constant to be 208 μM.

Binding Check Experiments

Preliminary LptA binding checks with various FDA approved compounds chosen based on computational analysis.

Compounds:

glecaprevir (GPR)
rapamycin (RPN)
proctolin (PTN)
venetoclax (VTX)

Conclusion and Discussion

Cloning of the E. coli LptA gene and the development of an expression strain was successfully achieved
The expression and purification of LptA led to a highly pure batch of the protein, enabling in vitro binding studies.
Preliminary experiments using microscale thermophoresis confirmed binding of E. coli LPS extracts to purified LptA in vitro.
Computational screens identified small molecules with promising LptA binding profiles, paving way for further exploration.

Future Research

Conduct additional computational screens to identify more candidate inhibitors of LptA.
Perform full dose response binding experiments to validate the binding observed for GPR, RPN and VTX to LptA by determining their dissociation constants.
Cell-based assays to determine how LptA expression level impacts cell survival when in the presence of various antibiotics
- Minimum Inhibitory Concentration assays (MICs)
- Minimum Inhibitory Concentration assays
- Disk-diffusion assays
LptA knockout and complementation experiments
- Knock out the WT LptA gene from the BW25113 ΔtolC genome and complement with the inducible pBAD24 plasmid so that the expression of the protein can be completely controlled by induction and repression.
- A pBAD24 plasmid containing the LptA gene for cell-based assays with the BW25113 ΔtolC E. coli strain has been developed.
Use LPS fluorescence labeling techniques in conjunction with microscopy to evaluate transport levels, inhibition, and localization of LptA on E. coli cell membranes.

Selected Bibliography

Pahil, Karanbir S., et al. Nature. 2024. 625, 572–577.
Tran, An X., et al. Journal of Biological Chemistry. 2008. 283, 20342–20349.
World Health Organization. Antimicrobial resistance. World Health Organization.
O’Neill, J. Review on Antibiotic Resistance, May 2016.
Schematics created with BioRender.

Acknowledgements

Taylor Barber, Morgan Price, Annalee Schmidt.
Dr. Alumasa Research Group.
Miami University, College of Arts and Science, Dean’s Scholar Program.
Department of Chemistry and Biochemistry, Miami University.

Funding

Miami University, College of Arts and Science, Dean’s Scholar Grant.
Department of Chemistry and Biochemistry, Miami University Start-Up Funds to JNA

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