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Analysis of Pseudomonas aeruginosa PAO1 Lipid A Changes During the Interaction with Model Organism, Caenorhabditis elegans

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Lipids

Abstract

Lipopolysaccharide (LPS) is the main surface constituent of Gram-negative bacteria. Lipid A, the hydrophobic moiety, outer monolayer of the outer cell membrane forms the major component of LPS. Immunogenic Lipid A is recognized by the innate immune system through the TLR 4/MD-2 complex. Pseudomonas aeruginosa PAO1, a Gram-negative bacterium is known to cause nosocomial infection and known for its adaptation to adverse environmental conditions. Pseudomonas aeruginosa can infect a broad host spectrum including Caenorhabditis elegans, a simple free living soil nematode. Here, we reveal that PAO1 modifies its Lipid A during the host interaction with C. elegans. The penta-acylated form of Lipid A was identified by using matrix assisted laser desorption ionization–time of flight analysis and the β-(1,6)-linked disaccharide of glucosamine with phosphate groups, 2 and 2′ amide linked fatty acid chain and 3 and 3′ ester linked fatty acids were investigated for the modification using the non destructive 1H NMR, spin–lattice (T 1) relaxation measurement, differential scanning calorimetry. T 1 relaxation measurements showed that the 2 and 2′ amide linked fatty acid chain, –CH in the glucosamine disaccharide of PAO1 lipid A, in an exposed host had a different spin lattice relaxation time compared to an unexposed host and the findings were reconfirmed using in vitro human corneal epithelial cells cell lines. Furthermore, scanning electron microscope and confocal laser scanning microscopy analysis revealed that the P. aeruginosa PAO1 biofilm formation was disturbed in the exposed host condition. The daf-12, daf-16, tol-1, pmk-1, ins-7 and ilys3 immune genes of C. elegans were examined with live bacterial and isolated lipid moiety infection and the expression was found to be highly specific. Overall, the present study revealed that PAO1 modified its 2 and 2′ amide linked fatty acid chain in the lipid A of PAO1 LPS during the exposed host condition.

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Abbreviations

°C:

Degree Celsius

cDNA:

Complementary deoxyribonucleic acid

CLSM:

Confocal laser scanning microscope

CO2 :

Carbon-di-oxide

CPA:

Common polysaccharide antigen

daf :

Abnormal dauer formation (gene)

DHB:

2,5-Dihydroxybenzoic acid

DSC:

Differential scanning calorimetry

FTIR:

Fourier transform infrared

h:

Hour

HCEC:

Human corneal epithelial cells

HEGF:

Human epithelial growth factor

ilys :

Invertebrate lysozyme (gene)

ins :

Insulin (gene)

KBr:

Potassium bromide

L4:

Larva stage 4

LPS:

Lipopolysaccharide

Lys:

Lysozyme

M:

Molar

MALDI–TOF:

Matrix assisted laser desorption ionization–time of flight

MAMP:

Microbe associated molecular pattern

m:

minute

mRNA:

Messenger ribonucleic acid

NGM:

Nematode growth medium

OD:

Optical density

OSA:

O-specific antigen(s)

PAMP:

Pathogen associated molecular pattern(s)

PBS:

Phosphate buffer saline

PCR:

Polymerase chain reaction

pmk :

P38 map kinase (gene)

qPCR:

Quantitative polymerase chain reaction

RNA:

Ribonucleic acid

rpm:

Rotation per minute

s:

Second

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEM:

Scanning electron microscope

tol :

Toll like receptor (gene)

UV–VIS:

Ultra violet–visible

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Acknowledgments

We sincerely acknowledge Dr. Michael Givskov, at the Faculty of Health Sciences, University of Copenhagen for providing the Pseudomonas aeruginosa PAO1 strain. We thank The Director, CECRI for permitting us to use the Central Instrumentation Facility for NMR analysis and also we thank Mr. V. M. Shanmugam, CECRI for his technical support. We are indebted to Prof. K. Dharmalingam, Director-Research and Dr. Chitra Thangavel, Aravind Medical Research Foundation, Madurai, India for providing human corneal epithelial cells for the study. Also we gratefully acknowledge the use of the Bioinformatics Infrastructure Facility, Alagappa University funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India (No- BT/BI/25/001/2006). This work was supported in part by the Grants from DBT, DST, ICMR (stipend to BV), ITC-AU collaborative project and CSIR of India to KB.

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Authors and Affiliations

  1. Department of Biotechnology, Alagappa University, Science Campus, Karaikudi, 630004, Tamil Nadu, India

    Balasubramanian Vigneshkumar & Krishnaswamy Balamurugan

  2. CSIR-Central Electro Chemical Research Institute, Karaikudi, 630003, India

    Srinivasan Radhakrishnan

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  1. Balasubramanian Vigneshkumar
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  2. Srinivasan Radhakrishnan
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  3. Krishnaswamy Balamurugan
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Correspondence to Krishnaswamy Balamurugan.

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Vigneshkumar, B., Radhakrishnan, S. & Balamurugan, K. Analysis of Pseudomonas aeruginosa PAO1 Lipid A Changes During the Interaction with Model Organism, Caenorhabditis elegans . Lipids 49, 555–575 (2014). https://doi.org/10.1007/s11745-014-3898-3

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  • DOI: https://doi.org/10.1007/s11745-014-3898-3

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