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Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes

ABSTRACT: Omega-3 polyunsaturated fatty acids (n-3 PUFA), such as the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are reported to beneficially affect intestinal immunity. The intestinal epithelial layer (IEL), made up of absorptive cells as the enterocytes, is the primary site of fatty acid absorption. So far, the biological pathways modulated by n-3 PUFA in IEL during infection remain elusive. In the present study, shotgun proteomics analysis integrated with RNA sequencing (RNA-seq) technology was employed to unveil the proteomic changes induced by n-3 PUFA in enterocytes, in the presence and absence of lipopolysaccharide (LPS) stress conditions. A total of 33, 85, and 88 differential proteins were identified in the IPEC-J2 cells exposed to (i) n-3 PUFA (DHA:EPA, 1:2, 10 µM, 24 h), (ii) LPS (10 µg/mL, 24 h), or (iii) n-3 PUFA pre-treatment followed by LPS stimulation, respectively. Functional annotation and pathway analysis of the differential proteins revealed the modulation of central carbon metabolism, including glycolysis/gluconeogenesis, pentose phosphate pathway, and oxidative phosphorylation. Specifically, LPS caused metabolic dysregulation in the IPEC-J2 cells, abated upon prior treatment with n-3 PUFA. Indeed, n-3 PUFA supplementation facilitated enterocyte development and lipid homeostasis. This work provided a comprehensive picture of the biological pathways modulated by n-3 PUFA in enterocytes, both in the absence and presence of a state of endotoxin-induced metabolic dysregulation. Our findings provide important insights into the role of n-3 PUFA in the intestinal barrier, which could support better planning of nutritional strategies in managing intestinal health and immunity.

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ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Cell Culture, Intestinal Epithelial Cell

SUBMITTER: Salvatore Pisanu

LAB HEAD: Salvatore Pisanu

PROVIDER: PXD036456 | Pride | 2023-12-11

REPOSITORIES: Pride

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Project description:Staphylococcus aureus (S. aureus) is a Gram-positive opportunistic bacterium commonly isolated on the skin and in the nasal cavity of many healthy individuals. It can cause infections, from local skin and soft tissue infections (SSTIs) like wound infections, abscesses, and cellulitis, to more severe conditions such as endocarditis, pneumonia, sepsis, and toxic shock syndrome. Its pathogenicity is due to its numerous virulence factors and its adaptability to environmental changes, including pH, oxygen, CO2, and temperature during host infections. Understanding metabolic adaptation is a complex step for identifying new therapeutic targets to combat antibiotic-resistant pathogens. Proteomic approaches are essential for studying microbial protein expression during environmental changes to understand metabolic pathways and antibiotic resistance mechanisms. We investigated the protein expression of two MRSA strains under three experimental conditions (ECs) to simulate the variations in pH, temperature, and NaCl concentration expected during a wound infection. One strain is Livestock-Associated (LA-MRSA) ST398, which colonizes the nasal cavity of pigs and has also been reported in other animals and used in the food industry. It poses a risk for farmers and people in direct contact with animals, such as veterinarians. The other strain is Community Associated (CA-MRSA), JE2 strain (USA300 isolate), one of the major causes of SSTI and the main cause of CA infections in the USA. The selected ECs mimic the stress conditions occurring on the skin during infection with S. aureus. These ECs were named experimental condition 1 (EC1), characterized by pH 7 at 37°C and considered as a control condition; experimental condition 2 (EC2), characterized by pH 6 at 35°C; and experimental condition 3 (EC3), characterized by pH 6 at 35°C with 5% NaCl added to the medium. The comparative analysis of bacterial proteomes under these diverse in vitro exposures provided evidence of metabolic reshaping and highlighted strain-specific and environmental-specific variations in critical bacterial functions.

			Action	DRS
	Control_C1.msf	Msf
	Control_C1.msfView	Msf
	Control_C1.raw	Raw
	Control_C2.msf	Msf
	Control_C2.msfView	Msf

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Comprehensive proteomic analysis reveals omega-3 fatty acids to counteract endotoxin-stimulated metabolic dysregulation in porcine enterocytes

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