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Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge in vitro.

ABSTRACT: AIM:To study the effect of Staphylococcus aureus cell wall antigens, peptidoglycan (PGN) and lipoteichoic acid (LTA) challenge on immune cells present in bovine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS:In this study, efforts have been made to investigate the effects of three combinations (10+10, 20+20 and 30+30 ?g/ml) of PGN and LTA obtained from S. aureus. These antigens were used to challenge the bovine PBMCs. After 6 h of incubation quantitative, real time-polymerase chain reaction was used to study toll-like receptor 2 (TLR-2) and major cytokine mRNA expression in bovine PBMC challenged with three different antigen blends. RESULTS:The results indicated that mRNA level of interferon gamma is influenced by the expression of TLR-2 gene. Tumor necrosis factor-alpha (TNF-?), interleukin 10 (IL-10), and IL-8 genes showed a maximum response at a dose of 10 ?g of PGN and 10 ?g of LTA challenge per ml of culture medium. The outcome also suggests that both IL-10 and IL-8 followed the expression pattern of TNF-?. CONCLUSION:A dose of 10 ?g of PGN and 10 ?g of LTA per ml of culture medium was found to be most suitable for challenging PBMC.

SUBMITTER: Sulabh S

PROVIDER: S-EPMC5057038 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge <i>in vitro</i>.

Sulabh Sourabh S Bhushan Bharat B Panigrahi Manjit M Verma Ankita A Baba Naseer Ahmad NA Kumar Pushpendra P

Veterinary world 20160917 9

<h4>Aim</h4>To study the effect of <i>Staphylococcus aureus</i> cell wall antigens, peptidoglycan (PGN) and lipoteichoic acid (LTA) challenge on immune cells present in bovine peripheral blood mononuclear cells (PBMCs).<h4>Materials and methods</h4>In this study, efforts have been made to investigate the effects of three combinations (10+10, 20+20 and 30+30 μg/ml) of PGN and LTA obtained from <i>S. aureus</i>. These antigens were used to challenge the bovine PBMCs. After 6 h of incubation quanti ...[more]

PMID: 27733800

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Project description:The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureussgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.IMPORTANCE The bacterial cell wall acts as a primary defense against environmental insults such as changes in osmolarity. It is also a vulnerable structure, as defects in its synthesis can lead to growth arrest or cell death. The important human pathogen Staphylococcus aureus has a typical Gram-positive cell wall, which consists of peptidoglycan and the anionic polymers LTA and wall teichoic acid. Several clinically relevant antibiotics inhibit the synthesis of peptidoglycan; therefore, it and teichoic acids are considered attractive targets for the development of new antimicrobials. We show that LTA is required for efficient peptidoglycan cross-linking in S. aureus and inactivation of a peptidoglycan glycosyltransferase can partially rescue this defect, together revealing an intimate link between peptidoglycan and LTA synthesis.

Project description:The mammary gland of the cow is particularly susceptible to infections of a wide range of pathogenic bacteria, including both Gram-positive and Gram-negative bacteria. The endotoxins of these pathogenic bacteria include peptidoglycan (PGN), lipoteichoic acid (LTA) and lipopolysaccharide (LPS), and they are the pathogen-associated molecular patterns (PAMPs) to induce mastitis. LPS can directly inhibit proliferation and milk fat synthesis of bovine mammary epithelial cells (BMECs) while inducing mastitis, but it is unclear whether PGN and LTA also have such effects. Furthermore, since the three PAMPs usually appear simultaneously in the udder of cows with mastitis, their synergistic effects on proliferation and milk fat synthesis of BMECs are worth investigating. The immortalized BMECs (MAC-T cells) were stimulated for 24 h using various concentrations of PGN, LTA and LPS, respectively, to determine the doses that could effectively cause inflammatory responses. Next, the cells were stimulated for 24 h with no endotoxins (CON), PGN, LTA, LPS, PGN + LTA, and PGN + LTA + LPS, respectively, with the predetermined doses to analyze their effects on proliferation and milk fat synthesis of BMECs. PGN, LTA and LPS successfully induced inflammatory responses of BMECs with doses of 30, 30 and 0.1 μg/mL, respectively. Although the proliferation of BMECs was significantly inhibited in the following order: LTA < PGN + LTA < PGN + LTA + LPS, there was no change in cell morphology and cell death. LTA significantly promoted the expression of fatty acid synthesis-related genes but did not change the content of intracellular triglyceride (TG), compared with the CON group. The mRNA expression of fatty acid synthesis-related genes in the LPS group was the lowest among all the groups. Meanwhile, LPS significantly decreased the content of intracellular non-esterified fatty acids (NEFAs) and TG, compared with the CON group. PGN had no effects on milk fat synthesis. Co-stimulation with PGN, LTA and LPS significantly increased the expression of fat acid synthesis-related genes and the intracellular NEFAs, but decreased intracellular TG, compared with sole LPS stimulation. Collectively, PGN, LTA and LPS showed an additive effect on inhibiting proliferation of BMECs. The promoting role of LTA in fatty acid synthesis might offset the negative effects of LPS in this regard, but co-stimulation with PGN, LTA and LPS significantly decreased intracellular TG content.

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Dataset Information

Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge in vitro.

Publications

Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge <i>in vitro</i>.

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