Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. reaction, the oxidation of glucose-6-phosphate to 6-phosphogluconolactone, in the pentose phosphate pathway, thereby providing reducing energy to all cells by maintaining the level of the reduced co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). NADPH plays an important role in maintaining the supply of reduced glutathione to counterbalance oxidant-induced oxidative stress [1]. Redox imbalance may induce cell MPI-0479605 manufacture apoptosis and MPI-0479605 manufacture necrosis, thus highlighting the role of G6PD in defending against oxidative damage [2,3]. G6PD deficiency is the most prevalent enzyme defect in humans and affects an estimated 400 million people worldwide, especially in populations historically exposed to endemic Rabbit polyclonal to DDX6 malaria [4]. The most common clinical manifestations are neonatal jaundice and acute hemolytic anemia, which is caused by the impairment of the erythrocytes ability to remove harmful oxidative stress triggered by exogenous agents such as drugs, infection, or fava bean ingestion [1,4]. Hemolytic anemia caused by infection and subsequent medication is a clinically important concern in patients with G6PD deficiency. This issue has been a primary focus for many decades in relation to efforts MPI-0479605 manufacture to understand the impact of infection (malaria) and antimalarial drugs [5,6]. Antimicrobial drug-induced hemolysis is considered the most common adverse clinical consequence of G6PD deficiency [7]. It has also been demonstrated that infections caused by certain viruses, such as hepatitis viruses (A, B, and E) and cytomegalovirus, were associated with hemolytic anemia in patients with G6PD deficiency [8,9]. Recently, it has been shown that infection by particular viruses, such as enterovirus 71, dengue virus, and coronavirus, was enhanced in G6PD-deficient cells [10C12]. However, the impact of bacterial infection on patients with G6PD deficiency still remains to be clarified. Most studies have focused on investigating the antibiotic-induced hemolysis after treatment for bacterial infection [7]. In addition, a case report showed that infection by may have triggered hemolysis and led to severe jaundice in a G6PD-deficient neonate, while another case report described hemolysis caused by infection [13,14]. Wilmanski and colleagues demonstrated that hyperinflammation (increasing cytokine levels) caused by acute endotoxemia (induced by the injection of lipopolysaccharide) resulted in increased mortality in G6PD-deficient mice [15]. Several studies also indicated that G6PD deficiency in leukocytes can result in chronic granulomatous disease (CGD) and possibly alter the host defense mechanisms for bacterial infections [16C18]. Thus far, the impact of bacterial infection on patients with G6PD deficiency has been found to primarily affect the blood cells, leading to hemolysis or immune weakness based upon the above studies. Bacterial infection or treatment with septic plasma might induce mitochondrial dysfunction by the accumulation of reactive oxygen species (ROS) and nitric oxide radical (NO) in lymphocytes or epithelial cells leading to cell apoptosis [19C21]. Therefore, we propose that cells with G6PD deficiency may be MPI-0479605 manufacture less tolerant to the oxidative stress caused by bacterial infection. In the present study, we investigate the direct impact of bacterial infection on G6PD-deficient epithelial cells using as a model pathogen. (MRSA). Our previous study demonstrated that the vancomycin-treated vancomycin-resistant (VRSA) strain did enhance cytotoxicity through the activation of B and alternation of virulence expression [22]. Whether such enhancement is even stronger in G6PD-deficient cells was also investigated in this study. Materials and Methods Bacterial strain and growth condition The vancomycin-resistant strain SJC1200 was generated by introducing a vancomycin resistance-carrying plasmid (pG1546) into strain ATCC 12598 as described previously [23]. Briefly, the gene cluster (the operon within TnHIP12467 MPI-0479605 manufacture was amplified and then cloned into pGHL6 from which the gene was removed to generate pG1546. All bacterial strains were routinely cultured at 37C with the specific required antibiotics (Sigma) in BHI broth or on agar plates. Cell culture and bacterial infection A lung carcinoma cell line (A549) obtained from the American Type Culture Collection (ATCC) and derivatives of this cell line were used in the present study. The stable G6PD-knockdown cell line, A549-5.20, and the control cell line transfected with pCI-neo vector only, A549-5S-5, were generated, approved and kindly given by Dr. Hung-Yao Ho [11]. Briefly, G6PD-RNAi plasmids were generated by the ligation of complementary oligonucleotides into the pCI-neo mammalian expression vector followed by transfection into A549 cells to generate A549-5.20. The cells were cultured in DMEM (Gibco BRL) supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 U/ml) at 37C in a humidified atmosphere.

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