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Introduction. Legionella pneumophila is a Gram-negative flagellated bacteria that can infect human lungs and cause a severe form of pneumonia named Legionnaires' disease. Hypothesis. We hypothesize that L. pneumophila infection induces methylomic changes in methylcytosine dioxygenases, ten-eleven translocation (TET) genes, and controls DNA methylation following infection. Aim. In the current research, we sought to further investigate DNA methylation changes in human lung epithelial cells upon L. pneumophila infection and determine how methylation inhibitor agents disturb L. pneumophila reproduction. Methodology. A549 cell line was used in L. pneumophila infection and inhibitors' treatment, including 5-azacytidine (5-AZA) and (-)-epigallocatechin-3-O-gallate (EGCG). Results. Interestingly, DNA methylation analysis of infected A549 using sodium bisulfite PCR and the methylation-sensitive HpaII enzyme showed potential methylation activity within the promoter regions of ten-eleven translocation (TET) genes located on CpG/397–8 and CpG/385–6 of TET1 and TET3, respectively. Such methylation changes in TET effectors decreased their expression profile following infection, indicated by quantitative real-time PCR (RT-qPCR), immunoblotting and flow cytometry. Furthermore, pre-treatment of A549 cells with 5-AZA or EGCG significantly decreased the bacterial reproduction characterized by the expression of L. pneumophila 16S ribosomal RNA and the c.f.u. ml−1 of bacterial particles. Moreover, both methylation inhibitors showed potent inhibition of methionine synthase (MS) expression, which was further confirmed by the docking analysis of inhibitor ligands and crystal structure of MS protein. Conclusion. These data provide evidence for the methylomic changes in the promoter region of TET1 and TET3 by L. pneumophila infection in the A549 cell line and suggest the anti-bacterial properties of 5-AZA and EGCG, as methylation inhibitors, are due to targeting the epigenetic effector methionine synthase.

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Thalassemia is a wide range hereditary disease with high incidence in Egypt along with the high frequency of consanguineous marriages. Investigation the heterogeneity, molecular basis, and natural history are the most effective methods to deal with the thalassemia to develop effective method for management and prevention including the prenatal diagnosis. The current study aims to detect the most common β-globin gene mutations in Egypt among β-thalassemic patients by using PCR based reverse hybridization method (StripAssay) for the most prevalent 22 β-globin gene mutations in the mediterranean population in an attempt to estimate the incidence of each mutation, and an attempt to improve our control strategy of βthalassemia. This study included a total of 37 confirmed βthalassemia ethnic Egyptian patients (23 males and 14 females) out of them 17 patients were a thalassemia major and 20 were a thalassemia intermediate. Evaluation of β-thalassemia mutations revealed that, the presence of 9 different β-globin mutations. The most frequent mutation were IVS 1-110[34%], IVS 1-6(23.5%), IVS 1-1(19%), Codon 27[6.5%], IVS 2-848[6.5%], IVS 2-745[2.1%] and IVS 2.1 [2.5%], Codon 39[4%]), and IVS 1.5 [1.5%]. IVS 1-110[G>A] is the commonest homozygous mutation found in 6 out of 14 homozygous cases accounting for about 43% of homozygous mutations while, IVS 1-110[G>A]/ IVS 1-6[T>C] is the commonest heterozygous mutation found in 6 out of 23 heterozygous cases accounting for about 26% of heterozygous mutations. In conclusion,β-Globin Strip Assay is useful rapid screening method for β-globin gene mutations which can provide an insight into the detection of individual patients, to take the proper measurement, and thus could be used as a basis for genetic counseling and prenatal diagnosis.