Ectoine Globally Hypomethylates DNA in Skin Cells and Suppresses Cancer Proliferation.

Epigenetic modifications, mainly aberrant DNA methylation, have been shown to silence the expression of genes involved in epigenetic diseases, including cancer suppression genes. Almost all conventional cancer therapeutic agents, such as the DNA hypomethylation drug 5-aza-2-deoxycytidine, have insurmountable side effects. To investigate the role of the well-known DNA protectant (ectoine) in skin cell DNA methylation and cancer cell proliferation, comprehensive methylome sequence analysis, 5-methyl cytosine (5mC) analysis, proliferation and tumorigenicity assays, and DNA epigenetic modifications-related gene analysis were performed. The results showed that extended ectoine treatment globally hypomethylated DNA in skin cells, especially in the CpG island (CGIs) element, and 5mC percentage was significantly reduced. Moreover, ectoine mildly inhibited skin cell proliferation and did not induce tumorigenicity in HaCaT cells injected into athymic nude mice. HaCaT cells treated with ectoine for 24 weeks modulated the mRNA expression levels of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l, Hdac1, Hdac2, Kdm3a, Mettl3, Mettl14, Snrpn, and Mest. Overall, ectoine mildly demethylates DNA in skin cells, modulates the expression of epigenetic modification-related genes, and reduces cell proliferation. This evidence suggests that ectoine is a potential anti-aging agent that prevents DNA hypermethylation and subsequently activates cancer-suppressing genes.

Metagenomic investigations on antibiotic resistance and microbial virulence in oil-polluted soils from China.

Engine oil spills have been associated with a wide range of human health problems. However, little is known about the effects of petroleum hydrocarbon pollution on soil microbial communities. In this study, three samples were collected from oil-polluted soils (OPS), and one control soil (CS) from Taolin town, China, near the old engine's scrapes was used. The aims of this study were to conduct metagenomic sequencing and subsequently perform resistome and virulome analysis. We also aimed to validate anti-microbial resistance and virulence genes and anti-bacterial sensitivity profiles among the isolates from oil-polluted soils. The OPS microbial community was dominated by bacterial species compared to the control samples which were dominated by metazoans and other organisms. Secondly, the resistosome and virulome analysis showed that ARGs and virulence factors were higher among OPS microbial communities. Antibiotic susceptibility assay and qPCR analysis for ARGs and virulence factors showed that the oil-polluted soil samples had remarkably enhanced expression of these ARGs and some virulence genes. Our study suggests that oil pollution contributes to shifting microbial communities to more resilient types that could survive the toxicity of oil pollution and subsequently become more resilient in terms of higher resistance and virulence potential.

Genomic and biotechnological potential of a novel oil-degrading strain Enterobacter kobei DH7 isolated from petroleum-contaminated soil.

In this study, a novel oil-degrading strain Enterobacter kobei DH7 was isolated from petroleum-contaminated soil samples from the industrial park in Taolin Town, Lianyungang, China. The whole genome of the strain was sequenced and analyzed to reveal its genomic potential. The oil degradation and growth conditions including nitrogen, and phosphorus sources, degradation cycle, biological dosing, pH, and oil concentration were optimized to exploit its commercial application. The genome of the DH7 strain contains 4,705,032 bp with GC content of 54.95% and 4653 genes. The genome analysis revealed that there are several metabolic pathways and enzyme-encoding genes related to oil degradation in the DH7 genome, such as the paa gene cluster which is involved in the phenylacetic acid degradation pathway, and complete degradation pathways for fatty acid and benzoate, genes related to chlorinated alkanes and olefins degradation pathway including adhP, frmA, and adhE, etc. The strain DH7 under the optimized conditions has demonstrated a maximum degradation efficiency of 84.6% after 14 days of treatment using synthetic oil, which comparatively displays a higher oil degradation efficiency than any Enterobacter species known to date. To the best of our knowledge, this study presents the first-ever genomic studies related to the oil degradation potential of any Enterobacter species. As Enterobacter kobei DH7 has demonstrated significant oil degradation potential, it is one of the good candidates for application in the bioremediation of oil-contaminated environments.

Cholesterol glucosylation-based survival strategy in Helicobacter pylori.

Helicobacter pylori is a major chronic health problem, infecting more than half of the population worldwide. H. pylori infection is linked with various clinical complications ranging from gastritis to gastric cancer. The resolution of gastritis and peptic ulcer appears to be linked with the eradication of H. pylori. However, resistance to antibiotics and eradication failure rates are reaching alarmingly high levels. This calls for urgent action in finding alternate methods for H. pylori eradication. Here, we discuss the recently identified mechanism of H. pylori known as cholesterol glucosylation, mediated by the enzyme cholesterol-α-glucosyltransferase, encoded by the gene cgt. Cholesterol glucosylation serves several functions that include promoting immune evasion, enhancing antibiotic resistance, maintaining the native helical morphology, and supporting functions of prominent virulence factors such as CagA and VacA. Consequently, strategies aiming at inhibition of the cholesterol glucosylation process have the potential to attenuate the potency of H. pylori infection and abrogate H. pylori immune evasion capabilities. Knockout of H. pylori cgt results in unsuccessful colonization and elimination by the host immune responses. Moreover, blocking cholesterol glucosylation can reverse antibiotic susceptibility in H. pylori. In this work, we review the main roles of cholesterol glucosylation in H. pylori and evaluate whether this mechanism can be targeted for the development of alternate methods for eradication of H. pylori infection.

Design of an inhibitor of Helicobacter pylori cholesteryl-α-glucoside transferase critical for bacterial colonization.

BACKGROUND: Fifty percent of the world's population surves as a host for Helicobacter pylori, gastric cancer causing bacteria, that colonizes the gastric region of digestive tract. It has a remarkable capacity to infect the host stomach for the entire lifetime despite an activated host immune response. METHODS: In this study, we have performed the virtual screening analysis of protein-inhibitor binding between the glycosyl transferase enzymes of Helicobacter pylori (CapJ or HP0421) and a corresponding library of inhibitors in the known substrate-binding pockets. We have docked our library of ligands consisting of cholesterol backbone with CapJ protein and identified several ligands' interacting amino acid residues present in active site pocket(s) of the protein. RESULTS: In most of the cases, the ligands showed an interaction with the residues of the same pocket of the enzyme. Top three (03) hits were filtered out from the whole data set, which might act as potent inhibitors of the enzyme-substrate reaction. CONCLUSIONS: This study describes a new possibility by which colonization of H. pylori can be limited. The reported evidence suggests that comprehensive knowledge and wet laboratory validation of these inhibitors are needed in order to develop them as lead molecules.

Mycobacterium tuberculosis DosR regulon gene Rv2004c contributes to streptomycin resistance and intracellular survival.

Tuberculosis (TB) is the deadly infectious disease challenging the public health globally and its impact is further aggravated by co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. In this study, we attempted to characterise the Rv2004c encoded protein, a member of DosR regulon, for its role in drug resistance. In silico docking analysis revealed that Rv2004c binds with streptomycin (SM). Phosphotransferase assay demonstrated that Rv2004c possibly mediates SM resistance through the aminoglycoside phosphotransferase activity. Further, E. coli expressing Rv2004c conferred resistance to 100µM of SM in liquid broth cultures indicating a mild aminoglycoside phosphotransferase activity of Rv2004c. Moreover, we investigated the role of MSMEG_3942 (an orthologous gene of Rv2004c) encoded protein in intracellular survival, its effect on in-vitro growth and its expression in different stress conditions by over expressing it in Mycobacterium smegmatis (M. smegmatis). MSMEG_3942 overexpressing recombinant M. smegmatis strains grew faster in acidic medium and also showed higher bacillary counts in infected macrophages when compared to M. smegmatis transformed with vector alone. Our results are likely to contribute to the better understanding of the involvement of Rv2004c in partial drug resistance, intracellular survival and adaptation of bacilli to stress conditions.

HBV triggers APOBEC2 expression through miR­122 regulation and affects the proliferation of liver cancer cells.

Hepatitis B virus (HBV) infection is responsible for 50% of liver cancer cases globally; this disease is one of the leading causes of cancer­associated mortality. One reported mechanism underlying the development of liver cancer is the mutation of tumor suppressor genes induced by the overexpression of apolipoprotein B mRNA­editing enzyme catalytic subunit 2 (APOBEC2) in hepatocytes. In addition, it has been observed that HBV inhibited microRNA (miR)­122 expression in hepatocytes; however, the molecular mechanisms involved in liver cancer development remain unknown and further investigations are required. In the present study, the mechanistic roles of HBV infection in modulating the expression of miR­122 and APOBEC2, and the development of liver cancer, were investigated. Reverse transcription­quantitative PCR and western blot analyses revealed that APOBEC2 expression was markedly upregulated following HBV infection. Of note, the expression profile of APOBEC2 in the Huh7 and HepG2 liver cancer cell lines opposed that of miR­122; this miR is the most abundant miRNA in the liver and has been associated with hepatocarcinogenesis. Mechanistically, it was demonstrated via a dual­luciferase assay that miR­122 could specifically bind to the 3'­untranslated region (3'UTR) of APOBEC2 mRNA, inhibiting its expression. Collectively, the findings of the present study may provide insight into the mechanistic role of HBV infection in modulating the expression of miR­122, which targets the 3'UTR of APOBEC2 mRNA, subsequently inducing liver carcinogenesis.

Roles of Cholesteryl-α-Glucoside Transferase and Cholesteryl Glucosides in Maintenance of Helicobacter pylori Morphology, Cell Wall Integrity, and Resistance to Antibiotics.

Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori, and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori, their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains (Hp26695Δ421 and Hp76Δ421) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics.IMPORTANCEHelicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics.