The mechanisms of metronidazole resistance of Helicobacter pylori: A transcriptomic and biochemical study.

Helicobacter pylori (H. pylori) is a bacterial pathogen in the stomach, causing gastritis, gastric ulcer, duodenal ulcer and even gastric cancer. The triple therapy containing one bismuth-containing compound or a proton-pump inhibitor with two antibiotics was the cornerstone of the treatment of H. pylori infections. However the drug resistance of Helicobacter pylori is more and more common, which leads to the continued decline in the radical cure rate. The purpose of this study was to investigate the mechanism of metronidazole resistance of H. pylori through transcriptomics and biochemical characterizations. In this study, a 128-time-higher metronidazole-resistant H. pylori strain compared to the sensitive strain was domesticated, and 374 significantly differential genes were identified by transcriptomic sequencing as compared to the metronidazole-sensitive strain. Through GO and KEGG enrichment analysis, antibiotic-resistance pathways were found to be mainly involved in redox, biofilm formation and ABC transportation, and the results were verified by qRT-PCR. The subsequent biochemical analysis found that the urease activity of the drug-resistant strain decreased, and whereas the capabilities of bacterial energy production, membrane production and diffusion ability increased. The work here will drop hints for the mechanisms of antibiotic-resistance of H. pylori and provide promising biomarkers for the further development of new-kind drugs to treat metronidazole-resistant H. pylori.

Crizotinib Shows Antibacterial Activity against Gram-Positive Bacteria by Reducing ATP Production and Targeting the CTP Synthase PyrG.

Infections caused by drug-resistant bacteria are a serious threat to public health worldwide, and the discovery of novel antibacterial compounds is urgently needed. Here, we screened an FDA-approved small-molecule library and found that crizotinib possesses good antimicrobial efficacy against Gram-positive bacteria. Crizotinib was found to increase the survival rate of mice infected with bacteria and decrease pulmonary inflammation activity in an animal model. Furthermore, it showed synergy with clindamycin and gentamicin. Importantly, the Gram-positive bacteria showed a low tendency to develop resistance to crizotinib. Mechanistically, quantitative proteomics and biochemical validation experiments indicated that crizotinib exerted its antibacterial effects by reducing ATP production and pyrimidine metabolism. A drug affinity responsive target stability study suggested crizotinib targets the CTP synthase PyrG, which subsequently disturbs pyrimidine metabolism and eventually reduces DNA synthesis. Subsequent molecular dynamics analysis showed that crizotinib binding occurs in close proximity to the ATP binding pocket of PyrG and causes loss of function of this CTP synthase. Crizotinib is a promising antimicrobial agent and provides a novel choice for the development of treatment for Gram-positive infections. IMPORTANCE Infections caused by drug-resistant bacteria are a serious problem worldwide. Therefore, there is an urgent need to find novel drugs with good antibacterial activity against multidrug-resistant bacteria. In this study, we found that a repurposed drug, crizotinib, exhibits excellent antibacterial activity against drug-resistant bacteria both in vivo and in vitro via suppressing ATP production and pyrimidine metabolism. Crizotinib was found to disturb pyrimidine metabolism by targeting the CTP synthase PyrG, thus reducing DNA synthesis. This unique mechanism of action may explain the decreased development of resistance by Staphylococcus aureus to crizotinib. This study provides a potential option for the treatment of drug-resistant bacterial infections in the future.

Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase.

Editing is a post-transcriptional process that changes the content of nucleic acids occurring on both DNA and RNA levels. Inosine at position 34 in tRNA is one such example, commonly produced via the deamination of A34, catalyzed by adenosine deaminase acting on tRNA (ADAT or Tad). The formation of inosine is essential for cell viability. The eukaryotic deaminases normally consist of the catalytic subunit Tad2 and the structural subunit Tad3, but the catalytic process is poorly understood. Despite the conservation of the (pseudo-) catalytic domains, the heterodimeric enzyme Tad2/3 also possesses additional domains that could exhibit novel functions. Here we present the structure of the N-terminal domain of the Schizosaccharomyces pombe Tad2/3 heterodimeric tRNA(A34) deaminase (N-SpTad2), which shares ~30% sequence identities with uridine-cytidine or pantothenate kinases, but lacks the predicted kinase functions. While biochemical assays indicated that the domain is not a nucleic-acid binder, it is able to significantly influence the A34-tRNA deamination activity of the holoenzyme. Through co-expression and purification analyses, we deduce that N-SpTad2 plays a role in mediating protein-protein contacts and enhancing the stability and solubility of SpTad2/3, without which the deaminase is not functional. Taken together, our structural and biochemical studies highlighted the importance of the additional domains to the intrinsic deaminase functions of heterodimeric Tad2/3 enzymes and promoted our understanding on this essential post-transcriptional tRNA modification.

Ciprofloxacin-Resistant Staphylococcus aureus Displays Enhanced Resistance and Virulence in Iron-Restricted Conditions.

The unreasonable misuse of antibiotics has led to the emergence of large-scale drug-resistant bacteria, seriously threatening human health. Compared with drug-sensitive bacteria, resistant bacteria are difficult to clear by host immunity. To fully explore the adaptive mechanism of resistant bacteria to the iron-restricted environment, we performed data-independent acquisition-based quantitative proteomics on ciprofloxacin (CIP)-resistant (CIP-R) Staphylococcus aureus in the presence or absence of iron. On bioinformatics analysis, CIP-R bacteria showed stronger amino acid synthesis and energy storage ability. Notably, CIP-R bacteria increased virulence by upregulating the expression of many virulence-related proteins and enhancing the synthesis of virulence-related amino acids under iron-restricted stress. This study will help us to further explain the adaptive mechanisms that lead to bacterial resistance to antibiotics depending on the host environment and provide insights into the development of novel drugs for the treatment of drug-resistant bacterial infections.

A Novel Iron Transporter SPD_1590 in Streptococcus pneumoniae Contributing to Bacterial Virulence Properties.

Streptococcus pneumoniae, a Gram-positive human pathogen, has evolved three main transporters for iron acquisition from the host: PiaABC, PiuABC, and PitABC. Our previous study had shown that the mRNA and protein levels of SPD_1590 are significantly upregulated in the ΔpiuA/ΔpiaA/ΔpitA triple mutant, suggesting that SPD_1590 might be a novel iron transporter in S. pneumoniae. In the present study, using spd1590-knockout, -complemented, and -overexpressing strains and the purified SPD_1590 protein, we show that SPD_1590 can bind hemin, probably supplementing the function of PiuABC, to provide the iron necessary for the bacterium. Furthermore, the results of iTRAQ quantitative proteomics and cell-infection studies demonstrate that, similarly to other metal-ion uptake proteins, SPD_1590 is important for bacterial virulence properties. Overall, these results provide a better understanding of the biology of this clinically important bacterium.

The mechanism of iron-compensation for manganese deficiency of Streptococcus pneumoniae.

Given their involvement in catalysis, infection, and biofilm formation, Fe and Mn are essential for bacterial survival and virulence. In this study, we found that Streptococcus pneumoniae (S. pneumoniae) could grow in the Mn-deficient medium (MDCM). Furthermore, findings showed that the Fe concentration in the bacterium increased when the Mn concentration decreased. In addition, it was noted that supplementing MDCM with Fe resulted in the recovery of bacterial growth. Quantitative proteomics using stable-isotope dimethyl labeling was performed to investigate the adaptive growth mechanism of S. pneumoniae under Mn-deficient conditions. It was found that the expression levels of 25 proteins were downregulated, whereas those of 54 proteins were upregulated in S. pneumoniae grown in MDCM. It was also noted that several of the downregulated proteins were involved in cell energy metabolism, amino acid synthesis, and reduction of oxidation products. More importantly, several ATP-binding cassette transporters related to Fe uptake, such as PiuA, PiaA, PitA, and SPD_1609, were overexpressed for increased Fe uptake from the MDCM. The results suggest that Mn deficiency disturbs multiple metabolic processes in S. pneumoniae. Furthermore, it causes a compensatory effect of Fe for Mn, which is beneficial for the survival of the bacterium in extreme environments. SIGNIFICANCE: The relationship between manganese and iron metabolism in S. pneumoniae has not been clearly revealed. In this paper, we suggest that Mn limitation disturbs multiple metabolic processes and evidently decreases the ATP level in the bacterium. In order to survive in this extreme environment, bacteria upregulated three type of Fe ion transporters PiuABC (heme), PiaABC (ferrichrome) and PitABC (Fe3+) to uptake enough Fe ions to response to Mn deficiency. Therefore, this study reveals a bacterial mechanism of Fe compensation for Mn, and provides new insight for investigating the relativeness of Fe and Mn metabolism of bacteria.

Comparison of Iron-Binding Ability Between Thr70-NapA and Ser70-NapA of Helicobacter pylori.

BACKGROUND: The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia. METHODS: To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA. RESULTS: Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability. CONCLUSIONS: Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration.

miR-340 suppresses glioblastoma multiforme.

Deregulation of microRNAs (miRs) contributes to tumorigenesis. Down-regulation of miR-340 is observed in multiple types of cancers. However, the biological function of miR-340 in glioblastoma multiforme (GBM) remains largely unknown. In the present study, we demonstrated that expression of miR-340 was downregulated in both glioma cell lines and tissues. Survival of GBM patients with high levels of miR-340 was significantly extended in comparison to patients expressing low miR-340 levels. Biological functional experiments showed that the restoration of miR-340 dramatically inhibited glioma cell proliferation, induced cell-cycle arrest and apoptosis, suppressed cell motility and promoted autophagy and terminal differentiation. Mechanistic studies disclosed that, miR-340 over-expression suppressed several oncogenes including p-AKT, EZH2, EGFR, BMI1 and XIAP. Furthermore, ROCK1 was validated as a direct functional target miR-340 and silencing of ROCK1 phenocopied the anti-tumor effect of mR-340. Our findings indicate an important role of miR-340 as a glioma killer, and suggest a potential prognosis biomarker and therapeutic target for GBM.

Iron acquisition and regulation systems in Streptococcus species.

Gram-positive Streptococcus species are responsible for millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Iron is essential for the growth and survival of Streptococcus in the host environment. Streptococcus species have developed various mechanisms to uptake iron from an environment with limited available iron. Streptococcus can directly extract iron from host iron-containing proteins such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly by relying on the employment of specialized secreted hemophores (heme chelators) and small siderophore molecules (high affinity ferric chelators). This review presents the most recent discoveries in the iron acquisition system of Streptococcus species - the transporters as well as the regulators.

Nickel trafficking system responsible for urease maturation in Helicobacter pylori.

Helicobacter pylori (H. pylori) is a common human pathogen responsible for various gastric diseases. This bacterium relies on the production of urease and hydrogenase to inhabit the acidic environment of the stomach. Nickel is an essential cofactor for urease and hydrogenase. H. pylori has to uptake sufficient nickel ions for the maturation of urease, and on the other way, to prevent the toxic effects of excessive nickel ions. Therefore, H. pylori has to strike a delicate balance between the import of nickel ions, its efficient intracellular storage, and delivery to nickel-dependent metalloenzymes when required. The assembly and maturation of the urease enzyme is a complex and timely ordered process, requiring various regulatory, uptake, chaperone and accessory proteins. In this review, we focus on several nickel trafficking proteins involved in urease maturation: NikR, NixA, HypAB, UreEFGH, HspA, Hpn and Hpnl. The work will deepen our understanding of how this pathogenic bacterium adapts to severe habitant environments in the host.

The actions of bismuth in the treatment of Helicobacter pylori infections: an update.

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations and gastric cancer. Bismuth-based triple or quadruple therapies have been commonly recommended for the treatment of H. pylori infections. Up to now, the molecular mechanisms by which bismuth inhibits the growth of H. pylori are far from clear. The present concise review intends to cover the most recent reports and discoveries in the field of the inhibitory mechanism of bismuth against H. pylori as well as the bacterial protective response to drug treatment, which will help us to further understand the molecular mechanisms underlying the actions of metal-based drugs and stimulate further development of effective anti-bacterial drugs.

Iron trafficking system in Helicobacter pylori.

Helicobacter pylori infections are closely associated with peptic ulcers, gastric malignancy and iron deficiency anemia. Iron is essential for almost all living organisms and the investigation of iron uptake and trafficking system is thus important to understand the pathological roles of H. pylori. Up to now, the iron trafficking system of H. pylori is not yet fully clear and merits further efforts in this regards. The available information about iron uptake and regulation has been discussed in this concise review, such as FeoB in ferrous transportation, FrpB2 in hemoglobin uptake, HugZ in heme processing, virulence factors (VacA and CagA) in transferrin utilization, Pfr and NapA in iron storage and Fur in iron regulation. The identified iron trafficking system will help us to understand the pathological roles of H. pylori in the various gastric diseases and iron deficiency anemia and stimulates further development of effective anti-bacterial drugs.

Inhibition of fumarase by bismuth(III): implications for the tricarboxylic acid cycle as a potential target of bismuth drugs in Helicobacter pylori.

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations and gastric cancer. Triple therapy combining bismuth compounds with two antibiotics is the cornerstone of the treatment of H. pylori infections. Up to now, the molecular mechanisms by which bismuth inhibits the growth of H. pylori are far from clear. In the bacterial tricarboxylic acid (TCA) cycle, fumarase catalyses the reversible hydration of fumarate to malic acid. Our previous proteomic work indicated that fumarase was capable of bismuth-binding. The interactions as well as the inhibitory effects of bismuth to fumarase have been characterized in this study. The titration of bismuth showed that each fumarase monomer binds one mol equiv of Bi(3+), with negligible secondary structural change. Bismuth-binding results in a near stoichiometric inactivation of the enzyme, leading to an apparent non-competitive mechanism as reflected by the Lineweaver-Burk plots. Our collective data indicate that the TCA cycle is a potential molecular target of bismuth drugs in H. pylori.

The in vivo functions of a histidine-rich protein Hpn in Helicobacter pylori: linking gastric and Alzheimer's diseases together?

Helicobacter pylori causes such gastric diseases as gastritis, peptic ulcerations, gastric cancer and MALT lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms amyloid-like oligomers in physiologically relevant conditions. Here we proposed the in vivo functions of this protein with relevance to its physical locations. The collective evidence presented here shed some light on the pathologic mechanisms of H. pylori infections, with emphasis on the bacterial colonization in the gastric environment, pathological effects to the gastric epithelial cells and the possible link to Alzheimer's disease.

Histidine-rich protein Hpn from Helicobacter pylori forms amyloid-like fibrils in vitro and inhibits the proliferation of gastric epithelial AGS cells.

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Hpn is a histidine-rich protein abundant in this bacterium and forms oligomers in physiologically relevant conditions. In this present study, Hpn oligomers were found to develop amyloid-like fibrils as confirmed by negative stain transition electron microscopy, thioflavin T and Congo red binding assays. The amyloid-like fibrils of Hpn inhibit the proliferation of gastric epithelial AGS cells through cell cycle arrest in the G2/M phase, which may be closely related to the disruption of mitochondrial bioenergetics as reflected by the significant depletion of intracellular ATP levels and the mitochondrial membrane potential. The collective data presented here shed some light on the pathologic mechanisms of H. pylori infections.

Iron-containing lipoprotein SiaA in SiaABC, the primary heme transporter of Streptococcus pyogenes.

The cell-surface lipoprotein SiaA, a component of the SiaABC transporter, acts as the primary receptor for heme in the infamous human pathogen Streptococcus pyogenes. However, little is known about the molecular mechanism of heme binding and release as well as the role of heme-binding ligands that contribute to the uptake of heme into the pathogenic bacteria. The present report aims to clarify the coordination properties of heme iron in SiaA. By substitution of either Met79 or His229 with alanine, the mutant M79A and H229A proteins display significantly decreased heme-binding affinity and substantially increased heme-release rates, as compared with wild-type SiaA protein. Both fluorescence and circular dichroism spectra indicated that heme binding results in alterations in the secondary structure of the protein. Heme release from SiaA is a stepwise process in which heme dissociates firstly from Met79 and then from His229 with distinct conformational changes. His229 may serve as an anchor for heme binding in SiaA and thus may play a major role in the stability of the coordination between heme and the protein.

Iron depletion decreases proliferation and induces apoptosis in a human colonic adenocarcinoma cell line, Caco2.

Iron is essential for maintaining cellular metabolism of most organisms. Iron chelators such as desferrioxamine have been used clinically in the treatment of iron overload diseases. In the present study, we used human colon adenocarcinoma cells as a proliferating cell model to validate that desferrioxamine inhibits cell proliferation and induces apoptosis. Proteomic analysis revealed that proteins involved in cell proliferation, signal transduction, metabolism and protein synthesis were significantly regulated by the availability of iron, rendering a close correlation between cell apoptosis and the disturbance of mitochondrial, signaling and metabolic pathways. These results provide new insights into the mechanisms of cell proliferation inhibition attributed to iron depletion.

Crystal structure and metal binding properties of the lipoprotein MtsA, responsible for iron transport in Streptococcus pyogenes.

An ability to acquire iron is essential for the viability and growth of almost all organisms and in pathogenic bacteria is strongly correlated with virulence. The cell surface lipoprotein MtsA, a component of the MtsABC transporter of Streptococcus pyogenes, acts as the primary receptor for inorganic iron by this significant human pathogen. Iron is bound as Fe(2+), with the participation of bicarbonate. The crystal structure of MtsA has been determined and refined at 1.8 A resolution (R = 0.167, and R(free) = 0.194). MtsA has the classic bacterial metal binding receptor (MBR) fold, with the Fe(2+) ion bound to the side chains of His68, His140, Glu206, and Asp281, at a totally enclosed site between the two domains of the protein. The absence of bicarbonate from the binding site suggests that it is displaced during the final stages of metal binding. Both the fold and metal binding site are most similar to those of the manganese receptors PsaA and MntC, consistent with the similar coordination requirements of Fe(2+) and Mn(2+). Binding studies confirm a 10-fold preference for Fe(2+) over Mn(2+), although both may be carried in vivo. Mutational analysis of the binding site shows that His140 is critical for a fully functional binding site but that Glu206 is dispensable. The crystal structure explains the distinct roles of these ligands and also reveals potential secondary binding sites that may explain the binding behavior of MtsA for metal ions other than Fe(2+).

microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs.

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, we identified miR-146b as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. We showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, we discovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, our findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

MicroRNA-15b regulates cell cycle progression by targeting cyclins in glioma cells.

MicroRNAs (miRNAs) are non-protein-coding RNAs that function as post-transcriptional gene regulators. Recent evidence has shown that miRNA plays a pivotal role in the development of many cancers including glioma, a lethal brain cancer. We have recently compared the miRNA expression profiles between normal brain and glioma tissues from Chinese patients by miRNA microarray and identified a panel of differentially expressed miRNAs. Here, we studied the function of one miRNA, miR-15b, in glioma carcinogenesis and elucidated its downstream targets. Over-expression of miR-15b resulted in cell cycle arrest at G0/G1 phase while suppression of miR-15b expression resulted in a decrease of cell populations in G0/G1 and a corresponding increase of cell populations in S phase. We further showed that CCNE1 (encoding cyclin E1) is one of the downstream targets of miR-15b. Taken together, our findings indicate that miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related molecules.