Zhan Yang

Shigella flexneri T3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-κB p65 protein

Shigella species possess a type III secretion system (T3SS), which is required for human infection and that delivers effector proteins into target host cells. Here, we show that the effector, IpaH4.5 dampens the pro‐inflammatory cytokine response. In both the Sereny test and a murine lung infection model, the Shigella ΔipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro‐inflammatory cytokine levels (MIP‐2 and TNF‐α) in the lung homogenates of wild type‐infected mice. Moreover, there was a threefold decrease in bacterial colonization of the mutant compared with the WT and ΔipaH4.5/ipaH4.5‐rescued strains. Yeast two‐hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF‐κB. Ten truncated versions of IpaH4.5 and p65 spanning different regions were constructed and expressed to further map the IpaH binding sites with p65. The results revealed thatthe p65 region spanning amino acids 1–190 of p65 interacted with the IpaH4.5/1–293 N‐terminal region. In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. Furthermore, the transcriptional activity of NF‐κB was shown to be inhibited by IpaH4.5 utilizing a dual‐luciferase reporter gene detection system containing NF‐κB promoter response elements. Thus, we conclude that the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF‐κB signalling pathway.

Abnormal fecal microbiota community and functions in patients with hepatitis B liver cirrhosis as revealed by a metagenomic approach

BackgroundAssessment and characterization of human colon microbiota is now a major research area in human diseases, including in patients with hepatitis B liver cirrhosis (HBLC).MethodsWe recruited 120 patients with HBLC and 120 healthy controls. The fecal microbial community and functions in the two groups were analyzed using high-throughput Solexa sequencing of the complete metagenomic DNA and bioinformatics methods.ResultsCommunity and metabolism-wide changes of the fecal microbiota in 20 HBLC patients and 20 healthy controls were observed and compared. A negative correlation was observed between the Child-Turcotte-Pugh scores and Bacteroidetes (P < 0.01), whereas a positive correlation was observed between the scores and Enterobacteriaceae and Veillonella (P < 0.01). Analysis of the additional 200 fecal microbiota samples demonstrated that these intestinal microbial markers might be useful for distinguishing liver cirrhosis microbiota samples from normal ones. The functional diversity was significantly reduced in the fecal microbiota of cirrhotic patients compared with in the controls. At the module or pathway levels, the fecal microbiota of the HBLC patients showed enrichment in the metabolism of glutathione, gluconeogenesis, branched-chain amino acid, nitrogen, and lipid (P < 0.05), whereas there was a decrease in the level of aromatic amino acid, bile acid and cell cycle related metabolism (P < 0.05).ConclusionsExtensive differences in the microbiota community and metabolic potential were detected in the fecal microbiota of cirrhotic patients. The intestinal microbial community may act as an independent organ to regulate the body’s metabolic balance, which may affect the prognosis for HBLC patients.

A Novel New Delhi Metallo-β-Lactamase Variant, NDM-14, Isolated in a Chinese Hospital Possesses Increased Enzymatic Activity against Carbapenems

ABSTRACT A novel New Delhi metallo-β-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem.

LuxS-Dependent AI-2 Regulates Versatile Functions in Enterococcus faecalis V583

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70min at an isothermal temperature of 63°C. The sensitivity of LAMP with the detection limit of 362fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.

Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method

In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5’ end (Nr and N), whereas their 3’ end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C–65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 109 copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes.

Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors.

To identify proteins with a potential role in the interaction of Bifidobacterium longum with intestinal epithelial cells, we profiled the protein response of B. longum NCC2705 following interaction with Caco-2 cells. Thirty-one protein spots, belonging to a total of 23 proteins, which exhibited a change in abundance of at least 3-fold were identified in B. longum NCC2705 following co-culture with Caco-2 cells, and were subsequently identified. Changes in expression were confirmed at the transcriptional level for a selection of these proteins. Enolase (Eno) and elongation factor Tu (EF-Tu) were amongst the proteins that showed the most prominent increase in abundance. Interaction of these proteins with plasminogen (Plg) was analyzed by Plg overlay assays, glutathione S-transferase (GST)-pull down, and western blot analysis. The results suggested that EF-Tu and Eno serve as surface receptors for B. longum NCC2705 binding to human plasminogen. Purified GST-EF-Tu and GST-Eno inhibited adhesion of B. longum NCC2705 to Caco-2 cells. Collectively, our data suggest that Eno and EF-Tu moonlight as adhesions, and are possibly involved in the protective role played by B. longum NCC2705 in defense against enteric pathogens. Biological significance The interaction of bifidobacteria with the human host plasminogen/plasmin system confirms the existence of a new component in the molecular cross-talk between bacteria and the host. Our study analyzed proteins EF-Tu and Eno with Plg binding activity, and they can inhibit adhesion of B. longum NCC2705 to Caco-2 cells, suggesting their role in the bacterial adherent to the enterocyte surface.

Prevalence and Detection of Stenotrophomonas maltophilia Carrying Metallo-β-lactamase blaL1 in Beijing, China

Intrinsic β-lactam resistance in Stenotrophomonas maltophilia is caused by blaL1 and/or blaL2, a kind of metallo-β-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of blaL1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of blaL1 in clinical samples by using two methods including a chromogenic method using calcein/Mn2+ complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65°C). The detection limit of LAMP was 3.79 pg/μl DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for blaL1, indicative of the high-specificity of the primers for the blaL1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of blaL1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these blaL1 genes were conservative with only a few sites mutated, and the strains had highly resistant to β-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of blaL1 and blaL2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain blaL1, blaL2, and blaNDM-1 genes, possessing an ability to hydrolyse all β-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying blaL1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

Proteomic Analysis of Clinical Isolate of Stenotrophomonas maltophilia with blaNDM-1,blaL1 and blaL2 β-Lactamase Genes under Imipenem Treatment

The co-occurrence of L1 and AmpR-L2 with bla(NDM-1) gene with an upstream 250-bp promoter was detected in a clinical isolate of Stenotrophomonas maltophilia DCPS-01, which was resistant to all β-lactams and sensitive only to colistin and fluoroquinolones. To investigate expression of resistance genes and the molecular mechanisms of bacteria resistance to carbapenems, proteomic profiles of the isolate was passaged with and without the drug by using 2D-PAGE. The results showed that 33 genes exhibiting a ≥3-fold change were identified as candidates that may help S. maltophilia survive drug selection. Strikingly, L1 was expressed more highly in cells grown with imipenem, and the abundant NDM-1 further increased, while very little L2 was detected even following induction. Specific activities for β-lactamase revealed that L2 remained at constitutive low levels (10.6 U/mg), while L1 and NDM-1 showed clear activity (69.8 U/mg). Our data support that imipenem could specifically and reversibly induce L1 and NDM-1, which together played key roles in drug resistance in DCPS-01. Although NDM-1 mediated resistance to carbapenems has been found in very few cases, to our knowledge, this is the first proteomics research of S. maltophilia with NDM-1, giving very broad-spectrum antibiotic resistance profiles.