Anaerobic Respiration of \u3cem\u3eEscherichia coli\u3c/em\u3e in the Mouse Intestine

The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in the intestine

Respiration of \u3cem\u3eEscherichia coli\u3c/em\u3e in the Mouse Intestine

Mammals are aerobes that harbor an intestinal ecosystem dominated by large numbers of anaerobic microorganisms. However, the role of oxygen in the intestinal ecosystem is largely unexplored. We used systematic mutational analysis to determine the role of respiratory metabolism in the streptomycin-treated mouse model of intestinal colonization. Here we provide evidence that aerobic respiration is required for commensal and pathogenic Escherichia coli to colonize mice. Our results showed that mutants lacking ATP synthase, which is required for all respiratory energy-conserving metabolism, were eliminated by competition with respiratory-competent wild-type strains. Mutants lacking the high-affinity cytochrome bd oxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo3 oxidase, which is used when oxygen tension is high, was found not to be necessary for colonization. Mutants lacking either nitrate reductase or fumarate reductase also had major colonization defects. The results showed that the entire E. coli population was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coli niche is alternately microaerobic and anaerobic, rather than static. The results indicate that success of the facultative anaerobes in the intestine depends on their respiratory flexibility. Despite competition for relatively scarce carbon sources, the energy efficiency provided by respiration may contribute to the widespread distribution (i.e., success) of E. coli strains as commensal inhabitants of the mammalian intestine

CCR5AS lncRNA variation differentially regulates CCR5, influencing HIV disease outcome.

Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

A study on limnological aspects of Ramsagar lake in Dinajpur district

Limnological aspects of the Ramsagar lake, a manmade lake of Dinajpur district was studied from January 2013 to August 2013. In this experiment, five sampling points inside the Ramsagar lake were selected and water quality parameters were analyzed fortnightly. Among different physico-chemical parameters, water temperature, dissolved oxygen, total alkalinity, total hardness, phosphate-phosphorus and concentration of Chlorophyll-a did not differ significantly except the water level and pH. A total of 29 species of plankton were recorded where phytoplankton comprised of 21 species and zooplankton comprised of 8 species. Chlorophyceae was found dominant throughout the study period followed by Bacillariophyceae, Cyanophyceae and Euglenophyceae. The highest plankton cell density was observed in the month of July and lowest plankton density was observed during the month of January. The study revealed that on the basis of physical, chemical, biological and environmental conditions the water of the lake was found to be suitable for survival of aquatic flora and fauna and also suitable for fish culture

Pharmacological inhibition of TPL2/MAP3K8 blocks human cytotoxic T lymphocyte effector functions.

CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens. During development, antigen-presenting cells secrete innate cytokines such as IL-12 and IFN-α, which drive CTL differentiation into diverse populations of effector and long-lived memory cells. Using whole transcriptome analyses, the serine/threonine protein kinase Tpl2/MAP3K8 was found to be induced by IL-12 and selectively expressed by effector memory (TEM) CTLs. Tpl2 regulates various inflammatory pathways by activating the ERK mediated MAP kinase pathway in innate immune cells such as macrophages and dendritic cells. In this study, we found that a specific small molecule Tpl2 inhibitor blocked IFN-γ and TNF-α secretion as well as cytolytic activity of human CTLs. This pathway was specific for human effector CTLs, as the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. Further, IL-12 failed to induce expression of Tpl2 in murine CTLs, and Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo in response to L. monocytogenes infection. In summary, we identified a species-specific role for Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors

Pharmacological blockade of Tpl2 significantly impairs human CTL cytokine secretion and lytic activity.

<p>CD8⁺CCR7^lo T_EM CTLs were sorted from healthy human PBMCs. (A) Sorted cells were stimulated with plate-bound anti-CD3 and rhIL-2 (200 U/mL)±indicated kinase inhibitors. Supernatants were collected at 24 h post stimulation. IFN-γ (top panel) and TNF-α (bottom panel) in the supernatant were measured using ELISA and mean±SEM is plotted for a representative of five similar experiments. (B) Sorted CD8⁺CCR7^lo T_EM CTLs were used in redirected lysis assay at the indicated Effector:Target ratios using anti-CD3 coated THP-1 target cells±Tpl2 inhibitor at the indicated concentrations. Killing of target cells is plotted as percent specific lysis, mean±SD plotted. Data shown are representative of two similar experiments from separate healthy adult donors. (C) Purified human CD8⁺ T cells were expanded for 5 days on anti-CD3/anti-CD28-coated plates in the presence of rhIL-12. Cells were rested for 24 h then restimulated with biotin-anti-CD8 + biotin-anti-CD3 with crosslinking streptavidin for 10 min in the absence or presence of the Tpl2 inhibitor at the indicated concentrations. Cell lysates were prepared and assessed for p-ERK and ERK by western blotting. Data are representative of 3 separate experiments.</p

Pharmacological inhibition of Tpl2 blocks effector cytokine secretion from human but not murine CTLs <i>in vitro</i>.

<p>(A) Total CD8⁺ T cells were isolated from WT C57BL/6 splenocytes and stimulated with plate-bound anti-CD3+anti-CD28 in the presence of rhIL-2 and rmIL-12. On day 3 post stimulation, the cells were split 1:10 in cIMDM+50 U/mL rhIL-2. On day 5 post stimulation, cells were harvested, counted, and re-stimulated for 24 h with plate-bound anti-CD3+50 U/mL rhIL-2 in the presence or absence of the indicated inhibitor conditions to test for effector cytokines secretion. IFN-γ (left panel) and TNF-α (right panel) in the supernatant were measured using ELISA. Mean±SEM is plotted and the error represents five individual mice, n = 5. (B) Naïve CD8⁺ (CD8⁺CD45RA⁺) T cells were isolated by negative selection or FACS from healthy human PBMC and stimulated with plate-bound anti-CD3+anti-CD28 and rhIL-12. Cells were split 1:10 with 100 U/mL IL-2 on d3 post stimulation. On d7, cells were re-stimulated with plate-bound anti-CD3+rhIL-2 (200 U/mL)±indicated kinase inhibitors. Supernatant was collected 24 h post 2^o stimulation. IFN-γ (left panel) and TNF-α (right panel) in the supernatant were measured by ELISA. Data shown are representative of five independent experiments and mean±SEM is plotted. *, p≤0.05 compared to control treated groups.</p

Differential Tpl2 mRNA expression in murine naïve versus effector/memory CTL subpopulations following <i>Listeria monocytogenes</i> infection.

<p>(A) WT C57BL/6 mice were infected intravenously with 2000 CFU of LM-OVA. One cohort of the mice was re-infected with 20,000 CFU of LM-OVA 48 days post infection. Spleen and LN from all mice were collected 51 days post 1° infection and surface receptor staining was performed for CD8, CD44, and CD62L. T_EM, T_CM, and T_N CTLs were defined as indicated for sorting and total RNA was isolated immediately after sorting. The cells were pooled from the mice under the same treatment group. (B) Tpl2 and IFN-γ mRNA were measured by qRT-PCR within the CD44^hiCD62L^lo (white bar, only from the mice with both 1° and 2° infections due to limiting cell numbers), CD44^hiCD62L^hi (black bar), and CD44^l°CD62L^hi (gray bar) sorted CTLs, and relative expression was determined by comparing to CD44^l°CD62L^hi population from each infection group. PPIA was used as the reference gene. This experiment was performed twice with 3–5 animals/group.</p