Identification of a Novel Mycobacterial Gene Involved in the Synthesis of a Phenolic Glycolipid and its Role in the Prevention of Phagosome Maturation

Pathogenic Mycobacteria persist in an early endosome-like compartment by interfering with late endosomal fusion mediating factors. Studies have unraveled some of the mechanisms employed by mycobacteria to create a niche for themselves in macrophages, but it is widely accepted that they possess an arsenal of weapons to impede phagosome genesis. M. marinum has gained importance in recent years, as a model organism to study mycobacterial pathogenesis due to its phylogenetic closeness to M. tuberculosis. The infection it causes in its natural hosts display characteristic features of tuberculosis, exhibiting blocking of phagosome maturation and granuloma formation. To gain insight into the genes required for the inhibition of phagosome maturation, M. marinum transposon mutant library representing knock outs covering the entire genome was sifted for mutants defective in inhibiting phagosome maturation by designing an elegant screen, which employs magnetic separation. In this process we identified a number of mutants unable to inhibit phagosome maturation and characterised in detail one of these mutants (mutant P1). The colony morphology and sequence analysis revealed that the interrupted gene of mutant P1 (pmiA) is likely to be involved in lipid metabolism. The mutant also had a reduced intracellular survival as inferred from the in vitro bacterial survival experiments in HMDM and using mice as an in vivo model. The mutant completely reverted to its wild-type phenotype when complemented with the respective gene from wild-type M. marinum. Thin layer chromatography on the lipids isolated from the mutant showed that the disruption of the gene pmiA in mutant P1 leads to the loss of a glycolipid of the outer envelope of M. marinum (Robinson N et al., Infect Immun. 2007 Feb;75(2):581-91). The missing glycolipid was further characterised to be a phenolic glycolicpid (PGL) using mass spectrometry and nuclear magnetic resonance spectroscopy. In order to prove that the lipid is capable of inhibiting phagosome maturation, it was extracted from wild-type M. marinum, coated on to hydrophobic beads and chased into human monocyte derived macrophages (HMDM). Characterising the phagosomes containing the beads by western blot analysis and immunofluorescence microscopy proved the lipid to be a key molecule employed by virulent mycobacteria to inhibit phagosome maturation. Phagosomes were characterised employing an efficient adenoviral transfection system harbouring Rab-GFP fusion proteins to transfect primary phagocytes. This transfection system enables phagosome maturation to be studied efficiently by fluorescence microscopy in live cells, in contrast to immunostaining which can be performed only on fixed cells. The gene pmiA involved in the biosynthesis of the phenolic glycolipid shows little homology with the gene sequences available through genome databases. It also does not display any signature sequences of proteins with known functions. Therefore, an attempt was made to study its interacting proteins by using Histidine-tag pull down assay. Proteins interacting with pmiA were analyzed by mass spectrometry. A methyl transferase and an isocitrate lyase, both enzymes critically involved in lipid biosynthesis were found to interact with pmiA. Our results prove that genes involved in the synthesis of this phenolic glycolipid are ideal pharmacological targets to design drug interventions against tuberculosis

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

2-(Pyrene-1-yl)-1,3-dithiane

In the title compound, C20H16S2, the pyrene ring is planar [maximum deviation 0.0144 (15) Å] and the dithiane ring adopts a chair conformation. The crystal packing is stabilized by C—H⋯π inter­actions. An intra­molecular C—H⋯S hydrogen bond generates an S(5) ring motif

Programmed necrotic cell death of macrophages: Focus on pyroptosis, necroptosis, and parthanatos

Macrophages are highly plastic cells of the innate immune system. Macrophages play central roles in immunity against microbes and contribute to a wide array of pathologies. The processes of macrophage activation and their functions have attracted considerable attention from life scientists. Although macrophages are highly resistant to many toxic stimuli, including oxidative stress, macrophage death has been reported in certain diseases, such as viral infections, tuberculosis, atherosclerotic plaque development, inflammation, and sepsis. While most studies on macrophage death focused on apoptosis, a significant body of data indicates that programmed necrotic cell death forms may be equally important modes of macrophage death. Three such regulated necrotic cell death modalities in macrophages contribute to different pathologies, including necroptosis, pyroptosis, and parthanatos. Various reactive oxygen and nitrogen species, such as superoxide, hydrogen peroxide, and peroxynitrite have been shown to act as triggers, mediators, or modulators in regulated necrotic cell death pathways. Here we discuss recent advances in necroptosis, pyroptosis, and parthanatos, with a strong focus on the role of redox homeostasis in the regulation of these events

Ethnicity and Dietary Practices as Colorectal Cancer Risk Predictors: A Retrospective Case-control Study in Sabah, Malaysia

Introduction: The association between colorectal cancer (CRC), ethnicity, and dietary practices have been well studied. However, limited studies have been conducted to assess dietary practices and ethnicity in Sabah on risk of CRC. This study aimed to assess the risk and protective factors in dietary practices and the inclusion of ethnicity and dietary practices as risk predictors for CRC. Methods: 148 CRC patients, 609 controls were recruited in this case-control study. Logistic regression analyses were performed to determine significant predictors of CRC. Prediction model was computed using Logistic Regression (LR) and C5 Decision Tree algorithms and compared. Results: Age 60-69 (aOR = 7.44, 95% CI = 3.69-15.00); male (aOR = 4.49, 95% CI = 2.67-7.54), Chinese (aOR = 32.32, 95% CI = 7.20-145.13); moderate physical activity (aOR = 3.67, 95% CI = 2.03-6.63), pickled mango (aOR = 5.66, 95% CI = 1.62-19.81), pork (aOR = 2.29, 95% CI = 1.09-4.79) increased the odds of developing CRC. No comorbidities (aOR = 0.53, 95% CI = 0.31-0.91), tertiary education attainment (aOR = 0.18, 95% CI = 0.07-0.43) were protective against CRC. Hosmer-Lemeshow test indicated good fit of the model (p =.946) and excellent discriminatory power (AUC=0.877). LR prediction model demonstrated better overall accuracy (89.2%), discriminatory power (AUC=0.82), sensitivity (77%), and specificity (91%) than the C5 Model. Conclusion: Frequent consumption of pickled mangoes and pork increased CRC risk among the Sabah population. Inclusion of ethnicity and dietary practices as predictors could potentially improve risk stratification of the Sabah population for early CRC screenin

Transport of Streptococcus pneumoniae Capsular Polysaccharide in MHC Class II Tubules

Bacterial capsular polysaccharides are virulence factors and are considered T cell–independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II–dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell–dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide–carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens

Study of the Structure of Hyperbranched Polyglycerol Coatings and Their Antibiofouling and Antithrombotic Applications

While blood‐contacting materials are widely deployed in medicine in vascular stents, catheters, and cannulas, devices fail in situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials can provide significant benefits for patients in terms of safety and patency as well as substantial cost savings. Herein, a novel but simple strategy is described for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma‐grafting of an ultrathin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O─C─O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface. In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3 log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG‐coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice shows no evidence of toxicity nor inflammation

Sex differences in immune responses to infectious diseases

Purpose The influence of sex hormones is recognized to account for the susceptibility and distinct outcomes of diverse infectious diseases. Methods This review discusses several variables including differences in behavior and exposure to pathogens, genetic, and immunological factors. Conclusion Understanding sex-based differences in immunity during different infectious diseases is crucial in order to provide optimal disease management for both sexes

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation