Partial Purification and Characterization of a Bacteriocin DT24 Produced by Probiotic Vaginal Lactobacillus brevis DT24 and Determination of its Anti-Uropathogenic Escherichia coli Potential

The emergence of antibiotic resistance has increased the interest for finding new antimicrobials in the past decade. Probiotic Lactic acid bacteria producing antimicrobial proteins like bacteriocin can be excellent agents for development as novel therapeutic agents and complement to conventional antibiotic therapy. Uropathogenic Escherichia coli, most causative agent of Urinary tract infection, has developed resistance to various antibiotics. In the present investigation, antibacterial substance like bacteriocin (Bacteriocin DT24) produced by probiotic Lactobacillus brevis DT24 from vaginal sample of healthy Indian woman was partially purified and characterized. It was efficiently working against various pathogens, that is, Uropathogenic E. coli, Enterococcus faecium, Enterococcus faecalis and Staphylococcus aureus. The antimicrobial peptide was relatively heat resistant and also active over a broad range of pH 2–10. It has been partially purified by ammonium sulfate precipitation and gel filtration chromatography and checked on reverse-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacteriocin DT24 was approximately 7-kDa protein. The peptide is inactivated by proteolytic enzymes, trypsin and lipase but not when treated with catalase, α-amylase and pepsin. It showed bacteriostatic mode of action against uropathogenic E. coli. Such characteristics indicate that this bacteriocin-producing probiotic may be a potential candidate for alternative agents to control urinary tract infections and other pathogens.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.     

Inner Nuclear Layer Thickening Is Inversley Proportional to Retinal Ganglion Cell Loss in Optic Neuritis

Aim

To examine the relationship between retinal ganglion cell loss and changes in the inner nuclear layer (INL) in optic neuritis (ON).

Methods

36 multiple sclerosis (MS) patients with a history of ON and 36 age and sex-matched controls underwent Optical Coherence Tomography. The paramacular retinal nerve fiber layer (RNFL), combined ganglion cell and inner plexiform layers (GCL/IPL) and inner nuclear layer (INL) thickness were measured at 36 points around the fovea. To remove inter-subject variability, the difference in thickness of each layer between the ON and fellow eye of each patient was calculated. A topographic analysis was conducted.

Results

The INL of the ON patients was thicker than the controls (42.9µm versus 39.6µm, p=0.002). ON patients also had a thinner RNFL (27.8µm versus 32.2µm, p<0.001) and GCL/IPL (69.3µm versus 98.1µm, p<0.001). Among the controls, there was no correlation between RNFL and GCL/IPL as well as RNFL and INL, but a positive correlation was seen between GCL/IPL and INL (r=0.65, p<0.001). In the ON group, there was a positive correlation between RNFL and GCL/IPL (r=0.80, p<0.001) but a negative correlation between RNFL and INL (r=-0.61, p<0.001) as well as GCL/IPL and INL (r=-0.44, p=0.007). The negative correlation between GCL/IPL and INL strengthened in the ON group when inter-subject variability was removed (r=-0.75, p<0.001). Microcysts within the INL were present in 5 ON patients, mainly in the superior and infero-nasal paramacular regions. While patients with microcysts lay at the far end of the correlation curve between GCL/IPL and INL (i.e. larger INL and smaller GCL/IPL compared to other patients), their exclusion did not affect the correlation (r= -0.76, p<0.001).

Conclusions

INL enlargement in MS-related ON is associated with the severity of GCL loss. This is a continuous relationship and patients with INL microcysts may represent the extreme end of the scale.     

Trends in inulinase production – a review

This article highlights the research work carried out in the production of inulinases from various inulin substrates using strains of bacteria, yeast and fungi. Inulin is one of the numerous polysaccharides of plant origin that contains glucose or fructose. It is used as a substrate in industrial fermentation processes and in food industries due to its relatively cheap and abundant source for the microbiological production of high-fructose syrups, ethanol and acetone–butanol. The various oligosaccharides derived from inulin also find their application in the medical and dietary sector. The inulinase acts on the β-(2,1)-D-fructoside links in inulin releasing D-fructose. Hence, this article illustrates the capability of various microbes in hydrolyzing the carbon at its optimum nutrient concentration and operating condition towards inulinase production.     

Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Covalent Modification Cycles through the Spatial Prism

Covalent modification cycles are basic units and building blocks of posttranslational modification and cellular signal transduction. We systematically explore different spatial aspects of signal transduction in covalent modification cycles by starting with a basic temporal cycle as a reference and focusing on steady-state signal transduction. We consider, in turn, the effect of diffusion on spatial signal transduction, spatial analogs of ultrasensitive behavior, and the interplay between enzyme localization and substrate diffusion. Our analysis reveals the need to explicitly account for kinetics and diffusional transport (and localization) of enzymes, substrates, and complexes. It demonstrates a complex and subtle interplay between spatial heterogeneity, diffusion, and localization. Overall, examining the spatial dimension of covalent modification reveals that 1), there are important differences between spatial and temporal signal transduction even in this cycle; and 2), spatial aspects may play a substantial role in affecting and distorting information transfer in modules/networks that are usually studied in purely temporal terms. This has important implications for the systematic understanding of signaling in covalent modification cycles, pathways, and networks in multiple cellular contexts.