Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 μg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.     

Functional Characterization of a Novel Outer Membrane Porin KpnO, Regulated by PhoBR Two-Component System in Klebsiella pneumoniae NTUH-K2044

Background

The diffusion of antibiotics through the outer membrane is primarily affected by the porin super family, changes contribute to antibiotic resistance. Recently we demonstrated that the CpxAR two-component signaling system alters the expression of an uncharacterized porin OmpC^KP, to mediate antimicrobial resistance in K. pneumoniae.

Principal Findings

In this study, functional characterization of the putative porin OmpC^KP (denoted kpnO) with respect to antimicrobial susceptibility and virulence was evaluated by generating an isogenic mutant, ΔkpnO in a clinical isolate of K. pneumoniae. Estimation of uronic acid content confirmed that ΔkpnO produced ∼2.0 fold lesser capsular polysaccharide than the wild-type. The ΔkpnO displayed higher sensitivity to hyper osmotic and bile conditions. Disruption of kpnO increased the susceptibility of K. pneumoniae to oxidative and nitrostative stress by ∼1.6 fold and >7 fold respectively. The loss of the Klebsiella porin led to an increase in the minimum inhibitory concentration of tetracycline (3-fold), nalidixic acid (4-fold), tobramycin (4-fold), streptomycin (10-fold), and spectinomycin (10-fold), which could be restored following complementation. The single deletion of kpnO reduced the survival of the pathogen by 50% when exposed to disinfectants. In Caenorhabditis elegans model, the kpnO mutant exhibited significantly (P<0.01) lower virulence. To dissect the role of PhoBR signaling system in regulating the expression of the kpnO, a phoB ^KP isogenic mutant was constructed. The phoB ^KP mutant exhibited impaired gastrointestinal stress response and decreased antimicrobial susceptibility. The mRNA levels of kpnO were found to be 4-fold less in phoB ^KP mutant compared to wild type. A regulatory role of PhoB^KP for the expression of kpnO was further supported by the specific binding of PhoB^KP to the putative promoter of kpnO.

Conclusions and Significance

Loss of PhoBR regulated porin KpnO resulted in increased antimicrobial resistance, increased susceptibility to gastrointestinal stress, and reduced virulence in K. pneumoniae NTUH-K2044.     

Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044

Background

Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobe belonging to the Enterobacteriaceae family of the γ-Proteobacteria class in the phylum Proteobacteria. Multidrug resistant K. pneumoniae have caused major therapeutic problems worldwide due to emergence of extended-spectrum β-lactamase producing strains. Two-component systems serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environmental conditions including antibiotic stress.

Principal Findings

In the present study, we investigated the role of an uncharacterized cpxAR operon in bacterial physiology and antimicrobial resistance by generating isogenic mutant (ΔcpxAR) deficient in the CpxA/CpxR component derived from the hyper mucoidal K1 strain K. pneumoniae NTUH-K2044. The behaviour of ΔcpxAR was determined under hostile conditions, reproducing stresses encountered in the gastrointestinal environment and deletion resulted in higher sensitivity to bile, osmotic and acid stresses. The ΔcpxAR was more susceptible to β-lactams and chloramphenicol than the wild-type strain, and complementation restored the altered phenotypes. The relative change in expression of acrB, acrD, eefB efflux genes were decreased in cpxAR mutant as evidenced by qRT-PCR. Comparison of outer membrane protein profiles indicated a conspicuous difference in the knock out background. Gel shift assays demonstrated direct binding of CpxR^KP to promoter region of ompC ^KP in a concentration dependent manner.

Conclusions and Significance

The Cpx envelope stress response system is known to be activated by alterations in pH, membrane composition and misfolded proteins, and this systematic investigation reveals its direct involvement in conferring antimicrobial resistance against clinically significant antibiotics for the very first time. Overall results displayed in this report reflect the pleiotropic role of the CpxAR signaling system and diversity of the antibiotic resistome in hyper virulent K1 serotype K. pneumoniae NTUH-K2044.     

Brain-derived neurotrophic factor enhances calcium regulatory mechanisms in human airway smooth muscle

Neurotrophins (NTs), which play an integral role in neuronal development and function, have been found in non-neuronal tissue (including lung), but their role is still under investigation. Recent reports show that NTs such as brain-derived neurotrophic factor (BDNF) as well as NT receptors are expressed in human airway smooth muscle (ASM). However, their function is still under investigation. We hypothesized that NTs regulate ASM intracellular Ca(2+) ([Ca(2+)](i)) by altered expression of Ca(2+) regulatory proteins. Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 h (overnight) in medium (control) or 1 nM BDNF in the presence vs. absence of inhibitors of signaling cascades (MAP kinases; PI3/Akt; NFκB). Measurement of [Ca(2+)](i) responses to acetylcholine (ACh) and histamine using the Ca(2+) indicator fluo-4 showed significantly greater responses following BDNF exposure: effects that were blunted by pathway inhibitors. Western analysis of whole cell lysates showed significantly higher expression of CD38, Orai1, STIM1, IP(3) and RyR receptors, and SERCA following BDNF exposure, effects inhibited by inhibitors of the above cascades. The functional significance of BDNF effects were verified by siRNA or pharmacological inhibition of proteins that were altered by this NT. Overall, these data demonstrate that NTs activate signaling pathways in human ASM that lead to enhanced [Ca(2+)](i) responses via increased regulatory protein expression, thus enhancing airway contractility.     

Antiangiogenic effects of butyric acid involve inhibition of VEGF/KDR gene expression and endothelial cell proliferation

The formation of new blood vessels from pre-existing ones is required for the growth of solid tumors and for metastasis. Interaction of tumor-secreted vascular endothelial growth factor (VEGF) with its receptor(s) on endothelial cells triggers endothelial cell proliferation and migration, which facilitate tumor angiogenesis. Butyric acid (BuA), a fermentation product of dietary fibers in the colon, is shown to alter gene expression and is postulated to be anticarcinogenic. The results presented in this paper indicate that BuA can be antiangiogenic in vivo by inhibiting angiogenesis in chorioallantoic membrane assay. BuA was not cytotoxic to endothelial cells but was a potent antiproliferative agent besides being proapoptotic to endothelial cells as verified by FACS analysis. Conditioned media from BuA-treated Ehrlich ascites tumor cells showed a 30% decrease in VEGF concentration when compared with untreated cells. The decrease in VEGF mRNA and its receptor, KDR mRNA levels in EAT and endothelial cells respectively, suggests that the VEGF-KDR system of angiogenesis is the molecular target for the antiangiogenic action of BuA.     

First evidence on induced topological changes in supercoiled DNA by an aluminium <Emphasis Type="SmallCaps">d</Emphasis>-aspartate complex

Aluminium is a debatable and suspected etiological factor in neurodegenerative disorders. Aluminium–amino acid complexes also play an important role in the complex biology of the metal. Recent reports indicate the presence of d-aspartate and d-glutamate in aging brain, human breast tumors, core amyloid plaques and neurofibrillary tangles of Alzheimer's brain. This stereoinversion from the l- to the d-enantiomer is enhanced by Al. Further, the observation that Al is localized in the chromatin region encouraged the present study of the interaction of Al–amino acid complexes with DNA. This study used circular dichroism of supercoiled DNA and showed that Al–d-Asp caused a native B-DNA to C-DNA conformational change, while Al–l-Asp, Al–l-Glu and Al–d-Glu did not alter the native B-DNA conformation. This differential DNA binding property of Al–amino acid complexes is assigned to the stereoisomerism and chirality of the complexes. Interestingly, polyamines like spermine further induced an asymmetric condensation of the "limit C-motif" induced by Al–d-Asp to a -DNA. The results were supported by computer modeling, gel studies and ethidium bromide binding. We also propose a mechanism of Al–d-Asp binding and its ability to modulate DNA topology.     

Synthesis of pyrrolo[2,1-c][1,4]benzodiazepines and their conjugates by azido reductive cyclization strategy as potential DNA-binding agents

Synthesis of pyrrolo[2,1-c][1,4]benzodiazepines via azido reductive cyclization process employing FeCl3-NaI reagent system. This methodology has been extended for the preparation of new nicotinamido-pyrrolobenzodiazepine hybrids linked through piperazino-alkane-oxy spacers that exhibit good DNA binding affinity.     

The protective effect of alpha-crystallin against acute inflammation in mice

Acute inflammation can activate macrophages or monocytes and subsequently release several inflammatory cytokines and reactive oxygen species (ROS). Oxidative stress triggered by the production of ROS plays deleterious role leading to multiple organ failure. This study was designed to investigate the prophylactic effect of alpha-crystallin, a major chaperone lens protein comprising of alpha-A and alpha-B subunits in inflammation-induced mice. Mice were divided into three groups (n=6 in each): control, inflammation and alpha-crystallin-treated. Results show that ROS was significantly higher in the lymphocytes, hepatocytes and astrocytes (P<0.05) of inflammation-induced mice when compared to control, but no significant changes were observed in the alpha-crystallin-treated group. Increased level of lipid peroxidation (LPO) and decreased activities of antioxidant such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione were observed in the inflammation-induced mice when compared to control, whereas the activities of these were found to be normal followed by alpha-crystallin treatment. We also observed a reduction in reduced glutathione levels in hepatocytes of inflammation-induced mice, which were normalized on alpha-crystallin treatment. The in vitro study has shown that alpha-crystallin treatment not only suppresses the increase in LPO levels but also inhibits the lipid breakdown resulting from autooxidation in mouse cerebral cortex homogenate, and strongly suggests that alpha-crystallin therapy may serve as a potent pharmacological agent in systemic inflammation.