54 research outputs found
Structural And Biochemical Elucidation Of The Mechanism Of C-Di-Gmp Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis.
No full textBacteria have developed multiple strategies to adapt to diverse ecological niches and hostile environments. One such strategy involves formation and maintenance of multicellular communities known as biofilms. In these microbial aggregates, sessile bacterial cells are encased in an extracellular matrix. It has now been established that c-di-GMP, a ubiquitous bacterial second messenger, is a central regulator of this developmental process in bacteria. It exerts its effects on transcriptional, translational and post-translational levels. While diguanylate cyclases and phosphodiesterases with conserved GGDEF and EAL (and HD-GYP) domains are responsible for the production and degradation of the dinucleotide, respectively, the receptors form a more diverse group with degenerate, catalytically inactive GGDEFEAL domain-containing proteins representing a major subfamily. One such protein, LapD from Pseudomonas fluorescens, uses an inside-out signaling mechanism to relay intracellular c-di-GMP concentration to control the localization of an outer-membrane anchored large adhesin protein LapA at the cell surface, by sequestering a periplasmic cysteine protease, LapG. When free, LapG cleaves the N-terminus of LapA, releasing it from the cell surface and ultimately leading to biofilm dispersal. Based on our structure-function analysis, here we propose a mechanism for the c-di-GMP-mediated, regulation of periplasmic proteolysis by LapD. We first elucidate the molecular basis of signal recognition and relay by P. fluorescens LapD and identify orthologous systems in multiple other bacteria including many pathogens such as Legionella pneumophila. This is followed by our work on L. pneumophila LapG, which provides us with the first atomic models of a bacterial protease of the DUF920 family and we are able to identify a highly conserved Ca2+-binding motif integral to its function. We then characterize the LapD-ortholog CdgS9 from L. pneumophila which confirms a common molecular mechanism. The crystal structure of the periplasmic output domain module reveals novel conformations and sheds new light on the mode of activation of the receptor. We finally report the structure of a complex between CdgS9output and P. fluorescens LapG which led to the discovery of the pharmacologically relevant binding interface between the output domain and LapG
Delineation of Innate Immune Response of Vibrio Cholerae and its Outer Membrane Vesicles in an Epithelial Cell-Dendritic Co-Culture Model
No full textCholera remains a devastating bacterial cause of human morbidity and mortality in India and other developing countries. The disease is produced by a Gram-negative,
motile organism Vibrio cholerae that colonizes in the human intestine and secretes a potent cholera toxin, which ultimately stimulates cellular adenylate cyclase to cause
massive intestinal fluid loss leading to profuse watery diarrhea. To combat the disease from newly emerging threats, besides the study of organisms V. cholerae, a thorough understanding of the host response following V. cholerae infection and the response generated by bacterial components such as lipopolysaccharide, flagellar proteins, CT as well as outer membrane vesicles (OMVs) is indispensable. Such findings will eventually improve the present vaccine strains as well as design drugs for appropriate targets. Vibrio cholerae induces acute inflammatory response at intestinal epithelial surface; the underlying cellular immune mechanisms for such effects are largely unexplored. Mucosal immune response is controlled by the crosstalk between the intestinal epithelial cells (ECs) and dendritic cells (DCs). ECs act as a sensor for
current environmental conditions and release a variety of mediators which instruct nearby DCs accordingly. The present study has established for the first time an ECDC
co-culture model for V. cholerae infection. Our studies revealed that an ECderived cytokine thymic stromal lymphopoietin (TSLP) is highly elevated in ECs stimulated with V. cholerae and its recombinant flagellin (rFlaA). V. cholerae treated human ECs produce DC-attracting chemokine MIP-3α (CCL20). Flagellin, a potent V. cholerae factor was responsible for maximum stimulation of epithelial CCL20
production and subsequent DC activation. Activated DCs express high levels of costimulatory molecules and secrete inflammatory cytokines TNF-α, IL-6 and IL-1β. Bacteria stimulated ECs conditioned DCs to produce Th2 cell-attracting chemokines CCL17 and CCL22. TSLP and other mediators present in the V. cholerae stimulated
EC-culture filtrate potently activated DCs, which subsequently primed CD4+T cells to differentiate into T helper type 2 (Th2) cells that produce high amounts of IL-4, IL-13 and TNF-α and low IFN-γ. TSLP-induced proinflammatory response in DCs involved the transcriptional mechanisms, MAPKs (ERK1/2, p38 and JNK) and STAT3 activation. This study suggests TSLP and other mediators released from ECs in response to V. cholerae colonization actively influence DCs in initiating inflammatory
response. Another important component of V. cholerae is outer membrane vesicles or OMVs which are naturally produced bacterial vesicles. They are discrete, closed outer
membrane blebs having an average diameter of 10-300 nm with a bilayer membrane and electron-dense luminal content and consist only of the protein and lipids of OM and periplasm. The existence of OMVs has been demonstrated in a variety of Gramnegative bacteria. Our study for the first time demonstrates that a fraction of cholera toxin (CT), the major virulence factor of pathogenic V. cholerae is associated with the outer membrane vesicles (OMVs). Atomic force microscopy (AFM) and also transmission electron microscopy (TEM) of purified OMVs from toxigenic V.
cholerae O395 strain revealed spherical shaped vesicles of size range 20–200 nm. Immunoblotting of purified OMVs with polyclonal anti-CT antibody and GM1 ganglioside dependent ELISA suggests that CT is associated with OMVs. CHO cell
assay indicated that OMV associated CT is physiologically active. OMVs labelled with fluorescent dye interacted with intestinal epithelial cells via the CT-receptor and
were internalized increasing the cAMP level. Thus OMVs may represent an important vehicle in delivering CT to epithelial cells. In pathogenic bacteria, as OMVs are associated with a mixture of different pathogen associated molecular patterns (PAMPs) like LPS, peptidoglycan and with
toxins as in V. cholerae, these OMVs are expected to elicit host immune response.The present study evaluated the interaction of OMVs with intestinal epithelial cells
and with EC-DC co-culture. It was observed that, cytosolic pattern recognition receptor (PRR) NOD1 significantly upregulated in epithelial cells during OMV stimulation. In contrast, another PRR NOD2 expression was constitutive in nature and not up regulated upon OMV treatment. Therefore we sought to investigate the production of proinflammatory cytokines in response to V. cholerae O395 OMV stimulation in NOD1 dependent manner. We observed that vesicles induced
expression of proinflammatory cytokines such as IL-8 and GM-CSF, chemokines like CCL2, CCL20 and also TSLP in ECs through activation of ERK1/2 and p38 MAPK and NF-kB pathways in NOD1 dependent manner. ECs stimulated with OMVs
activate DCs in a direct co-culture system. DC activation was indicated by an increased number of cells expressing high levels of the activation markers HLA-DR,
Synopsis CD80 and CD83. Activated DCs express high levels of costimulatory molecules and released inflammatory cytokines IL-1β, IL-6, TNF-α, IL-23 and chemokines CCL22,
CCL17 and subsequently primed CD4+T cells leading to IL-4, IL-13 and IL-17 expression. It was observed that neutralizing of IL-23p19 caused a significant decrease in IL-17 production. These results suggest that V. cholerae O395 OMVs modulate the epithelial pro-inflammatory response and activate DCs which promoted the T cells polarization towards an inflammatory Th2/Th17 response. Thus the overall
study suggest that V. cholerae and its component outer membrane vesicle modulate epithelial function and induce dendritic cell mediated Th2/Th17 proinflammatory response and OMV act as a potent vehicle in delivering a percentage of cholera toxin to intestinal epithelial cells
STATISTICAL METHODS FOR INTEGRATING DISPARATE DATA SOURCES
No full textMy thesis is about developing statistical methods by integrating disparate data sources with real data applications, and identifying gene-environment interactions (G x E) in more extensive studies using existing analytical methods. We propose a general and novel statistical framework for combining information on multivariate
regression parameters across multiple different studies which have varying level of covariate information . We illustrate the method using real data for developing a breast cancer risk prediction model. We propose a generalized method of moments (GMM) approach for analyzing two-phase studies where we take into account the dependent structure of the datasets across the two-phases. We illustrate the method using real data on Wilm's tumor, a common type of kidney cancer in children. We analyze the largest gene by smoking interaction study for pancreatic ductal adenocarcinoma risk conducted to date using existing statistical methods
LapG, Required for Modulating Biofilm Formation by Pseudomonas fluorescens Pf0-1, Is a Calcium-Dependent Protease
No full textBiofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro studies, explains the basis of this calcium-dependent regulation. Based on the crystal structure of LapG of Legionella pneumophila in the accompanying report by Chatterjee and colleagues (D. Chatterjee et al., J. Bacteriol. 194:4415–4425, 2012), we show that the calcium-binding residues of LapG, D134 and E136, which are near the critical C135 active-site residue, are required for LapG activity of P. fluorescens in vivo and in vitro. Furthermore, we show that mutations in D134 and E136 result in LapG proteins no longer able to interact with LapD, indicating that calcium binding results in LapG adopting a conformation competent for interaction with the protein that regulates its activity. Finally, we show that citrate, an environmentally relevant calcium chelator, can impact LapG activity and thus biofilm formation, suggesting that a physiologically relevant chelator of calcium can impact biofilm formation by this organism
Association of cholera toxin with Vibrio cholerae outer membrane vesicles which are internalized by human intestinal epithelial cells
No full textAbstractCholera toxin (CT) is the major virulence factor of pathogenic Vibrio cholerae. The present study demonstrates that a fraction of CT is associated with the outer membrane vesicles (OMVs) released by V. cholerae. Atomic force microscopy (AFM) and also transmission electron microscopy (TEM) of purified OMVs from toxigenic V. cholerae O395 revealed spherical shaped vesicles of size range 20–200nm. Immunoblotting of purified OMVs with polyclonal anti-CT antibody and GM1-ganglioside dependent ELISA suggest that CT is associated with OMVs. CHO cell assay indicated that OMV associated CT is physiologically active. OMVs labeled with fluorescent dye interacted with intestinal epithelial cells via the CT-receptor and were internalized increasing the cAMP level. Thus OMVs may represent an important vehicle in delivering CT to epithelial cells
Effect of non-normality and low count variants on cross-phenotype association tests in GWAS
No full textActive Power Regulation in Hybrid Power Plants (PV, Wind and Batteries)
No full textInverter Based Resources (IBRs) are becoming the majority in the energy mixleading to grid instabilities due to variable production. Hybrid Power Plants(HPPs) with Battery Energy Storage Systems (BESS) can provide frequency supportincreasing grid stability and reliability by participating in the AncillaryService (AS) market. The capability of the HPP with PV, WT and BESS, toprovide active power control is studied in this project using various scenarios.Firstly, complementing the mismatch between the forecasted and actual productionwith various battery sizes is dealt with. Thereafter, the provision of frequencyAS, through BESS, when there is frequency deviation in the power systemis validated. Lastly, the economic impact of negative spot prices is examined.This research provides an overview of the scope of some of the services that can beprovided with different BESS sizes and the significance of the impact of the marketconditions.Keywords: Active power regulation, HPP, Frequency AS, Power balance, BES
Vibrio cholerae O395 Outer Membrane Vesicles Modulate Intestinal Epithelial Cells in a NOD1 Protein-dependent Manner and Induce Dendritic Cell-mediated Th2/Th17 Cell Responses
No full textVibrio cholerae, the etiologic agent of cholera, secretes outer membrane vesicles (OMVs) that are internalized
into host cells. OMVs activate an inflammatory response in intestinal epithelial cells (ECs) via a NOD1-dependent pathway thatactivates dendritic cells (DCs) and promotes T cell polarization toward Th2/Th17 responses.
OMVs stimulate EC-DC cross-talk in generating an inflammatory response.Findings are important for the development of efficient vaccine strategies with OMV
Integrative and Transfer Learning Methods for Disparate Data with Applications in Single-cell Genomics and Statistical Genetics
No full textIn recent research of genomics and genetics, we face the challenges how to perform data analysis better by incorporating more information either internally from dataset itself or externally from other studies. My thesis will discuss about three approaches to addressing this problem.
The first part reviews existing benchmark works and introduces a new method, \texttt{mixhvg}, for selecting highly variable genes in single-cell RNA-sequencing. This process is vital due to the intrinsic characteristics of single-cell RNA-sequencing. Our work not only fills the gap in comprehensive benchmarks for selecting optimal methods but also proposes \texttt{mixhvg}, a hybrid approach that enhances performance robustly.
The second part builds on the first, examining the effects of highly variable gene selection on downstream analysis, specifically visualization. We find that selected genes depend on data, where local structures benefit from corresponding local gene selections. To illustrate local patterns more effectively, we propose an adaptive method, \texttt{SAVIS}, which integrates with \texttt{mixhvg} to further improve outcomes.
The third part discusses heterogeneous transfer learning for disparate datasets with unmatched feature sets. We address this by exploring transfer learning between two data types to construct high-dimensional generalized linear models. Here, the primary dataset has a smaller sample size but a comprehensive set of variables of interest, while the external dataset is larger but feature-limited. Our proposed HTL-GMM method utilizes the generalized method of moments (GMM) to enhance both prediction accuracy and post-selection inference effectively.
Overall, this thesis focuses on integrative and transfer learning methods applicable to single-cell genomics and genetic data, aiming to advance analytical capabilities in these fields
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