Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis.

We performed a genome-wide siRNA screen to identify host factors that regulated pathogen load in human macrophages infected with a virulent strain of Mycobacterium tuberculosis. Iterative rounds of confirmation, followed by validation, identified 275 such molecules that were all found to functionally associate with each other through a dense network of interactions. This network then yielded to a molecular description of the host cell functional modules that were both engaged and perturbed by the pathogen. Importantly, a subscreen against a panel of field isolates revealed that the molecular composition of the host interface varied with both genotype and the phenotypic properties of the pathogen. An analysis of these differences, however, permitted identification of those host factors that were invariantly involved, regardless of the diversification in adaptive mechanisms employed by the pathogen. Interestingly, these factors were found to predominantly function through the regulation of autophagy.

Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen.

Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage.

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.

Understanding PGE2, LXA4 and LTB4 balance during Mycobacterium tuberculosis infection through mathematical model.

Infection of humans with Mycobacterium tuberculosis (Mtb) results in diverse outcomes that range from acute disease to establishment of persistence and to even clearance of the pathogen. These different outcomes represent the combined result of host heterogeneity on the one hand, and virulence properties of the infecting strain of pathogen on the other. From the standpoint of the host, the balance between PGE2, LXA4 and LTB4 represents at least one of the factors that dictates the eventual pathophysiology. We therefore built an ODE model to describe the host-pathogen interaction and studied the local stability properties of the system, to obtain the parametric conditions that lead to different disease outcomes. We then modulated levels of the pro- and anti-inflammatory lipid mediators to better understand the convergence between host phenotype and factors that relate to virulence properties of the pathogen. Global sensitivity analysis, using the variance-based method of extended Fourier Amplitude Sensitivity Test (eFAST), revealed that disease severity was indeed defined by combined effects of phenotypic variability at the level of both host and pathogen. Interestingly here, [PGE2] was found to act as a switch between bacterial clearance and acute disease. Our mathematical model suggests that development of more effective treatments for tuberculosis will be contingent upon a better understanding of how the intrinsic variability at the level of both host and pathogen contribute to influence the nature of interactions between these two entities.

Mathematical model of mycobacterium-host interaction describes physiology of persistence.

Despite extensive studies on the interactions between Mycobacterium tuberculosis (M.tb) and macrophages, the mechanism by which pathogen evades anti-microbial responses and establishes persistence within the host cell remains unknown. In this study, we developed a four-dimensional ODE model to describe the dynamics of host-pathogen interactions in the early phase of macrophage infection. The aim was to characterize the role of host cellular regulators such as iron and lipids, in addition to the bactericidal effector molecule Nitric Oxide. Conditions for existence and stability of the equilibrium point were analysed by examining the behaviour of the model through numerical simulations. These computational investigations revealed that it was the ability of pathogen to interfere with iron and lipid homeostatic pathways of the host cell, which ensured a shift in balance towards pathogen survival and persistence. Interestingly, small perturbations in this equilibrium triggered the cell's bactericidal response, thereby producing an oscillatory dynamic for disease progression.

Differential proteomics approach to identify putative protective antigens of Mycobacterium tuberculosis presented during early stages of macrophage infection and their evaluation as DNA vaccines.

Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated.significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.

Proviral integration site for Moloney murine leukemia virus (PIM) kinases promote human T helper 1 cell differentiation.

The differentiation of human primary T helper 1 (Th1) cells from naïve precursor cells is regulated by a complex, interrelated signaling network. The identification of factors regulating the early steps of Th1 cell polarization can provide important insight in the development of therapeutics for many inflammatory and autoimmune diseases. The serine/threonine-specific proviral integration site for Moloney murine leukemia virus (PIM) kinases PIM1 and PIM2 have been implicated in the cytokine-dependent proliferation and survival of lymphocytes. We have established that the third member of this family, PIM3, is also expressed in human primary Th cells and identified a new function for the entire PIM kinase family in T lymphocytes. Although PIM kinases are expressed more in Th1 than Th2 cells, we demonstrate here that these kinases positively influence Th1 cell differentiation. Our RNA interference results from human primary Th cells also suggest that PIM kinases promote the production of IFNγ, the hallmark cytokine produced by Th1 cells. Consistent with this, they also seem to be important for the up-regulation of the critical Th1-driving factor, T box expressed in T cells (T-BET), and the IL-12/STAT4 signaling pathway during the early Th1 differentiation process. In summary, we have identified PIM kinases as new regulators of human primary Th1 cell differentiation, thus providing new insights into the mechanisms controlling the selective development of human Th cell subsets.

Delineation of key regulatory elements identifies points of vulnerability in the mitogen-activated signaling network.

Drug development efforts against cancer are often hampered by the complex properties of signaling networks. Here we combined the results of an RNAi screen targeting the cellular signaling machinery, with graph theoretical analysis to extract the core modules that process both mitogenic and oncogenic signals to drive cell cycle progression. These modules encapsulated mechanisms for coordinating seamless transition of cells through the individual cell cycle stages and, importantly, were functionally conserved across different cancer cell types. Further analysis also enabled extraction of the core signaling axes that progressively guide commitment of cells to the division cycle. Importantly, pharmacological targeting of the least redundant nodes in these axes yielded a synergistic disruption of the cell cycle in a tissue-type-independent manner. Thus, the core elements that regulate temporally distinct stages of the cell cycle provide attractive targets for the development of multi-module-based chemotherapeutic strategies.

The High Sensitivity of the Multi-Cancer Detection Test ONCOVERYX-F Offers a Promising Platform for Ovarian Cancer Screening.

We evaluated the potential relevance of our multi-cancer detection test, OncoVeryx-F, for ovarian cancer screening. For this, we compared its accuracy with that of CA125-based screening. We demonstrate here that, in contrast to CA125-based detection, OncoVeryx-F detected ovarian cancer with very high sensitivity and specificity. Importantly here, Stage I cancers too could be detected with an accuracy of >98%. Furthermore, again unlike CA 125, the detection accuracy of OncoVeryx-F remained comparable in both Caucasian and South Asian/Indian women. Thus, the robustness and accuracy of OncoVeryx-F, particularly for early-stage detection, underscores its potential utility for ovarian cancer screening.

A non-invasive method for concurrent detection of multiple early-stage cancers in women.

Untargeted serum metabolomics was combined with machine learning-powered data analytics to develop a test for the concurrent detection of multiple cancers in women. A total of fifteen cancers were tested where the resulting metabolome data was sequentially analysed using two separate algorithms. The first algorithm successfully identified all the cancer-positive samples with an overall accuracy of > 99%. This result was particularly significant given that the samples tested were predominantly from early-stage cancers. Samples identified as cancer-positive were next analysed using a multi-class algorithm, which then enabled accurate discernment of the tissue of origin for the individual samples. Integration of serum metabolomics with appropriate data analytical tools, therefore, provides a powerful screening platform for early-stage cancers.

Temporal Profiling of Host Proteome against Different M. tuberculosis Strains Reveals Delayed Epigenetic Orchestration.

Apart from being preventable and treatable, tuberculosis is the deadliest bacterial disease afflicting humankind owing to its ability to evade host defence responses, many of which are controlled by epigenetic mechanisms. Here, we report the temporal dynamics of the proteome of macrophage-like host cells after infecting them for 6, 18, 30, and 42 h with two laboratory strains (H37Ra and H37Rv) and two clinical strains (BND433 and JAL2287) of Mycobacterium tuberculosis (MTB). Using SWATH-MS, the proteins characterized at the onset of infection broadly represented oxidative stress and cell cytoskeleton processes. Intermediary and later stages of infection are accompanied by a reshaping of the combination of proteins implicated in histone stability, gene expression, and protein trafficking. This study provides strain-specific and time-specific variations in the proteome of the host, which might further the development of host-directed therapeutics and diagnostic tools against the pathogen. Also, our findings accentuate the importance of proteomic tools in delineating the complex recalibration of the host defence enabled as an effect of MTB infection. To the best of our knowledge, this is the first comprehensive proteomic account of the host response to avirulent and virulent strains of MTB at different time periods of the life span of macrophage-like cells. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD022352.

Extracting time-dependent obese-diabetic specific networks in hepatic proteome analysis.

Molecular mechanism governing biological processes leading to dietary obesity and diabetes are largely unknown. Here we study the liver proteome differentially expressed in a long-term high-fat and high-sucrose diet (HFHSD)-induced obesity and diabetes mouse model. Changes in mouse liver proteins were identified using iTRAQ, offline 2D LC (SCX and RP) and MALDI-TOF/TOF MS. A total of 1639 proteins was quantified during 3-15 weeks of disease progression and a pronounced proteome change was captured by incorporating the statistical analysis and network analysis. This underscores the importance of protein expression profiles involved in different biological processes that correlate well with the disease progression. The functionally important modules with key hub proteins such as Egfr, Pklr, Suclg1, and Pcx (Carbohydrate metabolism), Cyp2e1, Fasn, Acat1, and Hmgcs2 (Lipid metabolism and ketogenesis), and Gpx1, Mgst1, and Sod2 (ROS metabolism) can be linked to a physiological state of obesity and T2D. Multiple proteins involved in glucose catabolism and lipogenesis were down-regulated, whereas proteins involved in lipid peroxidation and oxidative phosphorylation were up-regulated. In conclusion, this proteomic study provides targets for future mechanistic and therapeutic studies in relation to development and prevention of obesity and Type 2 Diabetes.

A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation.

We demonstrate that the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 have a central role in mediating T-cell receptor-dependent induction of IL4 expression in human CD4(+) T cells. Significantly, this involved a novel mechanism wherein receptor cross-linking induced activated ERK to physically associate with a promoter element on the IL4 gene. The proximally localized ERK then facilitated recruitment of the key transcription factors necessary for initiating IL4 gene transcription. Although both ERK-1 and ERK-2 bound to the promoter, recruitment of either one alone was found to be sufficient. We thus identify a novel mode of function for ERK wherein its physical association with the promoter serves as a prerequisite for enhanceosome assembly. This unusual pathway is also indispensable for human Th2-cell differentiation.

Identification of host-dependent survival factors for intracellular Mycobacterium tuberculosis through an siRNA screen.

The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb) involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a "proof-of-concept" demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including multiple drug-resistance. Thus the host factors identified here may potentially be exploited for the development of anti-tuberculosis drugs.

Activating transcription factor 3 is a positive regulator of human IFNG gene expression.

IL-12 and IL-18 are essential for Th1 differentiation, whereas the role of IFN-alpha in Th1 development is less understood. In this microarray-based study, we searched for genes that are regulated by IFN-alpha, IL-12, or the combination of IL-12 plus IL-18 during the early differentiation of human umbilical cord blood CD4(+) Th cells. Twenty-six genes were similarly regulated in response to treatment with IL-12, IFN-alpha, or the combination of IL-12 plus IL-18. These genes could therefore play a role in Th1 lineage decision. Transcription factor activating transcription factor (ATF) 3 was upregulated by these cytokines and selected for further study. Ectopic expression of ATF3 in CD4(+) T cells enhanced the production of IFN-gamma, the hallmark cytokine of Th1 cells, whereas small interfering RNA knockdown of ATF3 reduced IFN-gamma production. Furthermore, ATF3 formed an endogenous complex with JUN in CD4(+) T cells induced to Th1. Chromatin immunoprecipitation and luciferase reporter assays showed that both ATF3 and JUN are recruited to and transactivate the IFNG promoter during early Th1 differentiation. Collectively, these data indicate that ATF3 promotes human Th1 differentiation.

A non-invasive method for concurrent detection of early-stage women-specific cancers.

We integrated untargeted serum metabolomics using high-resolution mass spectrometry with data analysis using machine learning algorithms to accurately detect early stages of the women specific cancers of breast, endometrium, cervix, and ovary across diverse age-groups and ethnicities. A two-step approach was employed wherein cancer-positive samples were first identified as a group. A second multi-class algorithm then helped to distinguish between the individual cancers of the group. The approach yielded high detection sensitivity and specificity, highlighting its utility for the development of multi-cancer detection tests especially for early-stage cancers.

GPS-Prot: a web-based visualization platform for integrating host-pathogen interaction data.

BACKGROUND: The increasing availability of HIV-host interaction datasets, including both physical and genetic interactions, has created a need for software tools to integrate and visualize the data. Because these host-pathogen interactions are extensive and interactions between human proteins are found within many different databases, it is difficult to generate integrated HIV-human interaction networks. RESULTS: We have developed a web-based platform, termed GPS-Prot http://www.gpsprot.org, that allows for facile integration of different HIV interaction data types as well as inclusion of interactions between human proteins derived from publicly-available databases, including MINT, BioGRID and HPRD. The software has the ability to group proteins into functional modules or protein complexes, generating more intuitive network representations and also allows for the uploading of user-generated data. CONCLUSIONS: GPS-Prot is a software tool that allows users to easily create comprehensive and integrated HIV-host networks. A major advantage of this platform compared to other visualization tools is its web-based format, which requires no software installation or data downloads. GPS-Prot allows novice users to quickly generate networks that combine both genetic and protein-protein interactions between HIV and its human host into a single representation. Ultimately, the platform is extendable to other host-pathogen systems.