Internalization of Pseudomonas aeruginosaStrain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction

Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity

Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF- B-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF- B signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity

The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response

TANK-binding kinase 1 (TBK1) is of central importance for the induction of type-I interferon (IFN) in response to pathogens. We identified the DEAD-box helicase DDX3X as an interaction partner of TBK1. TBK1 and DDX3X acted synergistically in their ability to stimulate the IFN promoter, whereas RNAi-mediated reduction of DDX3X expression led to an impairment of IFN production. Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction

Systems-pharmacology dissection of a drug synergy in imatinib-resistant CML

Occurrence of the BCR-ABL[superscript T315I] gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABL[superscript T315I]. To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABL[superscript T315I] CML cells on c-Myc through nonobvious off targets

Systems medicine and integrated care to combat chronic noncommunicable diseases

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems

Target 2035-update on the quest for a probe for every protein

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome