SUMMARY Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed.

Naïve CD4 + T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4 + T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

Legionella pneumophila, the causative agent of Legionnaires’ disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the Legionella-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the Escherichia coli biotin ligase BirA and infected the cells with L. pneumophila expressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni2+ nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity.

Abstract:The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.Key words: Legionella, type IV secretion system, effectors, Legionella-containing vacuole.Résumé : Le pathogène intracellulaire facultatif à Gram négatif Legionella pneumophila infecte une large gamme de différents protozoaires environnementaux de même que les macrophages alvéolaires humains des suites de l'inhalation d'aérosols contaminés. À l'intérieur de ses hôtes, il crée un compartiment précis et unique nommé vacuole contenant Legionella (« Legionella-containing vacuole », LCV) pour sa survie et sa multiplication. Afin d'établir une LCV, L. pneumophila utilise sont système de sécrétion de type IV (SST4) Dot/Icm, rendant possible la translocation de plus de 300 protéines effectrices dans la cellule hôte. Au cours des dernières années, on a établi que ces effecteurs subvertissent une multitude de processus cellulaires et permettent à Legionella de prendre le contrôle du trafic cellulaire, de la transcription et de la traduction. Or, la fonction exacte de la grande majorité des effecteurs est toujours inconnue. L'importante redondance fonctionnelle au sein des effecteurs explique en partie cette incertitude et invalide les approches génétiques conventionnelles adoptées pour élucider leur rôle. Dans le présent ouvrage, nous passons en revue les connaissances actuelles entourant les effecteurs SST4 de Legionella, faisons ressortir certaines interrogations, et abordons de nouvelles méthodes qui promettent de faciliter la caractérisation de la fonction des effecteurs SST4. [Traduit par la Rédaction] Mots-clés : Legionella, système de sécrétion de type IV, effecteurs, vacuole contenant Legionella.

AbstractNaïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. We used quantitative proteomics, bulk RNA-seq and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to generate a detailed map of cytokine-regulated gene expression programs. We demonstrated that cytokine response differs substantially between naïve and memory T cells and showed that memory cells are unable to differentiate into the Th2 phenotype. Moreover, memory T cells acquire a Th17-like phenotype in response to iTreg polarization. At the single-cell level, we demonstrated that T cells form a continuum which progresses from naïve to effector memory T cells. This continuum is accompanied by a gradual increase in the expression levels of chemokines and cytokines and thus represents an effectorness gradient. Finally, we found that T cell cytokine responses are determined by where the cells lie in the effectorness gradient and identified genes whose expression is controlled by cytokines in an effectorness-dependent manner. Our results shed light on the heterogeneity of T cells and their responses to cytokines, provide insight into immune disease inflammation and could inform drug development.