Search

T cell immunity is essential for the control of tuberculosis (TB), an important disease of the lung, and is generally studied in humans using peripheral blood cells. Mounting evidence, however, indicates that tissue-resident memory T cells (Trms) are superior at controlling many pathogens, including Mycobacterium tuberculosis (M. tuberculosis), and can be quite different from those in circulation. Using freshly resected lung tissue, from individuals with active or previous TB, we identified distinct CD4+and CD8+ Trm-like clusters within TB-diseased lung tissue that were functional and enriched for IL-17–producing cells. M. tuberculosis–specific CD4+ T cells producing TNF-α, IL-2, and IL-17 were highly expanded in the lung compared with matched blood samples, in which IL-17+ cells were largely absent. Strikingly, the frequency of M. tuberculosis–specific lung T cells making IL-17, but not other cytokines, inversely correlated with the plasma IL-1β levels, suggesting a potential link with disease severity. Using a human granuloma model, we showed the addition of either exogenous IL-17 or IL-2 enhanced immune control of M. tuberculosis and was associated with increased NO production. Taken together, these data support an important role for M. tuberculosis–specific Trm-like, IL-17–producing cells in the immune control of M. tuberculosis in the human lung.

Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum ( Cp) and Cryptosporidium hominis ( Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti- Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials.

Summary: Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses. : Recent outbreaks and lack of effective treatments against dengue and Zika virus have caused public concerns. Estoppey et al. have identified cavinafungin as exerting potent and selective antiviral activity by targeting the signal-binding cleft of the catalytic subunit of the endoplasmic reticulum signal peptidase. Keywords: Zika virus, dengue virus, cavinafungin, signal peptidase, SEC11A, SEC11, CRISPR/Cas9, chemogenomic profiling

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

There is strong evidence that immunotherapy-mediated tumor rejection can be driven by the reinvigoration of tumor-specific CD8+ T cells recognizing neoantigens derived from tumor somatic mutations (citations). Thus, it is possible that the relative abundance and/or characteristics of these tumor-reactive, mutation-specific CD8+ T cells can be used as predictive biomarkers of response to immunotherapy. However, only a fraction of these potential neoantigens are usually immunogenic and in addition, these tumor-reactive, mutation-specific CD8+ cells are present only at low frequencies in blood, making it difficult to reliably identify these effector cells. Here, mass cytometry and highly-multiplexed combinatorial tetramer staining together with cellular barcoding was used to profile immune cells in longitudinally collected PBMCs from 14 non-small cell lung cancer (NSCLC) patients treated with anti-PD-L1 (atezolizumab) antibody to compare patients with objective response (n=8) and progressive disease (n=6). Although no significant phenotypic differences were detected in bulk CD8+ T cells, greater insight was gained from a parallel analysis using highly multiplexed peptide-MHC multimer staining to screen and profile antigen-specific T cells. A longitudinal analysis was performed using peripheral blood CD8+ T cells for 800 candidate tumor neoantigens and 73 known viral-derived control peptides across all patient samples. In addition to virus antigen-specific T cells, a total of 20 different neoantigen-specific T cell populations were detected and their high dimensional profiles were compared. We found that neoantigen-specific T cells were more frequently detected in responding patients and their phenotypes were almost entirely distinct from non-responding patients. Neoantigen-specific T cells from responding patients showed a differentiated effector phenotype with high expression of KLRG1, 2B4 (CD244) and CD57, similar to CD8+ T cells associated with CMV and some types of EBV infection. In contrast, more memory-like phenotypic profiles, with high CD27 and CD127 expression, were observed for neoantigen-specific CD8+ T cells from patients with progressive disease. In addition to the utility of this approach for the ex vivo identification, characterization, and longitudinal tracking of rare tumor-specific T cells, this study supports further research into assessing whether the presence of late-differentiated neoantigen-specific T cells could be used as a predictor of response to checkpoint blockade. Citation Format: Mahesh Yadav, Michael Fehlings, Suchit Jhunjhunwala, Bill O'Gorman, Priti Hegde, Leesun Kim, Alessandra Nardin, Susan Flynn, Hermi Sumatoh, Marcus Ballinger, David Shames, Boon Heng Lee, Evan Newell. Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in non-small cell lung cancer patients responding to atezolizumab treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4055.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.

The aerobic saprophyte Mycobacterium smegmatis, like its pathogenic counterpart M. tuberculosis, has the ability to adapt to anaerobiosis by shifting down to a dormant state. Here, we report the identification and molecular genetic characterisation of the first dormancy-induced protein in M. smegmatis. Comparative SDS-polyacrylamide gel electrophoresis of protein extracts of aerobically growing and dormant anaerobic M. smegmatis cultures revealed the upregulation of a 27-kDa protein in the dormant state. Peptide sequencing showed that the induced protein is a homologue of the histone-like protein H1p, predicted by the M. tuberculosis genome project. The corresponding hlp gene was cloned from M. smegmatis and sequenced. Disruption of the hlp gene eliminated the histone-like protein but did not affect the viability of the dormant culture.

We report here that the physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a self generated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.

Poly(γ-glutamic acid) (γ-PGA) is a biocompatible, enzymatically-degradable, natural polymer with a higher resistance to hydrolysis than polyesters commonly used for tissue engineering scaffolds such as poly(L-lactide) (PLLA). Notably, γ-PGA's free carboxyl side groups allow for simple chemical functionalization, making it a versatile candidate for producing scaffolds. Here, a series of water-resistant fibrous scaffolds were engineered from ethyl (Et), propyl (Pr) and benzyl (Bn) esterifications of γ-PGA. All scaffolds were non-cytotoxic and γ-PGA-Bn showed an increase in cell adhesion of hMSCs compared to γ-PGA-Et and γ-PGA-Pr. Moreover, cells on γ-PGA-Bn showed three-fold higher viability at day 14 and significantly higher adhesion when compared with PLLA scaffolds, despite having a similar hydrophobicity. Cell attachment decreased by 40% when the polymer was only partially modified with benzyl groups (γ-PGA-Bn-77%), but was restored when integrin-binding RGD peptide was conjugated to the remaining free carboxylic groups, indicating the peptide was accessible and able to bind integrins. The mechanism behind the cell-material interactions on γ-PGA-Bn scaffolds was further investigated through protein adsorption and fibronectin conformation experiments. These results, in addition to the cell-adhesion studies, suggest an inherent effect of the benzyl modification in the mechanism of cell attachment to γ-PGA-Bn scaffolds. Finally, γ-PGA-Bn scaffolds cultured in osteogenic media were also efficient in supporting hMSCs differentiation towards an osteogenic lineage as determined by alkaline phosphatase and Runx2 gene expression. Taken together these data suggest that esterified γ-PGA polymer scaffolds are new and versatile candidates for tissue engineering applications and that, intriguingly, aromatic functionality plays a key role in the cell-scaffold interaction.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics., Candidate anti-tuberculosis drugs are often identified in whole-cell screens. Here, Pethe et al. show that inappropriate carbon-source selection can lead to the identification of compounds devoid of efficacy in vivo, underlining the importance of developing predictive in vitro screens.

By the use of the yeast two-hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co-immunoprecipitation analysis. The C-terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two-hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.