Sex-Linked Differences in Malaria Risk Across the Lifespan.

Despite the high burden of malaria worldwide, there is surprisingly scarce research on sex-based differences in malaria outside of pregnancy. A more thorough understanding of sexual dimorphism in malaria, and what underlies these sex-based differences, could elucidate the underlying mechanisms driving malaria pathogenesis and has the potential to inform malaria control efforts, including new vaccines. This review summarizes our current understanding of sex-based differences in the epidemiology of malaria across the lifespan, potential sex- or gender-based mechanisms driving these differences, and the knowledge gaps that need to be addressed.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

CD4+ T cells display a spectrum of recall dynamics during re-infection with malaria parasites.

Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4+ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4+ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic TEM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with TCM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4+ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4+ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.

Epigenetic signature and key transcriptional regulators of human antigen-specific type 1 regulatory T cells.

Human adaptive immunity is orchestrated by effector and regulatory T (Treg) cells. Natural Tregs arise in the thymus where they are shaped to recognize self-antigens, while type 1 Tregs or Tr1 cells are induced from conventional peripheral CD4 + T cells in response to peripheral antigens, such as alloantigens and allergens. Tr1 cells have been developed as a potential therapy for inducing antigen-specific tolerance, because they can be rapidly differentiated in vitro in response to a target antigen. However, the epigenetic landscape and the identity of transcription factors (TFs) that regulate differentiation, phenotype, and functions of human antigen-specific Tr1 cells is largely unknown, hindering Tr1 research and broader clinical development. Here, we reveal the unique epigenetic signature of antigen-specific Tr1 cells, and TFs that regulate their differentiation, phenotype and function. We showed that in vitro induced antigen-specific Tr1 cells are distinct both clonally and transcriptionally from natural Tregs and other conventional CD4 + T cells on a single-cell level. An integrative analysis of Tr1 cell epigenome and transcriptome identified a TF signature unique to antigen-specific Tr1 cells, and predicted that IRF4, BATF, and MAF act as their transcriptional regulators. Using functional genomics, we showed that each of these TFs play a non-redundant role in regulating Tr1 cell differentiation, suppressive function, and expression of co-inhibitory and cytotoxic proteins. By using the Tr1-specific TF signature as a molecular fingerprint, we tracked Tr1 cells in peripheral blood of recipients of allogeneic hematopoietic stem cell transplantation treated with adoptive Tr1 cell therapy. Furthermore, the same signature identified Tr1 cells in resident CD4 + T cells in solid tumors. Altogether, these results reveal the epigenetic signature and the key transcriptional regulators of human Tr1 cells. These data will guide mechanistic studies of human Tr1 cell biology and the development and optimization of adoptive Tr1 cell therapies.

Type I regulatory T cells in malaria: of mice and men.

Type I regulatory T (Tr1) cells are a population of regulatory CD4+ T cells implicated in the suppression of pathological immune responses across multiple diseases, but a unifying transcriptional signature of Tr1 identity across disease contexts has not been characterized. In this issue of the JCI, Edward, Ng, and colleagues identified a conserved transcriptional signature that distinguished Tr1 (IL-10+IFN-γ+) from Th1 (IL-10-IFN-γ+) cells in human and mouse malaria. This signature implicated genes encoding inhibitory receptors - including CTLA-4 and LAG-3 - and transcription factors - including cMAF. The authors identified coinhibitory receptor expression that distinguished Tr1 cells from other CD4+ T cell subsets. Furthermore, cMAF - and, to a lesser extent, BLIMP-1 - promoted IL-10 production in human CD4+ T cells. BLIMP-1 also played a role in supporting the expression of inhibitory receptors. These findings describe a few key features that seem to be conserved by Tr1 cells across multiple species, disease contexts, and marker definitions.

Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.

Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.

Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

A novel vibriophage exhibits inhibitory activity against host protein synthesis machinery.

Since the emergence of deadly pathogens and multidrug-resistant bacteria at an alarmingly increased rate, bacteriophages have been developed as a controlling bioagent to prevent the spread of pathogenic bacteria. One of these pathogens, disease-causing Vibrio parahaemolyticus (VPAHPND) which induces acute hepatopancreatic necrosis, is considered one of the deadliest shrimp pathogens, and has recently become resistant to various classes of antibiotics. Here, we discovered a novel vibriophage that specifically targets the vibrio host, VPAHPND. The vibriophage, designated Seahorse, was classified in the family Siphoviridae because of its icosahedral capsid surrounded by head fibers and a non-contractile long tail. Phage Seahorse was able to infect the host in a broad range of pH and temperatures, and it had a relatively short latent period (nearly 30 minutes) in which it produced progeny at 72 particles per cell at the end of its lytic cycle. Upon phage infection, the host nucleoid condensed and became toroidal, similar to the bacterial DNA morphology seen during tetracycline treatment, suggesting that phage Seahorse hijacked host biosynthesis pathways through protein translation. As phage Seahorse genome encodes 48 open reading frames with many hypothetical proteins, this genome could be a potential untapped resource for the discovery of phage-derived therapeutic proteins.