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Peroxisomes are versatile organelles mediating energy homeostasis and redox balance. While peroxisome dysfunction is linked to numerous diseases, the mechanisms regulating peroxisome dynamics during cellular stress remain elusive. Using yeast, we show that proteotoxic stress, including loss of endoplasmic reticulum (ER) or cytosolic chaperone function, impaired ER protein translocation, disrupted N-linked glycosylation, or reductive stress, triggers peroxisome proliferation. This occurs through increased de novo biogenesis from the ER, as well as growth and division, rather than impaired pexophagy. Peroxisome biogenesis is essential for cellular recovery from proteotoxic stress. Through comprehensive testing of major signaling pathways, we determine this response to be mediated by activation of the heat shock response and inhibition of Target of Rapamycin (TOR) signaling. Notably, the effects of proteotoxic stress and TOR inhibition on peroxisomes are also observed in human fibroblasts. Our findings reveal a critical and conserved role of peroxisomes in cellular response to proteotoxic stress. Here, the authors show that peroxisome proliferation is a conserved response to proteotoxic stress that occurs through stimulation of biogenesis and mediated by TOR and HSR signaling. Importantly, peroxisomes enable survival to proteotoxic stress.

Mice lacking apolipoprotein E (APOE, Apoe -/- mice) on a high cholesterol (HC) diet are highly susceptible to infection with Mycobacterium tuberculosis (Mtb) but the underlying immune dysregulation has been unclear. While neutrophils are often the predominant cell type in the lungs of humans with severe tuberculosis (TB), they are relatively scarce in the lungs of some strains of mice that are used to study the disease. The neutrophil levels in the lungs of Mtb-infected Apoe -/- HC mice are very high, and thus studies in this model offer the opportunity to examine the role of specific neutrophil functions in the pathology of severe TB. We determined that depleting neutrophils, depleting plasmacytoid dendritic cells (pDCs), or blocking type I interferon signaling improved the outcome of TB in Apoe -/- HC mice. We also demonstrated that blocking the activation of peptidylarginine deiminase 4 (PAD4), an enzyme critical to NET formation, leads to fewer NETs in the lungs and dramatically improves the outcome of TB in Apoe -/- HC mice without affecting the number of neutrophils in the lung. We found that the transcriptional profile of neutrophils in Mtb-infected Apoe -/- HC mice is biased towards a state that resembles the “N2” phenotype that has been defined in cancer models and has been implicated in matrix degradation and tissue destruction. Our observations strongly suggest that the state of the neutrophil when it encounters the Mtb-infected lung is one of the main drivers of severe disease and implies that targeted interventions that alter specific states or functions, such as the production of NETs, may improve outcome while preserving sufficient capacity for host-defense.

The mechanisms that underlie protective immunity to Mycobacterium tuberculosis (Mtb) remain incompletely defined. To identify immune correlates associated with protection, we performed single-cell RNA sequencing of lung immune cells after aerosol Mtb infection of naïve mice and mice with contained Mtb infection (CoMtb), a model of naturally acquired resistance, across multiple time points, mouse strains, and Mtb strains. Protection was associated with distinct temporal patterns of immune activation, cell recruitment, and resolution. Early after challenge, CoMtb mice exhibited rapid, transient recruitment and activation of CD4 ⁺ T cells, macrophages, NK, and NKT cells, accompanied by short-lived bursts of type I and II interferon signaling, increased oxidative phosphorylation, and enhanced chemokine-mediated cell-cell communication. In contrast, primary infection elicited delayed but sustained interferon and neutrophil responses and higher bacterial burdens. These data indicate that protection involves dynamically coordinated immune pathways rather than the magnitude of any single response. Transcriptional features of CoMtb overlapped with those observed in nonhuman primates following intravenous BCG vaccination, including enrichment of activated tissue-resident CD4 ⁺ T cells and innate effector populations. Together, these findings support a model in which effective immunity to Mtb depends on the timing and coordination of immune activation, providing a framework for vaccine strategies that reproduce protective lung immune dynamics.

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global tuberculosis (TB) control, yet the mechanisms enabling rapid evolution of resistance in Mtb remain poorly understood. Here, we show that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH R ), challenging the paradigm that resistance mutations must precede compensatory adaptation. Using Mycobacterium smegmatis mc 2 155 (Msm) as a model, we demonstrate that brief exposure to sublethal isoniazid (INH) enriches for “low-level resistance and tolerance” (LLRT) mutants in a single step. LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (>500× IC 50 ) at ~6-fold higher rates than wildtype, primarily through otherwise deleterious mycothiol biosynthesis mutations that become tolerable in an oxidative stress-buffered background. Crucially, sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH R evolution. Analysis of 1578 clinical Mtb isolates revealed significant enrichment of oxidative stress-related loci among those associated with INH R . Reanalysis of genome-wide CRISPRi data further linked oxidative stress response pathways to survival under multiple antibiotics. Together, these findings suggest that host-imposed oxidative stress and suboptimal drug exposure may prime Mtb populations for rapid resistance evolution, highlighting oxidative stress defenses as potential targets to limit resistance emergence. This study challenges the paradigm that resistance mutations precede fitness compensatory adaptations, showing that pre-existing alterations in mycobacterial oxidative stress responses can prime rapid evolution of resistance without fitness costs.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration : ClinicalTrials.gov NCT01994525 .

Asymptomatic and pre-symptomatic SARS-CoV-2 infections may contribute to ongoing community transmission, however, the benefit of routine screening of asymptomatic individuals in low-risk populations is unclear. To identify SARS-CoV-2 infections 553 seronegative individuals were prospectively followed for 52 weeks. From 4/2020-7/2021, participants submitted weekly self-collected nasal swabs for rtPCR and completed symptom and exposure surveys. Incident SARS2-CoV-2 infections were identified in 9/553 (1.6%) participants. Comparisons of SARS2-CoV-2(+) to SARS2-CoV-2(-) participants revealed significantly more close contacts outside the household (median: 5 versus 3; p = 0.005). The incidence of infection was higher among unvaccinated/partially vaccinated than among fully vaccinated participants (9/7,679 versus 0/6,845 person-weeks; p = 0.004). At notification of positive test result, eight cases were symptomatic and one pre-symptomatic. These data suggest that weekly SARS2-CoV2 surveillance by rtPCR did not efficiently detect pre-symptomatic infections in unvaccinated participants.

Key Points Peroxisomes are dynamic and diverse organelles that are found in nearly all eukaryotic cells, and the disruption of their function is linked to peroxisome-related diseases. During biogenesis, peroxisomal membrane and matrix proteins are imported into peroxisomes, and this is mediated by the capacity of peroxisomes to import protein complexes. Two mechanisms of peroxisome biogenesis, de novo generation and fission from pre-existing peroxisomes, have been characterized, and one possibility is that they are conditionally and coordinately regulated. The regulation of peroxisome dynamics is mediated by multifunctional peroxins, which have potential roles in coordinating peroxisome proliferation (by de novo generation and fission) with inheritance and degradation to control peroxisome size and abundance. Exciting roles of peroxins in peroxisome differentiation, motility and inheritance are also emerging. An important outstanding challenge is to elucidate how the processes that contribute to peroxisome dynamics are regulated and coordinated, and systems approaches such as mathematical modelling will be essential for tackling this. The control of peroxisome biogenesis by different mechanisms, including de novo generation or growth and fisson of existing peroxisomes, may be coordinated to control peroxisome size and number. Dissecting this process should aid our understanding of how peroxisome dynamics are regulated, with implications for peroxisome-related diseases. Peroxisomes carry out various oxidative reactions that are tightly regulated to adapt to the changing needs of the cell and varying external environments. Accordingly, they are remarkably fluid and can change dramatically in abundance, size, shape and content in response to numerous cues. These dynamics are controlled by multiple aspects of peroxisome biogenesis that are coordinately regulated with each other and with other cellular processes. Ongoing studies are deciphering the diverse molecular mechanisms that underlie biogenesis and how they cooperate to dynamically control peroxisome utility. These important challenges should lead to an understanding of peroxisome dynamics that can be capitalized upon for bioengineering and the development of therapies to improve human health.

Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM's enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells.

To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.