The rockefeller university

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains the deadliest human pathogen. Treatment is hampered by drug resistance and the persistence of slow-growing or non-replicating populations. Rifampicin, a cornerstone of first-line therapy, inhibits transcription during promoter escape, but resistance mutations undermine efficacy and drive resistance spread. We revisited the transcription cycle as an antibacterial target by characterizing AAP-SO2, an RNA polymerase inhibitor with whole-cell activity against Mtb. AAP-SO2 slows the nucleotide addition cycle, disrupting elongation and termination. Rifampicin-resistant mutations impose fitness costs by perturbing the balance of these steps, creating exploitable weaknesses. Inhibition of transcription with AAP-SO2 reduced the evolution of rifampicin resistance and was especially effective against the most common resistant mutant. Combination treatment with rifampicin and AAP-SO2 synergistically killed non-replicating Mtb in an ex vivo rabbit granuloma model. These findings show that exploiting functional vulnerabilities of the transcription cycle can counter rifampicin resistance and improve clearance of recalcitrant Mtb populations.

T cell-based immunotherapies have demonstrated effectiveness in treating diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) but predicting response and understanding resistance remains a challenge. To address this, we developed syngeneic models reflecting the genetics, epigenetics, and immunology of human FL and DLBCL. We show that EZH2 inhibitors reprogram these models to re-express T cell engagement genes and render them highly immunogenic. EZH2 inhibitors do not harm tumor-controlling T cells or CAR-T cells. Instead, they reduce regulatory T cells, promote memory chimeric antigen receptor (CAR) CD8 phenotypes, and reduce exhaustion, resulting in a decreased tumor burden. Intravital 2-photon imaging shows increased CAR-T recruitment and interaction within the tumor microenvironment, improving lymphoma cell killing. Therefore, EZH2 inhibition enhances CAR-T cell efficacy through direct effects on CAR-T cells, in addition to rendering lymphoma B cells immunogenic. This approach is currently being evaluated in two clinical trials, NCT05934838 and NCT05994235, to improve immunotherapy outcomes in B cell lymphoma patients.

Increased antibody affinity over time after vaccination, known as affinity maturation, is a prototypical feature of immune responses. Recent studies have shown that a diverse collection of B cells, producing antibodies with a wide spectrum of different affinities, is selected into the plasma cell (PC) pathway. How affinity-permissive selection enables PC affinity maturation remains unknown. We found that PC precursors (prePCs) expressing high-affinity antibodies received higher levels of T follicular helper cell (T-FH cell)-derived help and divided at higher rates compared with their lower-affinity counterparts once they left the germinal center. Our findings indicate that differential cell division by selected prePCs accounts for how diverse precursors develop into a PC compartment that mediates serological affinity maturation.

The thrombolytic protease tissue plasminogen activator (tPA) is expressed in the CNS, where it regulates diverse functions including neuronal plasticity, neuroinflammation, and blood-brain-barrier integrity. However, its role in different brain regions such as the substantia nigra (SN) is largely unexplored. In this study, we characterize tPA expression, activity, and localization in the SN using a combination of retrograde tracing and beta-galactosidase tPA reporter mice. We further investigate tPA's potential role in SN pathology in an alpha-synuclein mouse model of Parkinson's disease (PD). To characterize the mechanism of tPA action in alpha-synuclein-mediated pathology in the SN and to identify possible therapeutic pathways, we performed RNA-seq analysis of the SN and used multiple transgenic mouse models. These included tPA deficient mice and two newly developed transgenic mice, a knock-in model expressing endogenous levels of proteolytically inactive tPA (tPA Ala-KI) and a second model overexpressing proteolytically inactive tPA (tPA Ala-BAC). Our findings show that striatal GABAergic neurons send tPA+ projections to dopaminergic (DA)-neurons in the SN and that tPA is released from SN-derived synaptosomes upon stimulation. We also found that tPA levels in the SN increased following alpha-synuclein overexpression. Importantly, tPA deficiency protects DA-neurons from degeneration, prevents behavioral deficits, and reduces microglia activation and T-cell infiltration induced by alpha-synuclein overexpression. RNA-seq analysis indicates that tPA in the SN is required for the upregulation of genes involved in the innate and adaptive immune responses induced by alpha-synuclein overexpression. Overexpression of alpha-synuclein in tPA Ala-KI mice, expressing only proteolytically inactive tPA, confirms that tPA-mediated neuroinflammation and neurodegeneration is independent of its proteolytic activity. Moreover, overexpression of proteolytically inactive tPA in tPA Ala-BAC mice leads to increased neuroinflammation and neurodegeneration compared to mice expressing normal levels of tPA, suggesting a tPA dose response. Finally, treatment of mice with glunomab, a neutralizing antibody that selectively blocks tPA binding to the N-methyl-D-aspartate receptor-1 (NMDAR1) without affecting NMDAR1 ion channel function, identifies the tPA interaction with NMDAR1 as necessary for tPA-mediated neuroinflammation and neurodegeneration in response to alpha-synuclein-mediated neurotoxicity. Thus, our data identifies a novel pathway that promotes DA-neuron degeneration and suggests a potential therapeutic intervention for PD targeting the tPA-NMDAR1 interaction.

Enterococcus faecium is a gram-positive bacterium that is resident to the intestines of animals including humans. E. faecium is also an opportunistic pathogen that causes multidrug-resistant (MDR) infections. Bacteriophages (phages) have been proposed as therapeutics for the treatment of MDR infections; however, an obstacle for phage therapy is the emergence of phage resistance. Despite this, the development of phage resistance can impact bacterial fitness. Thus, understanding the molecular basis of fitness costs associated with phage resistance can likely be leveraged as an antimicrobial strategy. We discovered that phage-resistant E. faecium harbor mutations in the cell wall hydrolase gene sagA. SagA cleaves crosslinked peptidoglycan (PG) involved in PG remodeling. We show that mutations in sagA compromised E. faecium PG hydrolysis. One sagA mutant, with a defect in cell envelope integrity, increased cellular permeability, and aberrant distribution of penicillin-binding proteins, was also more sensitive to beta-lactam antibiotics. These changes correspond to a growth defect where cells have abnormal division septa, membrane blebbing, and aberrant cell shape. The dysregulation of the cell envelope caused by the sagA mutation alters the binding of phages to the E. faecium cell surface, where phage infection of E. faecium requires phages to localize to sites of peptidoglycan remodeling. Our findings show that by altering the function of a single PG hydrolase, E. faecium loses intrinsic beta-lactam resistance. This indicates that phage therapy could help revive certain antibiotics when used in combination.IMPORTANCEEnterococcus faecium causes hospital-acquired infections and is frequently resistant to frontline antibiotics, including those that target the cell wall. Bacteriophages represent a promising alternative to combat such infections. However, bacterial adaptation to phage predation often results in resistance. Such resistance is frequently accompanied by fitness trade-offs, most notably altered antibiotic susceptibility. This study provides mechanistic insights into phage resistance-associated antibiotic sensitivity in E. faecium. We show that phage-resistant E. faecium carrying a mutation in the peptidoglycan hydrolase SagA has compromised cell envelope integrity, mislocalized penicillin-binding proteins, and become sensitized to beta-lactam antibiotics. These findings highlight the potential of reviving antibiotics when used in combination with phages in the clinical setting.

BACKGROUND & AIMS: The role of goblet cells in small intestinal inflammation in Crohn's disease (CD) is unknown. Polymorphisms of NOD2 confer risk for CD and associate with small intestinal disease location. We previously showed in mice that Nod2 deficiency leads to overexpansion of Phocaeicola vulgatus in the gut and downstream goblet cell defects, which preceded small intestinal inflammation. In this study, we ask whether goblet cell defects occur in patients with CD with NOD2 polymorphisms and investigate in mice how P vulgatus signals through the intestinal epithelium. METHODS: We performed a retrospective study of patients with CD to assess clinical outcomes and goblet cell histology by NOD2 status. We evaluated the contribution of microbiota and MyD88 signaling in the intestinal epithelium to goblet cell defects in the setting of Nod2 deficiency using genetic mouse models and germ-free mice. RESULTS: In patients with CD who have undergone ileocolic resection, NOD2 risk alleles confer a risk for reoperation (odds ratio, 8.12; P 1/4 .047) and for increased phosphorylated extra-cellular signal-regulated kinase and goblet cell defects in uninflamed ileal tissue. We show that patients with CD with ileal involvement harbor P vulgatus regardless of NOD2 risk allele status. We show that intestinal epithelial MyD88 and TLR4 are required for goblet cell defects in Nod2-/-mice harboring P vulgatus. Finally, we show that P vulgatus requires complex microbiota to exert its effects in Nod2-deficient mice. CONCLUSIONS: Goblet cell defects may be a harbinger of small intestinal inflammation in patients with CD, particularly in the postoperative setting. Our findings in mice show that small intestinal goblet cell loss associated with Nod2 mutation is induced by microbiome dysbiosis and epithelial MyD88, in part due to TLR4 signaling. (Cell Mol Gastroenterol Hepatol 2025; 19:101533; https://doi.org/10.1016/j.jcmgh.2025.101533)

Approximately 3.4 million patients worldwide are diagnosed each year with cancers that have pathogenic mutations in one of three RAS proto-oncogenes (KRAS, NRAS and HRAS)1,2. These mutations impair the GTPase activity of RAS, leading to activation of downstream signalling and proliferation3, 4, 5-6. Long-standing efforts to restore the hydrolase activity of RAS mutants have been unsuccessful, extinguishing any consideration towards a viable therapeutic strategy7. Here we show that tri-complex inhibitors-that is, molecular glues with the ability to recruit cyclophilin A (CYPA) to the active state of RAS-have a dual mechanism of action: not only do they prevent activated RAS from binding to its effectors, but they also stimulate GTP hydrolysis. Drug-bound CYPA complexes modulate residues in the switch II motif of RAS to coordinate the nucleophilic attack on the gamma-phosphate of GTP in a mutation-specific manner. RAS mutants that were most sensitive to stimulation of GTPase activity were more susceptible to treatment than mutants in which the hydrolysis could not be enhanced, suggesting that pharmacological stimulation of hydrolysis potentiates the therapeutic effects of tri-complex inhibitors for specific RAS mutants. This study lays the foundation for developing a class of therapeutics that inhibit cancer growth by stimulating mutant GTPase activity.

Following transcript release during intrinsic termination, Escherichia coli RNA polymerase (RNAP) often remains associated with DNA in a post-termination complex (PTC). RNAPs in PTCs are removed from the DNA by the SWI2/SNF2 adenosine triphosphatase (ATPase) RapA. Here we determined PTC structures on negatively supercoiled DNA and with RapA engaged to dislodge the PTC. We found that core RNAP in the PTC can unwind DNA and initiate RNA synthesis but is prone to producing R-loops. Nucleotide binding to RapA triggers a conformational change that opens the RNAP clamp, allowing DNA in the RNAP cleft to reanneal and dissociate. We show that RapA helps to control cytotoxic R-loop formation in vivo, likely by disrupting PTCs. We suggest that analogous ATPases acting on PTCs to suppress transcriptional noise and R-loop formation may be widespread. These results hold importance for the bacterial transcription cycle and highlight a role for RapA in maintaining genome stability.