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Key Points All multicellular eukaryotes live in symbiotic associations with microorganisms, and the immune system accommodates host colonization by symbiotic microorganisms, maintains microbiota–host homeostasis and defends against pathogens. One family of antibacterial pattern recognition molecules — the peptidoglycan recognition proteins (PGRPs) — has evolved a variety of mechanisms to control host interactions with mutualistic, commensal and parasitic microorganisms to benefit both invertebrate and vertebrate hosts. PGRPs are antibacterial proteins of the innate immune system that are conserved from insects to mammals. In invertebrates, PGRPs function as soluble or cell-surface pattern recognition receptors and hydrolyse peptidoglycan, whereas in vertebrates they also directly kill bacteria. In Drosophila melanogaster , PGRPs are upstream pattern recognition molecules that activate the IMD and Toll pathways and induce the production of antimicrobial peptides, which control intestinal bacteria and defend against infections. PGRPs also control the level of pro-inflammatory peptidoglycan through their amidase activity. In mosquitoes, PGRPs not only defend the insect against bacterial infections, but also regulate symbiotic bacteria, as well as the host response to malaria parasites. In tsetse flies, PGRPs control endosymbiotic bacteria and trypanosome parasites. In squid, PGRPs control the winnowing and establishment of symbiotic luminescent bacteria in the squid light organ. In zebrafish, PGRPs protect the embryos from infections and enable their survival. In mammals, PGRPs control the acquisition and maintenance of normal gut microorganisms, which protect the host from enhanced inflammation, tissue damage and colitis. This Review discusses how invertebrate and vertebrate members of the PGRP family have developed an amazing variety of mechanisms to coordinate the host response to mutualistic, commensal and parasitic microorganisms. All animals, including humans, live in symbiotic association with microorganisms. The immune system accommodates host colonization by the microbiota, maintains microbiota–host homeostasis and defends against pathogens. This Review analyses how one family of antibacterial pattern recognition molecules — the peptidoglycan recognition proteins — has evolved a fascinating variety of mechanisms to control host interactions with mutualistic, commensal and parasitic microorganisms to benefit both invertebrate and vertebrate hosts.

Dysbiosis is a hallmark of inflammatory bowel disease (IBD), but it is unclear which specific intestinal bacteria predispose to and which protect from IBD and how they are regulated. Peptidoglycan recognition proteins (Pglyrps) are antibacterial, participate in maintaining intestinal microflora, and modulate inflammatory responses. Mice deficient in any one of the four Pglyrp genes are more sensitive to dextran sulfate sodium (DSS)-induced colitis, and stools from Pglyrp-deficient mice transferred to wild type (WT) germ-free mice predispose them to much more severe colitis than stools from WT mice. However, the identities of these Pglyrp-regulated bacteria that predispose Pglyrp-deficient mice to colitis or protect WT mice from colitis are not known. Here we identified significant changes in β-diversity of stool bacteria in Pglyrp-deficient mice compared with WT mice. The most consistent changes in microbiome in all Pglyrp-deficient mice were in Bacteroidales, from which we selected four species, two with increased abundance (Prevotella falsenii and Parabacteroides distasonis) and two with decreased abundance (Bacteroides eggerthii and Alistipes finegoldii). We then gavaged WT mice with stock type strains of these species to test the hypothesis that they predispose to or protect from DSS-induced colitis. P. falsenii, P. distasonis, and B. eggerthii all enhanced DSS-induced colitis in both WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora. By contrast, A. finegoldii (which is the most abundant species in WT mice) attenuated DSS-induced colitis both in WT mice with otherwise undisturbed intestinal microflora and in WT mice with antibiotic-depleted intestinal microflora, similar to the colitis protective effect of the entire normal microflora. These results identify P. falsenii, P. distasonis, and B. eggerthii as colitis-promoting species and A. finegoldii as colitis-protective species.

Candida glabrata is an opportunistic human pathogen, capable of causing severe systemic infections that are often resistant to standard antifungal treatments. To understand the importance of protein SUMOylation in the physiology and pathogenesis of C . glabrata , we earlier identified the components of SUMOylation pathway and demonstrated that the deSUMOylase CgUlp2 is essential for pathogenesis. In this work we show that the CgUlp2 is essential to maintain protein homeostasis via the SUMO-targeted ubiquitin ligase pathway. The dual loss of deSUMOylase and specific ubiquitin ligase, CgSlx8, results in heightened protein degradation, rendering the cells vulnerable to various stressors. This degradation affects crucial processes such as purine biosynthesis and compromises mitochondrial function in the mutants. Importantly, the absence of these ubiquitin ligases impedes the proliferation of C . glabrata in macrophages. These findings underscore the significance of SUMOylation and SUMO-mediated protein homeostasis as pivotal regulators of C . glabrata physiology and capacity to survive in host cells. Understanding these mechanisms could pave the way for the development of effective antifungal treatments.

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O⁶-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells. DNA polymerase epsilon has a critical role in DNA replication initiation. Here, the authors show that in human cancer cells POLE is dispensable for the replicative helicase assembly but not for replication initiation, which requires the non-catalytic domain of POLE1.

Objective Hepatocellular carcinoma (HCC) disproportionately affects socially disadvantaged individuals. While tumor stage and liver function predict survival, biologic stress from chronic adversity, measured by allostatic load (AL), may provide additional prognostic insight. We aimed to evaluate the association between AL and mortality in patients with HCC and assess whether health literacy (HL) modifies this relationship. Results We conducted a secondary analysis of 139 adults with HCC at two hospitals in Indianapolis (2019–2022). AL was calculated from six physiologic biomarkers, with scores ≥ 3 indicating high AL. HL and cumulative social disadvantage were measured using validated tools. Higher AL (HR 1.27; 95% CI 1.05–1.55) and lower HL (HR 1.83; 95% CI 1.10–3.05) were independently associated with mortality. After adjustment, AL remained significant but was attenuated when HL was included. Stratified analyses showed that AL predicted mortality only among patients with high HL. These exploratory findings suggest AL may help identify higher-risk patients, warranting validation in prospective, multi-center studies.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome. BRCA2 has essential roles in suppressing genome instability at stalled replication forks, but how this is achieved remains unclear. Here, the authors apply Okazaki-fragment sequencing to predict sites of genomic instability caused by head-on transcription-replication conflicts upon BRCA2 loss.

Nod2 is a pattern recognition receptor that modulates host innate immune responses and protects from inflammation, steatosis, and obesity. Obesity and inflammation are risk factors for hepatocellular carcinoma, however, the role of Nod2 in obesity-dependent hepatic tumorigenesis is not known. Here we tested the hypothesis that Nod2 protects from high fat diet (HFD)-dependent hepatic cancer. We used an obesity-dependent hepatic tumor model. WT and Nod2 −/− mice were treated with the carcinogen dimethylbenz[a]anthracene (DMBA) and maintained on HFD. Nod2 −/− mice treated with DMBA and maintained on HFD gain significantly more weight and develop more liver tumors than similarly treated WT mice. Livers of Nod2 −/− tumorigenic mice had increased expression of genes involved in cell proliferation, immune responses, and cholesterol biosynthesis, increased infiltration of neutrophils, inflammatory monocytes, and T cells, and increased activation of STAT3 and ERK during the later stages of tumorigenesis. Bioinformatic analyses of genes with differential expression predicted an increase in cancer, immune, and cholesterol biosynthesis pathways. In summary, we have identified a novel role for Nod2 and demonstrate that Nod2 protects from HFD-dependent liver malignancy and this protection is accompanied by decreased cell proliferation, inflammation, steroid biosynthesis, neutrophils and macrophages infiltration, and STAT3 and MAPK signaling in the liver.

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. Our results unveil a non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. Here the authors describe that the human origin recognition complex subunit 1 (ORC1) binds to RNAs transcribed from genes with origins of replication at their TSS impacting origin activation.

Genetics plays a central role in susceptibility to obesity and metabolic diseases. BALB/c mice are known to be resistant to high fat diet (HFD)-induced obesity, however the genetic cause remains unknown. We report that deletion of the innate immunity antibacterial gene Nod2 abolishes this resistance, as Nod2 −/− BALB/c mice developed HFD-dependent obesity and hallmark features of metabolic syndrome. Nod2 −/− HFD mice developed hyperlipidemia, hyperglycemia, glucose intolerance, increased adiposity, and steatosis, with large lipid droplets in their hepatocytes. These changes were accompanied by increased expression of immune genes in adipose tissue and differential expression of genes for lipid metabolism, signaling, stress, transport, cell cycle, and development in both adipose tissue and liver. Nod2 −/− HFD mice exhibited changes in the composition of the gut microbiota and long-term treatment with antibiotics abolished diet-dependent weight gain in Nod2 −/− mice, but not in wild type mice. Furthermore, microbiota from Nod2 −/− HFD mice transferred sensitivity to weight gain, steatosis, and hyperglycemia to wild type germ free mice. In summary, we have identified a novel role for Nod2 in obesity and demonstrate that Nod2 and Nod2 -regulated microbiota protect BALB/c mice from diet-induced obesity and metabolic dysfunction.