UNLABELLED: Wolbachia species are endosymbiotic bacteria that regulate the sexual development and reproduction of their insect hosts. Despite their biological importance, functional analyses of Wolbachia genes remain limited, largely due to the absence of a robust genetic manipulation system for these intracellular bacteria. Here, we examined the impact of wFur, a Wolbachia strain originally isolated from Ostrinia furnacalis, on host cell proliferation. Inoculation of wFur onto Wolbachia-free O. furnacalis cultured cells resulted in pronounced inhibition of cell proliferation, accompanied by cytotoxic effects. To identify the genetic determinants of this phenotype, we conducted functional screening of wFur-derived genes in O. furnacalis cultured cells. This approach identified two candidate genes whose overexpression markedly suppressed cell proliferation. One gene, designated w52, encodes a protein with strong sequence and structural homology to TomO (toxic manipulator of oogenesis), previously reported to inhibit Drosophila S2 cell proliferation. The second gene, designated w75, encodes a putative membrane-associated protein widely conserved among Wolbachia genomes. Moreover, overexpression of w52 or w75 also impaired cell proliferation in Bombyx mori cultured cells, whereas no effect was observed in Spodoptera frugiperda Sf-9 cells. These results suggest that the cytostatic activity of these Wolbachia genes is likely to be insect species- and/or cell type-specific. IMPORTANCE: Wolbachia are maternally inherited intracellular bacteria notable for their exceptionally broad distribution among insect species. Despite their importance, particularly as reproductive manipulators, only a limited number of studies have addressed the functions of Wolbachia genes, largely due to the absence of well-established transinfection systems and reliable genetic engineering tools for these intracellular bacteria. In this study, we developed a transinfection system using Ostrinia furnacalis cultured cells and its male-killing strain wFur. Moreover, we identified two wFur genes, whose expression inhibited cell proliferation and exhibited cytotoxicity, through an in-house functional screening system of wFur genes. This strategy can be readily extended to investigations of other Wolbachia species, thereby contributing to a more comprehensive understanding of Wolbachia gene functions.

Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knockdown Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes, such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total neutral lipids upon Nnmt RNAi. Treating Nnmt RNAi AML12 cells with cycloleucine, an inhibitor of SAM biogenesis suppresses SAM accumulation and rescues the decrease of neutral lipids. MNAM also shows activity to elevate neutral lipids. These results suggest that NNMT contributes to lipid metabolism by maintaining proper SAM and MNAM homeostasis. This study provides an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism.

Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause inflammation in various host organs. Despite the well-known coincidence of acute phase response and inflammation, the direct roles of SAA proteins in inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in liver inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. Consistently, 4T1-induced gene expression changes in bone marrow do not require Saa1-2. This study clarifies the negligible contribution of SAA1-2 proteins in liver inflammation in the 4T1 breast cancer model.

Cancers disrupt host homeostasis in various manners but the identity of host factors underlying such disruption remains largely unknown. Here we show that nicotinamide-N-methyltransferase (NNMT) is a host factor that mediates metabolic dysfunction in the livers of cancer-bearing mice. Multiple solid cancers distantly increase expression of Nnmt and its product 1-methylnicotinamide (MNAM) in the liver. Multi-omics analyses reveal suppression of the urea cycle accompanied by accumulation of amino acids, and enhancement of uracil biogenesis in the livers of cancer-bearing mice. Importantly, genetic deletion of Nnmt leads to alleviation of these metabolic abnormalities, and buffers cancer-dependent weight loss and reduction of the voluntary wheel-running activity. Our data also demonstrate that MNAM is capable of affecting urea cycle metabolites in the liver. These results suggest that cancers up-regulate the hepatic NNMT pathway to rewire liver metabolism towards uracil biogenesis rather than nitrogen disposal via the urea cycle, thereby disrupting host homeostasis.

Antisense (as)lncRNAs can regulate gene expression but the underlying mechanisms and the different cofactors involved remain unclear. Using Native Elongating Transcript sequencing, here we show that stabilization of antisense Exo2-sensitivite lncRNAs (XUTs) results in the attenuation, at the nascent transcription level, of a subset of highly expressed genes displaying prominent promoter-proximal nucleosome depletion and histone acetylation. Mechanistic investigations on the catalase gene ctt1 revealed that its induction following oxidative stress is impaired in Exo2-deficient cells, correlating with the accumulation of an asXUT. Interestingly, expression of this asXUT was also activated in wild-type cells upon oxidative stress, concomitant to ctt1 induction, indicating a potential attenuation feedback. This attenuation correlates with asXUT abundance, it is transcriptional, characterized by low RNAPII-ser5 phosphorylation, and it requires an histone deacetylase activity and the conserved Set2 histone methyltransferase. Finally, we identified Dicer as another RNA processing factor acting on ctt1 induction, but independently of Exo2. We propose that asXUTs could modulate the expression of their paired-sense genes when it exceeds a critical threshold, using a conserved mechanism independent of RNAi.