Hongjian Zhou

Vagococcus fluvialis is an emerging opportunistic Gram-positive bacterium isolated from environmental, animal, and human clinical sources, yet host responses to this pathogen remain poorly understood. Here, we established a systemic V. fluvialis infection model in Drosophila melanogaster to investigate the immune and metabolic consequences of infection. Survival analysis showed that V. fluvialis caused dose-dependent acute lethality, whereas a sublethal dose allowed characterization of host responses over time. Transcriptomic profiling revealed extensive infection-induced gene expression changes, including activation of innate immune and oxidative stress responses and suppression of metabolic regulatory genes. Antimicrobial peptide analysis demonstrated robust induction of multiple AMP families, including Toll and Imd associated effectors, suggesting a broad humoral immune response rather than a strictly pathway-restricted immune program. Functional enrichment analyses further revealed coordinated immune–metabolic remodeling, characterized by activation of immune and secretory pathways together with suppression of central carbon metabolism. Consistently, infected flies exhibited progressive depletion of glycogen, trehalose, and triglyceride stores, accompanied by reduced expression of genes involved in glucose utilization, glycogen synthesis, and fatty acid biosynthesis. Together, these findings demonstrate that V. fluvialis infection induces strong innate immune activation while disrupting systemic energy homeostasis in Drosophila. This study establishes Drosophila as a useful model for studying host responses to V. fluvialis and provides new insight into the immune–metabolic trade-offs associated with systemic bacterial infection.

Innate immune response is the first line of host defense against pathogenic microorganisms, and its excessive or insufficient activation is detrimental to the organism. Many individual microRNAs (miRNAs) have emerged as crucial post-transcriptional regulators of immune homeostasis in Drosophila melanogaster. However, the synergistical regulation of miRNAs located within a cluster on the Imd-immune pathway remains obscured. In our study, a genetic screening with 52 transgenic UAS-miRNAs was performed to identify ten miRNAs or miRNA clusters, including the miR310~313 cluster, which may function on Imd-dependent immune responses. The miRNA RT-qPCR analysis showed that the expression of miR-310~313 cluster members exhibited an increase at 6–12 h post E. coli infection. Furthermore, the overexpression of the miR-310~313 cluster impaired the Drosophila survival. And the overexpression of miR-310/311/312 reduced Dpt expression, an indication of Imd pathway induced by Gram-negative bacteria. Conversely, the knockdown of miR-310/311/312 led to increases in Dpt expression. The Luciferase reporter expression assays and RT-qPCR analysis confirmed that miR-310~313 cluster members directly co-targeted and inhibited Imd transcription. These findings reveal that the members of the miR-310~313 cluster synergistically inhibit Imd-dependent immune responses by co-targeting the Imd gene in Drosophila.

The Drosophila Toll signaling pathway mainly responds to Gram-positive (G⁺) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, our results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulated after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G⁺ bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G⁺ bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity. Key Points Drosophila lncRNA-CR33942 is mainly expressed in the nucleus. lncRNA-CR33942 enhances Toll immune responses and the survival of Drosophila. lncRNA-CR33942 facilitates antimicrobial peptide transcriptions via interacting with Dif/Dorsal.

Drosophila Myc (dMyc), as a broad-spectrum transcription factor, can regulate the expression of a large number of genes to control diverse cellular processes, such as cell cycle progression, cell growth, proliferation and apoptosis. However, it remains largely unknown about whether dMyc can be involved in Drosophila innate immune response. Here, we have identified dMyc to be a negative regulator of Drosophila Imd pathway via the loss- and gain-of-function screening. We demonstrate that dMyc inhibits Drosophila Imd immune response via directly activating miR-277 transcription, which further inhibit the expression of imd and Tab2-Ra/b. Importantly, dMyc can improve the survival of flies upon infection, suggesting inhibiting Drosophila Imd pathway by dMyc is vital to restore immune homeostasis that is essential for survival. Taken together, our study not only reports a new dMyc-miR-277-imd/Tab2 axis involved in the negative regulation of Drosophila Imd pathway, and provides a new insight into the complex regulatory mechanism of Drosophila innate immune homeostasis maintenance. Innate immunity is the first line of defense against pathogenic microorganisms. Both overactivation and depression of immune response are detrimental to the organism. It is indispensable to regulate the duration and intensity of immune response. In this work, we find that Drosophila Myc (dMyc) as a transcription factor activates the transcription of miR-277 to negatively regulate the Imd pathway via inhibiting the expression of imd/Tab2 gene in the middle and later stage of Drosophila innate immune. dMyc is required to restore Drosophila immune homeostasis for the survival of flies upon infection. Since dMyc is well conserved in animals, our findings will be important in understanding the complex regulatory mechanisms of innate immune responses in animals.