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Dr. Feng Shao’s laboratory reveals a novel ubiquitination and degradation mechanism used by an enteric bacterial pathogen Shigella flexneri to counteract cell-autonomous innate immune defense

Publication Date:2017/10/23

Oct. 11, 2017 - Studies from Dr. Feng Shao’s laboratory reveals a novel ubiquitination and degradation mechanism used by an enteric bacterial pathogen Shigella flexneri to counteract cell-autonomous innate immune defense. The work entitled “Ubiquitination and degradation of GBPs by a Shigella effector to suppress host defense” is published in the journal Nature as Accelerated Article Preview.

Innate immunity is the first line of defense against microbial infection. Interferon, as a critical component in innate immunity, stimulates the transcription of a large set of genes to mount host defenses against infection. Among interferon-stimulated genes, guanylate-binding proteins (GBPs) are the large family and conserved in mammals. Human has seven GBPs while mouse has eleven. GBPs are emerging players in cell-autonomous defense against virus, parasites and bacteria. Bacterial pathogens are also under positive selection by host immune clearance. Successful pathogens have evolved virulence mechanisms to paralyze key host defense pathways to be adapted to the harsh host environment. A common and important virulence strategy employed by pathogenic bacteria is to deliver into host cells toxins and effectors that are often endowed with potent and unique biochemical activities. However, it is not known whether bacterial pathogens have evolved any mechanism to deal with or counteract GBPs-mediated immune defenses.

S. flexneri is a cytoplasmic free-living bacterium and its infection causes bacillary dysentery and gastrointestinal disorders in human. The Shao laboratory has long been using S. flexneri as a model to study bacteria-host interaction, and made several novel and important discoveries. The present study comes from an accidental observation when they were studying the mechanism for GBPs-mediated antibacterial effect, in which they found that infection of host cells with S. flexneri induces rapid degradation of hGBP1. This phenomenon is unique to S. flexneri and does not occur with other enteric bacterial infections. They then performed an unbiased genome-wide transposon mutant screen in S. flexneri and identified IpaH9.8 that is required and sufficient for hGBP1 degradation. IpaH9.8 belongs to the IpaH family of ubiquitin-ligase effectors in Shigella that contain an N-terminal domain composed of variable amounts of Leucine-Rich Repeats (LRRs) and a C-terminal ubiquitin ligase domain. Early in 2008, the Shao laboratory reported the crystal structure of IpaH3 of S. flexneri, revealing a distinct class of ubiquitin ligases widely present in pathogenic and commensal bacteria. Despite these, the biological function and physiological substrate of the IpaH family are still not known. In this study, researchers in the Shao laboratory then performed a series of cell culture infection and in vitro biochemical studies, and demonstrated IpaH9.8 directly binds to hGBP1 to catalyze Lys48-linked polyubiquitin chain, leading to proteasomal degradation of hGBP1. Interestingly, IpaH9.8 can target multiple GBPs; the targeting occurs in host cell cytoplasm, and is independent of the nucleotide-bound state of the GTPases and their differential functionality in antibacterial defense. When IpaH9.8 is absent or the recognition of GBPs is disrupted by point mutations in IpaH9.8, the GBPs translocate to intracellular S. flexneri to limit their survival. In mouse infections, IpaH9.8 and its targeting of GBPs are critical for S. flexneri multiplication and causing mouse lethality. Mice lacking several IpaH9.8-targeted GBPs respond similarly as wild-type mice to ipaH9.8-positive infection, but are susceptible to ipaH9.8-deficient S. flexneri ΔipaH9.8. These findings emphasize the functional importance of IpaH9.8 degradation of GBPs in S. flexneri infection.

The study for the first time identifies GBPs as the physiological substrate of the IpaH family of ubiquitin-ligase effectors during S. flexneri infection. The mechanism of IpaH9.8 action illustrates a new bacterial virulence strategy and highlights the functional importance of GBPs in antibacterial defenses. The study also shows that GBPs cause morphological abnormality of the bacteria to inhibit their growth, thereby establishing a unique and valuable system for further studying the mechanisms of action for GBPs in cell-autonomous immunity. The unique activity of IpaH9.8 can also be used as a useful inhibitor for GBPs.

Peng Li, a PhD student of the PTN program in the Shao laboratory, is the first author of the study. PhD student Wei Jiang also made significant contribution; other contributors include Dr. Qin Yu, Wang Liu, and Dr. Ping Zhou from the Shao laboratory, Dr. Jun Li from Lilin Du laboratory, the undergraduate thesis student Junjie Xu, as well as Bo Xu and Dr. Fengchao Wang from the NIBS transgenic facility. Dr. Feng Shao is the corresponding author. The research was supported by the Basic Science Center of the National Science Foundation, the National Key Research and Development Project, the Beijing Scholar Program of Beijing Municipal Government, and the Strategic Priority Research Program of the Chinese Academy of Science, and carried out at National Institute of Biological Sciences, Beijing.