Session MOC. There are 4 abstracts in this session.

Session: Immunity and the Microbiome, time: 09:50 - 10:15 am

Proteomic scatomancy:  Towards stool-based personalized health forecasts

Joshua Elias
Stanford University, Stanford, CA

The intestinal microbiome profoundly influences immune responses within the gut, and can have far-reaching effects throughout the body.  Complex, dynamic interactions between host, microbe and diet underlie these effects, but are only partially reflected by 16S and metagenomic sequencing which remain foundations of microbiome research.  Towards building a more mechanistic understanding of how our resident microbes influence health and disease, our “host-centric proteomics of stool” method directly measures proteins secreted or shed into the gut in response to changing intestinal ecosystems. Where microbe sequencing data can correlate certain taxa with possible health effects, well-annotated host proteins more directly implicate specific host processes in health maintenance and disease.  These kinds of pathway-level observations can be important for assessing health before the onset of symptoms, when interventions may be most effective.  In this talk, I will describe our recent efforts in understanding pre-symptomatic changes to host innate immune responses in a mouse model of multiple sclerosis, and with an exploration of natural variation in self-reported healthy human subjects.  I will also touch upon how our unconstrained database search software (TagGraph) can incorporate metaproteomic information into these assessments --  even when protein sequence databases are incomplete.  With it, we can characterize dynamic host, microbial and dietary interactions directly from stool.  This is a fundamental step towards understanding the forces that shape the gut microbiome, and how it in turn can shift the balance between health and disease.

Tips and Tricks (if submitted):

Session: Immunity and the Microbiome, time: 10:15 - 10:35 am

Metaproteomics to study drug effects on the gut microbiome

Daniel Figeys
Ottawa Institute of Systems Biology, University of, Ottawa, ON

We are interested in understanding the role of the microbiome in pediatric inflammatory bowel disease.  In particular, we will present some of our recent results on the changes in the metaproteome occuring in the microbiome during the disease.  

We are also interested in the development of in vitro assays to rapidly assess the effects of compounds on individual microbiome.  We will also present recent development of an in vitro microbiome assay coupled to metaproteomics to understand the effects of different compounds on individual human microbiomes.

Tips and Tricks (if submitted):

Session: Immunity and the Microbiome, time: 10:40 - 10:55 am

Interactome and Proteome Dynamics Uncover Immune Modulatory Associations of the Pathogen Sensing Factor cGAS

Krystal Lum; Bokai Song; Joel Federspiel; Benjamin Diner; Timothy Howard; Ileana Cristea
Princeton University, Princeton, NJ

Detection of virus DNA genomes is an essential component of the mammalian innate immune response. One prominent host defense factor is the DNA sensor cyclic-GMP-AMP synthase (cGAS). Upon direct binding to viral DNA, cGAS catalyzes the generation of cyclic dinucleotides, which activate the adaptor protein STING to induce expression of type I interferons. Our lab further established that cGAS additionally induces apoptosis in a STING-dependent manner under certain infection conditions with herpes simplex virus 1 (HSV-1). Despite the critical role for cGAS in sensing viral DNA, little is known about how cGAS activity is regulated upon infection. How cGAS is homeostatically maintained in an inactive state and whether these rely on certain protein interactions remain unclear. Additionally, our understanding of the global impact of HSV-1 infection on the cellular proteome is limited. Here, we provide the first unbiased characterization of cytoplasmic cGAS protein interactions during cellular states of active immune signaling via infection with HSV-1 in primary human fibroblasts. We place cGAS interactions in the context of temporal proteome alterations using isobaric-labeling (TMT) mass spectrometry (MS). We compare several HSV-1 strains that induce varying cytokine responses and apoptosis, presenting the most in-depth characterization of proteomes during HSV-1 replication. Via domain construction, CRISPR-mediated knockouts, antiviral cytokine measurements, and targeted MS (PRM) quantification, we establish a functional interaction between cGAS and an RNA pathogen-sensing factor, 2ʹ-5ʹ-oligoadenylate synthase-like protein OASL. We demonstrate that this interaction occurs in different types of cells. We further establish that the OAS-like domain of OASL interacts with the cGAS Mab21 domain, while the OASL ubiquitin-like domain inhibits cGAS-mediated interferon response. Therefore, we uncover a regulatory crosstalk between the traditional RNA and DNA sensing pathways. Our findings explain how cGAS may be inactively maintained in cellular homeostasis, with OASL functioning as a negative feedback loop for cytokine induction.

Tips and Tricks (if submitted):

Session: Immunity and the Microbiome, time: 10:55 - 11:10 am

Influence of the gut microbiota on histone acetylation through butyrate oxidation

Peder Lund1; Sarah Smith1; Johayra Simithy1; Lillian Chau1; Elliot Friedman1; Yedidya Saiman1; Sophie Trefely2; Mariana Lopes1; Zuo-Fei Yuan1; Kevin Janssen1; Nathaniel Snyder2; Gary Wu1; Benjamin A. Garcia1
1University of Pennsylvania, Philadelphia, PA; 2A.J Drexel Autism Institute, Drexel University, Philadelphia, PA

The gut microbiota is a diverse microbial community, residing primarily in the human colon, that accomplishes functions related to host defense, digestion, and immunoregulation.  Given that disruptions to the microbiota are associated with pathological conditions, including inflammatory bowel disease (IBD), investigating the molecular interactions between the microbiota and host is important for understanding how the microbiota impacts host health.  One mode of interaction involves small molecules, such as butyrate.  Butyrate is a product of bacterial fermentation and has long been known to inhibit histone deacetylases (HDACs), which represent one class of chromatin-modifying enzymes that regulate gene expression by controlling histone post-translational modifications.  Thus, the microbiota has the potential to broadly modulate host cell epigenetics and gene expression.  Accordingly, using quantitative mass spectrometry, we have observed that germ-free mice, which lack a microbiota, have reduced levels of histone H4 acetylation in the colon.  Sequencing analyses suggest that the loss of acetylation occurs across the genome rather than at specific loci, though transcription start sites appear protected.  While the reduced acetylation in germ-free mice may stem from elevated HDAC activity in the absence of butyrate, an alternative possibility is that germ-free mice have lower histone acetyltransferase activity because of less oxidation of butyrate to acetyl-CoA, the necessary cofactor for histone acetylation.  Indeed, through isotope tracing analyses performed in cell culture and in mice, we have demonstrated that butyrate and inulin, a fermentable plant polysaccharide, provide carbon for histone acetylation reactions.  Ongoing work is focused on determining the relative contribution of butyrate to histone acetylation as a source of acetyl-CoA versus an HDAC inhibitor.  Overall, our findings will advance insight into how the microbiota influences host cell epigenetics and gene expression programs, which may be relevant to the pathogenesis of inflammatory disorders, such as IBD.

Tips and Tricks (if submitted):