Periodic Reporting for period 1 - GUT-CHECK (Deciphering commensal-host-pathogen metabolic interactions to combat intestinal infections)
Período documentado: 2022-09-01 hasta 2025-02-28
Bacteroides are key gut commensals, as they produce diffusible intermediates as part of their metabolism that are utilized by pathogens and the host [PMID: 34893402]. These metabolites arise from the action of ~100 polysaccharide utilization loci (PUL) [PMID: 28138099]. While the field has focused mostly on transcriptional PUL control, recent work from my group and others suggested that Bacteroides employ also post-transcriptional means, brought about by noncoding RNAs, to regulate metabolic genes [PMID: 27353652; 34251866; 32678091].
In GUT-CHECK, I hypothesize that PUL regulation integrates transcriptional and RNA-mediated post-transcriptional control, which in turn shape the outcome of host-pathogen interactions. To test this hypothesis, I will address three specific objectives (Fig. 1):
1) How is PUL expression regulated?
2) How does PUL regulation impact interactions with pathogens and the host?
3) To what extent can PUL regulation be manipulated to thwart pathogen invasion?
In GUT-CHECK, I hypothesize that PUL regulation integrates transcriptional and RNA-mediated post-transcriptional control, which in turn shape the outcome of host-pathogen interactions. To test this hypothesis, I will address three specific objectives (Fig. 1):
1) How is PUL expression regulated?
2) How does PUL regulation impact interactions with pathogens and the host?
3) To what extent can PUL regulation be manipulated to thwart pathogen invasion?
Regulatory architecture controlling PUL expression in B. thetaiotaomicron
Each individual PUL processes a specific substrate. Thus, PUL expression has to be tightly controlled and tied to the nutritional fluctuations that are commonly associated with the gut environment. Only recently did RNA-seq studies from my group [PMID: 32678091] and others [PMID: 27353652] map the Bacteroides transcriptome at single-nucleotide resolution. This included the identification of a family of sRNAs encoded in antisense orientation to PUL operons. Strikingly, RNA-seq revealed an anti-correlation between several PUL operons and their corresponding sRNA, suggesting an sRNA may repress its cognate PUL genes. Indeed, repression has been confirmed for one example in Bacteroides fragilis [PMID: 27353652], yet the underlying mechanism is unknown.
Through an in-depth transcriptomics study of B. thetaiotaomicron grown in vitro in minimal medium containing a single, defined carbon source, we have since obtained deeper insight into the conditional expression of PUL asRNAs [PMID: 38528147]. This comprehensive metabolic expression data now allowed us to probe the anti-correlation phenomenon on a more global scale. We again found examples where an asRNA’s expression inversely mirrored that of its cognate PUL operon, but also observed counter-examples of positive correlation in individual asRNA/PUL pairs. In other words, the extended transcriptomic data revealed a more nuanced picture of PUL-associated asRNAs than anticipated and further enhances the need for functional characterization of this specialized class of noncoding RNAs. Based on the collective expression data we have now derived at a short list of PUL/asRNA pairs, for in-depth characterization.
Bacteroides PUL-dependent effects on its interaction with pathogens and the host
Accumulating evidence suggests PUL systems as central hubs in host-pathogen interactions in the human intestine [PMID: 28657886; 27393306]. GUT-CHECK uses Salmonella enterica serovar Typhimurium as a surrogate model for Gram-negative enteric pathogens. Meanwhile, we monitored the in vitro growth of B. thetaiotaomicron in a defined medium supplemented with a panel of dietary and host-derived polysaccharides. We further characterized gene expression profiles during carbohydrate breakdown and revealed specific PULs to be induced during growth in the presence of single polysaccharides. In contrast to B. thetaiotaomicron, Salmonella was unable to thrive on the majority of dietary polysaccharides when provided as sole carbon source. Strikingly however, combining co-culture with spent media assays revealed the pre-degradation of these polysaccharides by B. thetaiotaomicron to support pathogen outgrowth. Using mutants of B. thetaiotaomicron, we could pinpoint specific PUL genes responsible for the cross-feeding process in vitro. GC-MS-based metabolomics enabled us to pinpoint PUL-derived metabolites that constitute major substrates for Salmonella. Together, our data suggest a metabolic interplay between B. thetaiotaomicron and S. Typhimurium, in which B. thetaiotaomicron facilitates the utilization of dietary and host-derived carbon sources by Salmonella.
We have established a human epithelial cell culture model for colonization/infection experiments under hypoxic conditions. The model can be pre-colonized with B. thetaiotaomicron and infected with enteric pathogens, and may be coupled to diverse transcriptomics readouts. For example, we have successfully established Triple RNA-seq to measure host-pathogen-commensal gene expression simultaneously as they interact. We will devise a comparative Triple RNA-seq experiment, based on either B. thetaiotaomicron wild-type or defined PUL knockouts. Inter-species expression correlation will pinpoint genes and pathways in the respective interaction partners (Salmonella, human) whose expression depends on specific PUL activities, thereby further allowing us to narrow in on PUL systems involved in Salmonella cross-feeding under more in vivo-like conditions, as potential drug targets.
Exploitation of PUL regulation to interfere with infection
Alternatives to broad-spectrum antibiotics could help reduce the collateral damage imposed by the treatment of infectious diseases on the gut microbiota. As one step in this direction, the goal of the final objective is to evaluate the potential of Bacteroides PUL control mechanisms for medical interventions of enteric infections. Specifically, can individual PUL systems be inhibited (or their inhibition be relieved) to block S. enterica outgrowth without compromising the balance between B. thetaiotaomicron and the host? Through delivery of exogenous PUL repressors complying with the rules of sRNA-mediated PUL repression, even PUL systems without endogenous sRNAs would become subject to manipulation. To this end, we have made progress in establishing antisense oligonucleotide (ASO) technology [PMID: 32185839] in B. thetaiotaomicron.
We set out with a global screen for bacteria-penetrating peptides (BPPs), and identifies MK2i (mitogen-activated protein kinase-activated protein kinase 2 inhibitory peptide) as a promising carrier peptide with high uptake efficiency into—and without overt toxicity to—B. thetaiotaomicron cells. We further evaluated the influence of media composition, growth phase, and alternative cell surface capsules on BPP uptake. Building on these results, we designed specific MK2i-coupled peptide nucleic acids (PNAs) against fabG, an essential Bacteroides gene involved in envelope fatty acid synthesis. Low micromolar concentrations of this BPP-PNA conjugate evoked a specific, dose-dependent growth inhibition. Targeting a nonessential reporter gene, we then traced the fate of the targeted transcript and cognate protein in the bacterial cell. This lays the ground for leveraging ASO technology to deliberately switch off or on certain PUL systems.
Each individual PUL processes a specific substrate. Thus, PUL expression has to be tightly controlled and tied to the nutritional fluctuations that are commonly associated with the gut environment. Only recently did RNA-seq studies from my group [PMID: 32678091] and others [PMID: 27353652] map the Bacteroides transcriptome at single-nucleotide resolution. This included the identification of a family of sRNAs encoded in antisense orientation to PUL operons. Strikingly, RNA-seq revealed an anti-correlation between several PUL operons and their corresponding sRNA, suggesting an sRNA may repress its cognate PUL genes. Indeed, repression has been confirmed for one example in Bacteroides fragilis [PMID: 27353652], yet the underlying mechanism is unknown.
Through an in-depth transcriptomics study of B. thetaiotaomicron grown in vitro in minimal medium containing a single, defined carbon source, we have since obtained deeper insight into the conditional expression of PUL asRNAs [PMID: 38528147]. This comprehensive metabolic expression data now allowed us to probe the anti-correlation phenomenon on a more global scale. We again found examples where an asRNA’s expression inversely mirrored that of its cognate PUL operon, but also observed counter-examples of positive correlation in individual asRNA/PUL pairs. In other words, the extended transcriptomic data revealed a more nuanced picture of PUL-associated asRNAs than anticipated and further enhances the need for functional characterization of this specialized class of noncoding RNAs. Based on the collective expression data we have now derived at a short list of PUL/asRNA pairs, for in-depth characterization.
Bacteroides PUL-dependent effects on its interaction with pathogens and the host
Accumulating evidence suggests PUL systems as central hubs in host-pathogen interactions in the human intestine [PMID: 28657886; 27393306]. GUT-CHECK uses Salmonella enterica serovar Typhimurium as a surrogate model for Gram-negative enteric pathogens. Meanwhile, we monitored the in vitro growth of B. thetaiotaomicron in a defined medium supplemented with a panel of dietary and host-derived polysaccharides. We further characterized gene expression profiles during carbohydrate breakdown and revealed specific PULs to be induced during growth in the presence of single polysaccharides. In contrast to B. thetaiotaomicron, Salmonella was unable to thrive on the majority of dietary polysaccharides when provided as sole carbon source. Strikingly however, combining co-culture with spent media assays revealed the pre-degradation of these polysaccharides by B. thetaiotaomicron to support pathogen outgrowth. Using mutants of B. thetaiotaomicron, we could pinpoint specific PUL genes responsible for the cross-feeding process in vitro. GC-MS-based metabolomics enabled us to pinpoint PUL-derived metabolites that constitute major substrates for Salmonella. Together, our data suggest a metabolic interplay between B. thetaiotaomicron and S. Typhimurium, in which B. thetaiotaomicron facilitates the utilization of dietary and host-derived carbon sources by Salmonella.
We have established a human epithelial cell culture model for colonization/infection experiments under hypoxic conditions. The model can be pre-colonized with B. thetaiotaomicron and infected with enteric pathogens, and may be coupled to diverse transcriptomics readouts. For example, we have successfully established Triple RNA-seq to measure host-pathogen-commensal gene expression simultaneously as they interact. We will devise a comparative Triple RNA-seq experiment, based on either B. thetaiotaomicron wild-type or defined PUL knockouts. Inter-species expression correlation will pinpoint genes and pathways in the respective interaction partners (Salmonella, human) whose expression depends on specific PUL activities, thereby further allowing us to narrow in on PUL systems involved in Salmonella cross-feeding under more in vivo-like conditions, as potential drug targets.
Exploitation of PUL regulation to interfere with infection
Alternatives to broad-spectrum antibiotics could help reduce the collateral damage imposed by the treatment of infectious diseases on the gut microbiota. As one step in this direction, the goal of the final objective is to evaluate the potential of Bacteroides PUL control mechanisms for medical interventions of enteric infections. Specifically, can individual PUL systems be inhibited (or their inhibition be relieved) to block S. enterica outgrowth without compromising the balance between B. thetaiotaomicron and the host? Through delivery of exogenous PUL repressors complying with the rules of sRNA-mediated PUL repression, even PUL systems without endogenous sRNAs would become subject to manipulation. To this end, we have made progress in establishing antisense oligonucleotide (ASO) technology [PMID: 32185839] in B. thetaiotaomicron.
We set out with a global screen for bacteria-penetrating peptides (BPPs), and identifies MK2i (mitogen-activated protein kinase-activated protein kinase 2 inhibitory peptide) as a promising carrier peptide with high uptake efficiency into—and without overt toxicity to—B. thetaiotaomicron cells. We further evaluated the influence of media composition, growth phase, and alternative cell surface capsules on BPP uptake. Building on these results, we designed specific MK2i-coupled peptide nucleic acids (PNAs) against fabG, an essential Bacteroides gene involved in envelope fatty acid synthesis. Low micromolar concentrations of this BPP-PNA conjugate evoked a specific, dose-dependent growth inhibition. Targeting a nonessential reporter gene, we then traced the fate of the targeted transcript and cognate protein in the bacterial cell. This lays the ground for leveraging ASO technology to deliberately switch off or on certain PUL systems.
In summary, as evidenced by three publications and several manuscripts that are currently in preparation, GUT-CHECK is making good progress. The functional insights gained will establish fundamental understanding of host-microbiota-pathogen interaction and may lead to novel RNA-based treatments for intestinal infections.