Tumor Microenvironment Profiles Reveal Distinct Therapy-Oriented Proteogenomic Characteristics in Colorectal Cancer.

Advances in immunotherapy have made an unprecedented leap in treating colorectal cancer (CRC). However, more effective therapeutic regimes need a deeper understanding of molecular architectures for precise patient stratification and therapeutic improvement. We profiled patients receiving neoadjuvant chemotherapy alone or in combination with immunotherapy (PD-1 checkpoint inhibitor) using Digital Spatial Profiler (DSP), a high-plex spatial proteogenomic technology. Compartmentalization-based high-plex profiling of both proteins and mRNAs revealed pronounced immune infiltration at tumor regions associated with immunotherapy treatment. The protein and the corresponding mRNA levels within the same selected regions of those patient samples correlate, indicating an overall concordance between the transcriptional and translational levels. An elevated expression of PD-L1 at both protein and the mRNA levels was discovered in the tumor compartment of immunotherapy-treated patients compared with chemo-treated patients, indicating potential prognostic biomarkers are explorable in a spatial manner at the local tumor microenvironment (TME). An elevated expression of PD-L1 was verified by immunohistochemistry. Other compartment-specific biomarkers were also differentially expressed between the tumor and stromal regions, indicating a dynamic interplay that can potentiate novel biomarker discovery from the TME perspectives. Simultaneously, a high-plex spatial profiling of protein and mRNA in the tumor microenvironment of colorectal cancer was performed.

Spatial transcriptomics and proteomics technologies for deconvoluting the tumor microenvironment.

The tumor microenvironment (TME) harbors heterogeneous contents and plays critical roles in tumorigenesis, metastasis, and drug resistance. Therefore, the deconvolution of the TME becomes increasingly essential to every aspect of cancer research and treatment. Novel spatially-resolved high-plex molecular profiling technologies have been emerging rapidly as powerful tools to obtain in-depth understanding from TME perspectives due to their capacity to allow high-plex protein and RNA profiling while keeping valuable spatial information. Based on our practical experience, we review a variety of available spatial proteogenomic technologies, including 10X Visium, GeoMx Digital Spatial Profiler (DSP), cyclic immunofluorescence-based CODEX and Multi-Omyx, mass spectrometry (MS)-based imaging mass spectrometry (IMS) and multiplex ion-beam imaging (MIBI). We also discuss FISSEQ, MERFISH, Slide-seq, and HDST, some of which may become commercially available in the near future. In particular, with our experience, we elaborate on DSP for spatial proteogenomic profiling and discuss its unique features designed for immuno-oncology and propose anticipation towards its future direction. The emerging spatially technologies are rapidly reshaping the magnitude of our understanding of the TME.

Rid Enhances the 6-Hydroxypseudooxynicotine Dehydrogenase Reaction in Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine through a hybrid of the pyridine and pyrrolidine pathways. The oxidation of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoyl-semialdehyde-pyridine by 6-hydroxypseudooxynicotine dehydrogenase (Pno) is an important step in the breakdown of the N-heterocycle in this pathway. Although Pno has been characterized, the reaction is not fully understood; what is known is that it starts at a high speed followed by a rapid drop in the reaction rate, leading to the formation of a very small amount of product. In this study, we speculated that an unstable imine intermediate that is toxic with regard to the metabolism is produced in the reaction. We found that a Rid protein (designated Rid-NC) encoded by a gene in the nicotine-degrading gene cluster enhanced the reaction. Rid is a widely distributed family of small proteins with various functions, and some subfamilies have deaminase activity to eliminate the toxicity of the reactive intermediate, imine. Biochemical analyses showed that Rid-NC relieved the toxicity of the presumed imine intermediate produced in the Pno reaction and that, in the presence of Rid-NC, Pno maintained a high level of activity and the amount of the reaction product was increase by at least 5-fold. Disruption of the rid-NC gene led to slower growth of strain S33 on nicotine. The mechanism of Rid-NC-mediated detoxification of the imine intermediate was discussed. A phylogenetic analysis indicated that Rid-NC belongs to the rarely studied Rid6 subfamily. These results further our understanding of the biochemical mechanism of nicotine degradation and provide new insights into the function of the Rid6 subfamily proteins.IMPORTANCE Rid is a family of proteins that participate in metabolite damage repair and is widely distributed in different organisms. In this study, we found that Rid-NC, which belongs to the Rid6 subfamily, promoted the 6-hydroxypseudooxynicotine dehydrogenase (Pno) reaction in the hybrid of the pyridine and pyrrolidine pathways for nicotine degradation by Agrobacterium tumefaciens S33. Rid-NC hydrolyzed the presumed reactive imine intermediate produced in the reaction to remove its toxicity on Pno. The finding furthers our understanding of the metabolic process of the toxic N-heterocyclic aromatic compounds in microorganisms. This study demonstrated that the Rid family of proteins also functions in the metabolism of N-heterocyclic aromatic alkaloids, in addition to the amino acid metabolism, and that Rid6-subfamily proteins also have deaminase activity, similar to the RidA subfamily. The ability of reactive imines to damage a non-pyridoxal-5'-phosphate-dependent enzyme was reported. This study provides new insights into the function of the Rid family of proteins.

6-Hydroxypseudooxynicotine Dehydrogenase Delivers Electrons to Electron Transfer Flavoprotein during Nicotine Degradation by Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways. The hybrid pathway consists of at least six steps involved in oxidoreductive reactions before the N-heterocycle can be broken down. Collectively, the six steps allow electron transfer from nicotine and its intermediates to the final acceptor O2 via the electron transport chain (ETC). 6-Hydroxypseudooxynicotine oxidase, renamed 6-hydroxypseudooxynicotine dehydrogenase in this study, has been characterized as catalyzing the fourth step using the artificial electron acceptor 2,6-dichlorophenolindophenol. Here, we used biochemical, genetic, and liquid chromatography-mass spectrometry (LC-MS) analyses to determine that 6-hydroxypseudooxynicotine dehydrogenase utilizes the electron transfer flavoprotein (EtfAB) as the physiological electron acceptor to catalyze the dehydrogenation of pseudooxynicotine, an analogue of the true substrate 6-hydroxypseudooxynicotine, in vivo, into 3-succinoyl-semialdehyde-pyridine. NAD(P)+, O2, and ferredoxin could not function as electron acceptors. The oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was verified to be derived from H2O. Disruption of the etfAB genes in the nicotine-degrading gene cluster decreased the growth rate of A. tumefaciens S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine, an intermediate downstream of the hybrid pathway, indicating the requirement of EtfAB for efficient nicotine degradation. The electrons were found to be further transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase. These results aid in an in-depth understanding of the electron transfer process and energy metabolism involved in the nicotine oxidation and provide novel insights into nicotine catabolism in bacteria.IMPORTANCE Nicotine has been studied as a model for toxic N-heterocyclic aromatic compounds. Microorganisms can catabolize nicotine via various pathways and conserve energy from its oxidation. Although several oxidoreductases have been characterized to participate in nicotine degradation, the electron transfer involved in these processes is poorly understood. In this study, we found that 6-hydroxypseudooxynicotine dehydrogenase, a key enzyme in the hybrid pyridine and pyrrolidine pathway for nicotine degradation in Agrobacterium tumefaciens S33, utilizes EtfAB as a physiological electron acceptor. Catalyzed by the membrane-associated electron transfer flavoprotein:ubiquinone oxidoreductase, the electrons are transferred from the reduced EtfAB to coenzyme Q, which then could enter into the classic ETC. Thus, the route for electron transport from the substrate to O2 could be constructed, by which ATP can be further sythesized via chemiosmosis to support the baterial growth. These findings provide new knowledge regarding the catabolism of N-heterocyclic aromatic compounds in microorganisms.

Green route to synthesis of valuable chemical 6-hydroxynicotine from nicotine in tobacco wastes using genetically engineered Agrobacterium tumefaciens S33.

BACKGROUND: Tobacco is widely planted as an important nonfood economic crop throughout the world, and large amounts of tobacco wastes are generated during the tobacco manufacturing process. Tobacco and its wastes contain high nicotine content. This issue has become a major concern for health and environments due to its toxicity and complex physiological effects. The microbial transformation of nicotine into valuable functionalized pyridine compounds is a promising way to utilize tobacco and its wastes as a potential biomass resource. Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways, in which several intermediates, such as 6-hydroxynicotine, can be used as renewable precursors to synthesize drugs and insecticides. This provides an opportunity to produce valuable chemical 6-hydroxynicotine from nicotine via biocatalysis using strain S33. RESULTS: To accumulate the intermediate 6-hydroxynicotine, we firstly identified the key enzyme decomposing 6-hydroxynicotine, named 6-hydroxynicotine oxidase, and then disrupted its encoding gene in A. tumefaciens S33. With the whole cells of the mutant as a biocatalyst, we tested the possibility to produce 6-hydroxynicotine from the nicotine of tobacco and its wastes and optimized the reaction conditions. At 30 °C and pH 7.0, nicotine could be efficiently transformed into 6-hydroxynicotine by the whole cells cultivated with glucose/ammonium/6-hydroxy-3-succinoylpyridine medium. The molar conversion and the specific catalytic rate reached approximately 98% and 1.01 g 6-hydroxynicotine h-1 g-1 dry cells, respectively. The product could be purified easily by dichloromethane extraction with a recovery of 76.8%, and was further confirmed by UV spectroscopy, mass spectroscopy, and NMR analysis. CONCLUSIONS: We successfully developed a novel biocatalytic route to 6-hydroxynicotine from nicotine by blocking the nicotine catabolic pathway via gene disruption, which provides an alternative green strategy to utilize tobacco and its wastes as a biomass resource by converting nicotine into valuable hydroxylated-pyridine compounds.

Genomic and transcriptomic analyses of Agrobacterium tumefaciens S33 reveal the molecular mechanism of a novel hybrid nicotine-degrading pathway.

Agrobacterium tumefaciens S33 is able to degrade nicotine via a novel hybrid of the pyridine and pyrrolidine pathways. It can be utilized to remove nicotine from tobacco wastes and transform nicotine into important functionalized pyridine precursors for some valuable drugs and insecticides. However, the molecular mechanism of the hybrid pathway is still not completely clear. Here we report the genome analysis of strain S33 and its transcriptomes grown in glucose-ammonium medium and nicotine medium. The complete gene cluster involved in nicotine catabolism was found to be located on a genomic island composed of genes functionally similar but not in sequences to those of the pyridine and pyrrolidine pathways, as well as genes encoding plasmid partitioning and replication initiation proteins, conjugal transfer proteins and transposases. This suggests that the evolution of this hybrid pathway is not a simple fusion of the genes involved in the two pathways, but the result of a complicated lateral gene transfer. In addition, other genes potentially involved in the hybrid pathway could include those responsible for substrate sensing and transport, transcription regulation and electron transfer during nicotine degradation. This study provides new insights into the molecular mechanism of the novel hybrid pathway for nicotine degradation.

Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33.

Agrobacterium tumefaciens S33 can grow with nicotine as the sole source of carbon, nitrogen, and energy via a novel hybrid of the pyridine pathway and the pyrrolidine pathway. Characterization of the enzymes involved in the hybrid pathway is important for understanding its biochemical mechanism. Here, we report that the molybdenum-containing nicotine dehydrogenase (NdhAB), which catalyzes the initial step of nicotine degradation, is located in the periplasm of strain S33, while the 6-hydroxynicotine oxidase and 6-hydroxypseudooxynicoine oxidase are in the cytoplasm. This is consistent with the fact that NdhA has a Tat signal peptide. Interestingly, an open reading frame (ORF) adjacent to the ndhAB gene was verified to encode a copper-containing electron carrier, pseudoazurin (Paz), which has a signal peptide typical of bacterial Paz proteins. Both were transported into the periplasm after being produced in the cytoplasm. We purified NdhAB from the periplasmic fraction of strain S33 and found that with Paz as the physiological electron acceptor, NdhAB catalyzed the hydroxylation of nicotine at a specific rate of 110.52 ± 8.09 µmol · min-1 · mg of protein-1, where the oxygen atom in the hydroxyl group of the product 6-hydroxynicotine was derived from H2O. The apparent Km values for nicotine and Paz were 1.64 ± 0.07 µM and 3.61 ± 0.23 µM, respectively. NAD(P)+, O2, and ferredoxin could not serve as electron acceptors. Disruption of the paz gene disabled the strain for nicotine degradation, indicating that Paz is required for nicotine catabolism in the strain. These findings help our understanding of electron transfer during nicotine degradation in bacteria.IMPORTANCE Nicotine is a toxic and addictive N-heterocyclic aromatic alkaloid produced in tobacco. Its catabolism in organisms and degradation in tobacco wastes have become major concerns for human health and the environment. Bacteria usually decompose nicotine using the classical strategy of hydroxylating the pyridine ring with the help of activated oxygen by nicotine dehydrogenase, which binds one molybdopterin, two [2Fe2S] clusters, and usually one flavin adenine dinucleotide (FAD) as well. However, the physiological electron acceptor for the reaction is still unknown. In this study, we found that the two-component nicotine dehydrogenase from Agrobacterium tumefaciens S33, naturally lacking an FAD-binding domain, is located in the periplasmic space and uses a copper-containing electron carrier, pseudoazurin, as its physiological electron acceptor. We report here the role of pseudoazurin in a reaction catalyzed by a molybdopterin-containing hydroxylase occurring in the periplasmic space. These results provide new biochemical knowledge on microbial degradation of N-heterocyclic aromatic compounds.