The Colorectal Cancer Microbiota Alter Their Transcriptome To Adapt to the Acidity, Reactive Oxygen Species, and Metabolite Availability of Gut Microenvironments

Abstract

The gut microbiome is implicated in the pathology of colorectal cancer (CRC). However, the mechanisms by which the microbiota actively contribute to disease onset and progression remain elusive. In this pilot study, we sequenced fecal metatranscriptomes of 10 non-CRC and 10 CRC patient gut microbiomes and conducted differential gene expression analyses to assess any changed functionality in disease. We report that oxidative stress responses were the dominant activity across cohorts, an overlooked protective housekeeping role of the human gut microbiome. However, expression of hydrogen peroxide and nitric oxide-scavenging genes was diminished and augmented, respectively, positing that these regulated microbial responses have implications for CRC pathology. CRC microbes enhanced expression of genes for host colonization, biofilm formation, genetic exchange, virulence determinants, antibiotic, and acid resistances. Moreover, microbes promoted transcription of genes involved in metabolism of several beneficial metabolites, suggesting their contribution to patient metabolite deficiencies previously solely attributed to tumor cells. We showed in vitro that expression of genes involved in amino acid-dependent acid resistance mechanisms of meta-gut Escherichia coli responded differently to acid, salt, and oxidative pressures under aerobic conditions. These responses were mostly dictated by the host health status of origin of the microbiota, suggesting their exposure to fundamentally different gut conditions. These findings for the first time highlight mechanisms by which the gut microbiota can either protect against or drive colorectal cancer and provide insights into the cancerous gut environment that drives functional characteristics of the microbiome. IMPORTANCE The human gut microbiota has the genetic potential to drive colorectal cancer onset and progression; however, the expression of this genetic potential during the disease has not been investigated. We found that microbial expression of genes that detoxify DNA-damaging reactive oxygen species, which drive colorectal cancer, is compromised in cancer. We observed a greater activation of expression of genes involved in virulence, host colonization, exchange of genetic material, metabolite utilization, defense against antibiotics, and environmental pressures. Culturing gut Escherichia coli of cancerous and noncancerous metamicrobiota revealed different regulatory responses of amino acid-dependent acid resistance mechanisms in a health-dependent manner under environmental acid, oxidative, and osmotic pressures. Here, for the first time, we demonstrate that the activity of microbial genomes is regulated by the health status of the gut in vivo and in vitro and provides new insights for shifts in microbial gene expression in colorectal cancer.

Keywords: acidity; colorectal cancer; gut microbiota; metatranscriptome; reactive oxygen species; virulence.

FIG 1

The core transcriptome of the gut microbiota is mostly maintained in colorectal cancer. (A) Threshold of subsystems considered core, 20 subsystems of 900 identified contribute 41% of total transcriptome activity. Asterisks denote statistically significant differences between the health and CRC cohorts. **, P_adj < 0.008. (B) Metatranscriptional profile of the most prevalently expressed, “core” subsystems across all samples in both colorectal cancer (CRC) and non-CRC cohorts. The gut microbiota generate biomass primarily through glycolysis-gluconeogenesis, the serine-glyoxylate cycle, purine metabolism, amino acids (Gln/Glu and Asn/Asp) biosynthesis and ions, vitamins, and iron transport. Microbial metabolism of sialic acid, a terminal modification of host colonocytes and mucus, also appears to be a common housekeeping activity of the human gut microbiome. We also observed that oxidative stress responses (Ton and Tol transport systems, thioredoxin reduction, heat shock dnaK gene cluster subsystems) featured within the core transcriptome of both healthy and CRC-associated microbiota. The individual subsystem contribution to the overall transcriptome is displayed as a percentage above gray bars.

FIG 2

The microbiome response to H₂O₂ is diminished, and the response to NO is increased in colorectal cancer despite high background levels of oxidative stress activities in health and disease. (A) Activity of subsystems involved in modulation of oxidant levels are repressed in CRC. These subsystems involve sensors of oxidative stress (87), reduction of quinones (88), and c-type cytochrome and the antioxidant riboflavin (vitamin B₂) synthesis (89). (B) Expression of specific genes related to oxidative damage in CRC. The expression of RNA polymerase sigma factor, a universal regulator of microbial oxidative stress response, the DNA-binding protein HU-α, a bacterial histone-like protein which displays high affinity to damaged DNA and plays a part in the oxidative DNA damage response (90), was also significantly downregulated. The expression of 4Fe-4S ferredoxin, thiol oxidoreductase and putative cytochrome c-type biogenesis protein genes, prominent regulators of redox status and global nitrogen and sulfur cycles, was also significantly diminished. Transcription of the riboflavin synthase and alkyl hydroperoxide reductase genes was also downregulated. (C) CRC gut microbiota express genes for the utilization and oxidation of several nonenzymatic antioxidants such as ectoine and l-ascorbate. (D) Microbiota in CRC maintains a reduced gut environment. Expression of cytochrome c₅₅₁/c₅₅₂ and regulatory protein SoxS, a superoxide response regulon transcriptional regulator (91), was upregulated. The CRC microbiota showed a high uptake of Se (selenate and selenite), an essential element that is critical for production and activity of antioxidative selenoproteins. Selenoproteins are vital for host immunity and antiviral defense, which enhanced levels of the inner membrane transport protein YbaT, and selenoproteins O synthesis have been observed (92), correlating with higher Se uptake. (E) The CRC gut contains elevated O₂⁻ and NO levels, and the expression of genes the activity of which is implicated in their removal was elevated. Transcription of cytochrome c oxidase, CcoO subunit, with high NO reductase activity and MerR, a transcriptional factor that regulates NO defense (93), was significantly overactive in CRC. Synthesis of NO-induced universal stress proteins D, E, and F (94) was significantly enhanced. Aconitate hydratase 2 and 2-methylisocitrate dehydratase, the expression of which is negatively regulated by NO, are also transcribed to a higher degree. (F) A high level of reactive oxygen species (ROS)-reducing activity appears to be a housekeeping characteristic of the gut microbiome. Expression of major ROS-reducing genes was maintained in a health status-independent manner. (L3) denotes a subsystem. *, P ≤ 0.05; **, P ≤ 0.01. FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.

FIG 3

The CRC microbiome are adapted to the high acidity of the gut and metabolize host-required metabolites more readily. (A) Activity of glutamate-dependent acid resistance mechanisms through transcriptional activator GadE, glutamate transport membrane-spanning protein, and inner membrane transport protein YbaT (Fig. 2D), were all enhanced in CRC alongside the acid stress chaperone HdeB. Basic compounds such as ammonia (NH₃⁺) can be produced by bacteria to offset low cellular pH, particularly from urea (95); the higher transcription of nickel transport ATP-binding protein NikE observed may be critical in providing the nickel for the activity of ureases that catalyze this conversion. Production of l-malate via expression of malate synthase and its conversion to L-lactate and CO₂ by malolactic enzyme were also prominent features of the CRC microbiome, the activity of which is triggered at a pH of <2.3. Levels of ethanolamine permease transcription and acid stress-induced transcriptional regulators SpxA1 and SpxA2, which are virulence determinants in pathogens, were over-represented. Conversely, alkali pH-induced genes 4-hydroxybenzoyl-CoA thioesterase and putative helix-turn-helix (HTH)-type transcriptional regulator YdjF and YdjL oxidoreductase exhibited lower expression during cancer. (B) Iron uptake and transport-related genes are upregulated by the gut microbiota in CRC. Expression of EfeO and EfeB, iron acquisition yersiniabactin synthesis enzyme, outer membrane protein C precursor, ferric hydroxamate ABC transporter (a chelating mechanism of ferric iron [Fe³⁺] uptake), and two-component sensor kinase SPy1061 homolog that respond to iron availability and acid stress was more active. (C) The CRC gut microbiota actively metabolize exogenous DNA. Transcription of dihydropyrimidinase, N-methylhydantoinases A and B, guanine-hypoxanthine permease, d-serine/d-alanine/glycine transporter, phage-associated cell wall hydrolase, and PotB genes was increased in CRC. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 4

Amino acid Arg- and Lys-dependent acid defense mechanisms in E. coli are regulated by both environmental factors and the health status origin of bacteria. The level of expression of E. coli speA (Arg-decarboxylase), adiA (biosynthetic Arg-decarboxylase), and cadA (Lys-decarboxylase) genes was quantified by quantitative reverse transcription (qRT)-PCR in response to pH 3.5 adjusted by either dl-lactate or HCl, osmotic (5% NaCl), and oxidative (1.5 mM H₂O₂) pressures. Microbiota derived from CRC (untreated CRC and treated CRC) and control (untreated control and treated control) were aerobically cultured. Error bars denote standard deviation (treated, n = 9; untreated, n = 6). Asterisks represent statistically significance. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 5

The microbiome in colorectal cancer colonize the host and form biofilms, exchange DNA, and overexpress numerous virulence determinants. (A) Transcription of genes that are important for colonization, flagellin and pilin modifications, and the formation/remodeling of the cell wall (96) was elevated in the CRC microbiome. Higher transcription of BolA and the curli production subsystem (which play roles in biofilm formation) and lower transcription of the possible hypoxanthine oxidase XdhD and the bifunctional PLP-dependent enzyme with β-cystathionase and maltose regulon repressor activities (which facilitate biofilm disassembly) suggest increased biofilm formation in the CRC-associated microbiome. (B) Quorum sensing (QS) and motility were regulated in CRC. Gram-negative QS-associated genes were overrepresented in CRC, and expression of the secY gene, translocase, and DegP/HtrA serine proteases was higher in CRC. Gram-positive QS mechanisms were, however, attenuated in cancer. Transcription of several chemotaxis and flagellar production/function genes (CheY, FliI, FliG, and CheD) was reduced in the CRC niche. (C) The CRC microbiome activate expression of virulence factors. Production of capsular polysaccharide synthesis enzyme Cap5L; heteropolysaccharide repeat unit export protein, Irp2, which encodes the iron acquisition yersiniabactin synthesis enzyme (Fig. 3B); hemolysin III; and the LPXTG-containing motif internalin D was increased. Expression of R-alcohol forming, (R)- and (S)-acetoin-specific 2,3-butanediol dehydrogenase (Fig. 3A), which reduces acetoin to 2,3-butanediol, was enhanced in CRC, suggesting a potentially high supply of acetoin, promoting a pro-cancerous phenotype of the CRC-specific microbiota. (D) The CRC gut microbiota are prone to the exchange of genetic information, protective against pervasive bacteriophages and repair errors in their genome. Transcription of a DNA-entry nuclease (a competence-specific nuclease) was increased in CRC. Expression of the CRISPR-associated RAMP Cmr2 gene, a part of the type III system, and retron-type reverse transcriptase was amplified. However, transcription of the CRISPR-associated protein CT1974, a member of the CRISPR subtype I-E of E. coli (97), was decreased. There was increase in transcription of genes for helicase YoaA (involved in the repair of replication forks), domain clustered with uracil-DNA glycosylase and FIG137864:putative endonuclease domain (involved in releasing damaged pyrimidines from double-stranded DNA [dsDNA]). Higher expression of cysteinyl-tRNA synthetase-related protein in CRC suggests that the RecA-mediated recombinational repair mechanism and hence the SOS response were increased under cancerous conditions. (E) Antibiotic resistance activities of the microbiome are positively regulated in CRC. Increased transcription of the two-component regulatory system VanR/VanS (98), which senses either the presence of extracellular vancomycin and/or cell wall disruption by, e.g., bacitracin, was observed. Vex2, encoding an ATP transporter that is important for a vancomycin-tolerant phenotype, was overexpressed. The CRC gut microbiota showed an enhanced expression of MarB, a periplasmic protein that may indirectly repress the expression of MarA, a trigger of bacterial response to different toxic compounds, including antibiotics (99). β-Lactam resistance of the CRC microbiome appears to be significantly enhanced, as it is seen via greater activity of the BlaR1 family regulatory sensor-transducer disambiguation subsystem. The expression of BlaR1/MecR1 family genes (100) that sense β-lactams and activate expression of β-lactamase PC1/blaZ and penicillin-binding proteins 1A/1B and 3 (poorly acylated by β-lactam antibiotics) that confer resistance to the antibiotic was elevated. Activity of the subsystem, phosphoenolpyruvate phosphomutase, and expression of the phosphonopyruvate decarboxylase gene, involved in biosynthesis of fosfomycin, were increased in CRC. Lactacin F ABC transporter permease component, a bacteriocin, was transcribed less in CRC. Horizontal gene transfer facilitated through expression of ComA, a member of bacteriocin-associated ATP-binding transporter family, was repressed. However, higher conjugative activity was likely a feature of the microbiome through enhanced transcription of TraM and TraN genes, as well as the TraI gene, encoding IncF plasmid conjugative transfer DNA-nicking and unwinding protein. This would enhance genome plasticity and confer more adaptive traits to the microbiota in CRC. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.