The Microbiome Colorectal Cancer Puzzle: Initiator, Propagator, and Avenue for Treatment and Research.

The human gut microbiome has an ever-increasing role in the instigation and progression of colorectal cancer (CRC). Recent investigations have focused on identifying the key causative bacterial species and the composition and structure of the microbiome as a whole that ultimately lead to tumorigenesis in the colon. Understanding the bacterial mechanisms that promote CRC provides a rich area for the development of new screening modalities and therapeutics that may improve patient outcomes. This article reviews the various mechanisms that bacteria in the gut use to induce and/or promote tumor formation, discusses the application of the microbiome in the prevention and therapy of CRC, and provides directions for future research endeavors aiming to develop a more complete understanding of this complex phenomenon.

Heme sensing and detoxification by HatRT contributes to pathogenesis during Clostridium difficile infection.

Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.

Human Colon Cancer-Derived Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice.

Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient-derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. SIGNIFICANCE: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825.

The Cancer Microbiome: Recent Highlights and Knowledge Gaps.

Knowledge of the human microbiome, which is likely a critical factor in the initiation, progression, and prognosis of multiple forms of cancer, is rapidly expanding. In this review, we focus on recent investigations to discern putative, causative microbial species and the microbiome composition and structure currently associated with procarcinogenesis and tumorigenesis at select body sites. We specifically highlight forms of cancer, gastrointestinal and nongastrointestinal, that have significant bacterial associations and well-defined experimental evidence with the aim of generating directions for future experimental and translational investigations to develop a clearer understanding of the multifaceted mechanisms by which microbiota affect cancer formation. SIGNIFICANCE: Emerging and, for some cancers, strong experimental and translational data support the contribution of the microbiome to cancer biology and disease progression. Disrupting microbiome features and pathways contributing to cancer may provide new approaches to improving cancer outcomes in patients.

ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity.

Clostridioides difficile is a spore-forming bacterium that causes severe colitis and is a major public health threat. During infection, C. difficile toxin production results in damage to the epithelium and a hyperinflammatory response. The immune response to CDI leads to robust neutrophil infiltration at the sight of infection and the deployment of numerous antimicrobials. One of the most abundant host immune factors associated with CDI is calprotectin, a metal-chelating protein with potent antimicrobial activity. Calprotectin is essential to the innate immune response to C. difficile and increasing levels of calprotectin correlate with disease severity in both adults and children with CDI. The fact that C. difficile persists in the presence of high levels of calprotectin suggests that this organism may deploy strategies to compete with this potent antimicrobial factor for essential nutrient metals during infection. In this report, we demonstrate that a putative zinc (Zn) transporter, ZupT, is employed by C. difficile to survive calprotectin-mediated metal limitation. ZupT is highly expressed in the presence of calprotectin and is required to protect C. difficile against calprotectin-dependent growth inhibition. When competing against wild-type C. difficile, zupT mutants show a defect in colonization and persistence in a murine model of infection. Together these data demonstrate that C. difficile utilizes a metal import system to combat nutritional immunity during CDI and suggest that strategies targeting nutrient acquisition in C. difficile may have therapeutic potential.IMPORTANCE During infection, pathogenic organisms must acquire essential transition metals from the host environment. Through the process of nutritional immunity, the host employs numerous strategies to restrict these key nutrients from invading pathogens. In this study, we describe a mechanism by which the important human pathogen Clostridioides difficile resists transition-metal limitation by the host. We report that C. difficile utilizes a zinc transporter, ZupT, to compete with the host protein calprotectin for nutrient zinc. Inactivation of this transporter in C. difficile renders this important pathogen sensitive to host-mediated metal restriction and confers a fitness disadvantage during infection. Our study demonstrates that targeting nutrient metal transport proteins in C. difficile is a potential avenue for therapeutic development.

Characterization of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases from Borrelia burgdorferi: Antibiotic targets for Lyme disease.

BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4 µg/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.

Clostridioides difficile Senses and Hijacks Host Heme for Incorporation into an Oxidative Stress Defense System.

Clostridioides difficile infection of the colon leads to severe inflammation and damage to the gastrointestinal epithelium due to the production of potent toxins. This inflammatory tissue damage causes the liberation of high concentrations of host heme at infection sites. Here, we identify the C. difficile heme-sensing membrane protein system (HsmRA) and show that this operon induces a protective response that repurposes heme to counteract antimicrobial oxidative stress responses. HsmR senses vertebrate heme, leading to increased expression of the hsmRA operon and subsequent deployment of HsmA to capture heme and reduce redox damage caused by inflammatory mediators of protection and antibiotic therapy. Strains with inactivated hsmR or hsmA have increased sensitivity to redox-active compounds and reduced colonization persistence in a murine model of relapse C. difficile infection. These results define a mechanism exploited by C. difficile to repurpose toxic heme within the inflamed gut as a shield against antimicrobial compounds.

The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation.

The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is critical to establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In medium containing CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism. To identify CP-responsive genes important during infection, we measured the abundance of select C. difficile transcripts in a mouse CDI model relative to expression in vitro Gene transcripts involved in selenium (Se)-dependent proline fermentation increased during infection and in response to CP. Increased proline fermentation gene transcription was dependent on CP Zn binding and proline availability, yet proline fermentation was only enhanced when Se was supplemented. CP-deficient mice could not restrain C. difficile proline fermentation-dependent growth, suggesting that CP-mediated Zn sequestration along with limited Se restricts C. difficile proline fermentation. Overall, these results highlight how C. difficile colonization depends on the availability of multiple nutrients whose abundances are dynamically influenced by the host response.IMPORTANCEClostridioides difficile infection (CDI) is the leading cause of postantibiotic nosocomial infection. Antibiotic therapy can be successful, yet up to one-third of individuals suffer from recurrent infections. Understanding the mechanisms controlling C. difficile colonization is paramount in designing novel treatments for primary and recurrent CDI. Here, we found that limiting nutrients control C. difficile metabolism during CDI and influence overall pathogen fitness. Specifically, the immune protein CP limits Zn availability and increases transcription of C. difficile genes necessary for proline fermentation. Paradoxically, this leads to reduced C. difficile proline fermentation. This reduced fermentation is due to limited availability of another nutrient required for proline fermentation, Se. Therefore, CP-mediated Zn limitation combined with low Se levels overall reduce C. difficile fitness in the intestines. These results emphasize the complexities of how nutrient availability influences C. difficile colonization and provide insight into critical metabolic processes that drive the pathogen's growth.

Immunogenicity of a West Nile virus DIII-cholera toxin A2/B chimera after intranasal delivery.

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.