The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation

Lopez, Christopher A.; Beavers, William N.; Weiss, Amy L.; Knippel, Reece J.; Zackular, Joseph P.; Chazin, Walter J.; Skaar, Eric P.

doi:10.1128/mbio.02289-19

Cited by 30 publications

(38 citation statements)

References 69 publications

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“…Previously, our group demonstrated that calprotectin is an essential component of the innate immune response to CDI and calprotectin-mediated transition metal limitation is inhibitory to C. difficile growth (7). Furthermore, we showed that subinhibitory concentrations of calprotectin dramatically alter the transcriptional profile and metabolism of C. difficile in vitro (19).…”

Section: Discussionmentioning

confidence: 81%

“…ZupT has been reported in several studies to be one of the most highly upregulated genes in C. difficile during metal stress, suggesting this importer plays a key role in metal homeostasis for this important pathogen (19,21,22). However, despite this strong transcriptional response, inactivation of zupT does not completely inhibit the ability of C. difficile to colonize the host or grow in the presence of lower concentrations of calprotectin ( Fig.…”

Section: Discussionmentioning

confidence: 90%

“…Here, we explored the role of one of the most highly upregulated genes in the presence of calprotectin, zupT. Previous studies have shown that zupT is upregulated under metal stress, but this putative metal transporter has not been studied experimentally in C. difficile (19,21,22). We demonstrated that zupT expression was robustly induced in the presence of calprotectin or a chemical chelator, and that inactivation of zupT decreased the ability of C. difficile to grow in the presence of subinhibitory concentrations of calprotectin.…”

Section: Discussionmentioning

confidence: 99%

“…Calprotectin is essential to the immune response to CDI and calprotectin-mediated metal limitation is antimicrobial to C. difficile (3,7). An RNA sequencing study by our group showed that the putative Zn transporter ZupT is one of the most highly upregulated genes in the presence of calprotectin in vitro (19). To begin to assess the contribution of ZupT to the response to host-mediated metal starvation, the transcriptional induction of zupT during treatment with recombinant calprotectin was validated using reverse transcription-quantitative PCR (qRT-PCR).…”

Section: Difficile Upregulates the Putative Zn Transporter Zupt Dumentioning

confidence: 95%

“…ZupT imports Zn during metal limitation. ZupT is highly expressed during competition with calprotectin and zinc uptake is essential to C. difficile survival during calprotectin-mediated metal starvation; however, it remains unknown if this putative metal transporter imports Zn into the cell (7,19). To investigate the ability of ZupT to take up Zn, cell cultures were grown in Zn-limited media and pulsed with a minor Zn stable isotope ( 70 Zn) prior to analysis of intracellular 70 Zn levels by inductively coupled plasma mass spectrometry (ICP-MS).…”

Section: Difficile Upregulates the Putative Zn Transporter Zupt Dumentioning

confidence: 99%

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ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

Zackular

Knippel

Lopez

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Difficile Upregulates the Putative Zn Transporter Zupt Dumentioning

confidence: 95%

Section: Difficile Upregulates the Putative Zn Transporter Zupt Dumentioning

confidence: 99%

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ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

Zackular

Knippel

Lopez

et al. 2020

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Bionic Regulators Break the Ecological Niche of Pathogenic Bacteria for Modulating Dysregulated Microbiome in Colitis

et al. 2022

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of first-line therapeutic agents. [1] IBD is tightly associated with dysbiosis of the gut microbiome, characterized by an increase in facultative anaerobic bacteria of Enterobacteriaceae and a decrease in beneficial bacteria. [2] Extensive studies have shown that gut microbiome dysbiosis plays a critical role in the etiology of IBD, and some commensal microbiomes also manifest anti-inflammatory activity to an extent. [3] Consequently, the exploitation of therapies that can reprogram the gut microbiome to treat IBD has been suggested. Various therapies have been applied to reprogram the microbiome, including enteral nutrition, fecal microbiota transplantation, prebiotics, probiotics, and postbiotic supplements. [4] Although considerable efforts have been made, the therapeutic effects of these treatments remain hampered by the non-specificity of the treatment and the enormous growth of pathogenic microbiota. [2c,5] As Escherichia coli occupies several ecological niches during IBD owing to abnormal expansion, beneficial bacteria can establish ecological niches by restricting the proliferation of E. coli. [2c,6] Traditional medical therapies for IBD have centered on broad-spectrum antibiotics to prevent the expansion of pathogenic bacteria. [4a] However, the use of antibiotics also destroys probiotics and increases the risk of antibiotic-induced Therapeutic approaches that reprogram the gut microbiome are promising strategies to alleviate and cure inflammatory bowel disease (IBD). However, abnormal expansion of Escherichia coli during inflammation can promote pathogenic bacteria occupying ecological niches to resist reprogramming of the microbiome. Herein, a bionic regulator (CaWO 4 @YCW) is developed to efficiently and precisely regulate the gut microbiome by specifically suppressing the abnormal expansion of E. coli during colitis and boosting probiotic growth. Inspired by the binding of E. coli strains to the mannoserich yeast cell wall (YCW), YCW is chosen as the bionic shell to encapsulate CaWO 4 . It is demonstrated that the YCW shell endows CaWO 4 with superior resistance to the harsh environment of the gastrointestinal tract and adheres to the abnormally expanded E. coli in colitis, specifically as a positioner. Notably, the high expression of calprotectin at the colitis site triggers the release of tungsten ions through calcium deprivation in CaWO 4 , thus inhibiting E. coli growth by replacing molybdenum in the molybdopterin cofactor. Moreover, YCW functions as a prebiotic and promotes probiotic growth. Consequently, CaWO 4 @YCW can efficiently and precisely reprogram the gut microbiome by eliminating pathogenic bacteria and providing prebiotics, resulting in an extraordinary therapeutic advantage for DSS-induced colitis.

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Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model

Martinez,

Berg,

Rodriguez

et al. 2024

MicrobiologyOpen

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Clostridioides difficile is an anaerobic, spore‐forming, Gram‐positive pathogenic bacterium. This study aimed to analyze the effect of two samples of healthy fecal microbiota on C. difficile gene expression and growth using an in vitro coculture model. The inner compartment was cocultured with spores of the C. difficile polymerase chain reaction (PCR)‐ribotype 078, while the outer compartment contained fecal samples from donors to mimic the microbiota (FD1 and FD2). A fecal‐free plate served as a control (CT). RNA‐Seq and quantitative PCR confirmation were performed on the inner compartment sample. Similarities in gene expression were observed in the presence of the microbiota. After 12 h, the expression of genes associated with germination, sporulation, toxin production, and growth was downregulated in the presence of the microbiota. At 24 h, in an iron‐deficient environment, C. difficile activated several genes to counteract iron deficiency. The expression of genes associated with germination and sporulation was upregulated at 24 h compared with 12 h in the presence of microbiota from donor 1 (FD1). This study confirmed previous findings that C. difficile can use ethanolamine as a primary nutrient source. To further investigate this interaction, future studies will use a simplified coculture model with an artificial bacterial consortium instead of fecal samples.

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The Immune Protein Calprotectin Impacts Clostridioides difficile Metabolism through Zinc Limitation

Cited by 30 publications

References 69 publications

ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

Bionic Regulators Break the Ecological Niche of Pathogenic Bacteria for Modulating Dysregulated Microbiome in Colitis

Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model

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