ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

Abstract

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.

Zinc import mediated by AdcABC is critical for colonization of the dental biofilm by Streptococcus mutans in an animal model

“…Although AdcAII and Pht‐encoding genes are absent in S. mutans , a gene ( smu2069 ) coding for a hypothetical protein with 37% identity and 60% similarity to the Escherichia coli ZupT, a member of the zinc import ZIP family commonly found in gram‐negative and in selected gram‐positive bacteria (Grass et al., 2002; Zackular et al., 2020), was identified through BLAST searches. To probe the possible role of Smu2069 in zinc uptake, we created a strain bearing a smu2069 deletion in UA159 (Δ smu2069 ) and in the Δ adcCB background thereby generating a Δ adcCB Δ smu2069 triple mutant.…”

“…Growth curves of UA159, ΔadcA, ΔadcCB, or ΔadcCB comp in (a) FMC, (b) FMC supplemented with 5 μM ZnSO 4 , (c) BHI, (d) CP/BHI medium containing 200 μg/ml of human calprotectin, and (e) BHI containing 10 μM TPEN. Curves shown represent average and SD of at least five independent biological replicates [Colour figure can be viewed at wileyonlinelibrary.com]import ZIP family commonly found in gram-negative and in selected gram-positive bacteria(Grass et al, 2002;Zackular et al, 2020), was identified through BLAST searches. To probe the possible role of Smu2069 in zinc uptake, we created a strain bearing a smu2069 deletion in UA159 (Δsmu2069) and in the ΔadcCB background thereby generating a ΔadcCBΔsmu2069 triple mutant.…”

RNA-based thermoregulation of a Campylobacter jejuni zinc resistance determinant

“…The inflammatory S100 protein calprotectin also contributes to bacterial killing at certain sites of infection by sequestering metals [including Zn(II) and Mn(II)] following its copious release from neutrophils [ 13 ]. Nonetheless many bacterial pathogens can overcome this host mediated metal-limitation via Zn(II) uptake systems (for example [ 14 – 16 ]), and notably C . jejuni possesses an ATP-binding cassette (ABC) Zn(II)-importer needed for intestinal survival [ 17 ].…”

Zinc Import Mediated by AdcABC is Critical for Colonization of the Dental Biofilm byStreptococcus mutansin an Animal Model

“…While AdcAII and Pht-encoding genes are absent in S. mutans, a gene (smu2069) coding for a hypothetical protein with 37% identity and 60% similarity to the E.coli ZupT, a member of the zinc import ZIP family commonly found in gram-negative and in selected gram-positive bacteria (Grass, Wong, Rosen, Smith, & Rensing, 2002;Zackular et al, 2020) was identified through BLAST searches. To probe the possible role of Smu2069 in zinc uptake, we created a strain bearing a smu2069 deletion in UA159 (Δsmu2069) and in the Δ adcCB background thereby generating a Δ adcCBΔsmu2069 triple mutant.…”