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Previous work documented the general antibacterial mechanism of iron containing clays that involved hydroxyl radical (•OH) production from soluble Fe, and attack of cell membrane and intracellular proteins. Here we explore the role of clay structural Fe(II) in •OH production at near neutral pH and identify a lipid involved in the antibacterial process. Structural Fe(III) in nontronite NAu-2 was reduced (rNAu-2) and E. coli, a model bacterium, was exposed to rNAu-2 in oxic suspension. The antibacterial activity of rNAu-2 was dependent on pH and Fe(II) concentration, where E. coli were completely killed at pH 6, but survived at pH 7 and 8. In the presence of a •OH scavenger or in anaerobic atmosphere, E. coli survived better, suggesting that cell death may be caused by •OH generated from oxidation of structural Fe(II) in rNAu-2. In-situ imaging revealed damage of a membrane lipid, cardiolipin, in the polar region of E. coli cells, where reactive oxygen species and redox-active labile Fe were enriched. Our results advance the previous antibacterial model by demonstrating that the structural Fe(II) is the primary source of •OH, which damages cardiolipin, triggers the influx of soluble Fe into the cell, and ultimately leads to cell death.

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Reduced nontronite has been demonstrated to be antibacterial through the production of hydroxyl radical (•OH) from the oxidation of structural Fe­(II). Herein, we investigated the antibacterial activity of more common smectite–illite (S–I) clays toward Escherichia coli cells, including montmorillonite SWy-3, illite IMt-2, 50–50 S–I rectorite RAr-1, 30–70 S–I ISCz-1, and nontronite NAu-2. Under an oxic condition, reduced clays (with a prefix r before mineral names) produced reactive oxygen species (ROS), and the antibacterial activity followed the order of rRAr-1 > rSWy-3 ≥ rNAu-2 ≫ rIMt-2 ≥ rISCz-1. The strongest antibacterial activity of rRAr-1 was contributed by a combination of •OH and Fe­(IV) generated from structural Fe­(II)/adsorbed Fe2+ and soluble Fe2+, respectively. Higher levels of lipid and protein oxidation, intracellular ROS accumulation, and membrane disruption were consistent with this antibacterial mechanism of rRAr-1. The antibacterial activity of other S–I clays depended on layer expandability, which determined the reactivity of structural Fe­(II) and the production of •OH, with the expandable smectite being the most antibacterial and nonexpandable illite the least. Our results provide new insights into the antibacterial mechanisms of clay minerals.

Antibody-mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of pre-existing donor-reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that anti-donor immune responses following treatment with rabbit anti-mouse thymocyte globulin (mATG) were dominated by T cells derived from the pre-existing memory compartment. Administration of mATG one week prior to transplantation (pre-TP) was more efficient in targeting pre-existing donor-reactive memory T cells, inhibiting overall anti-donor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri-TP). The failure of peri-TP mATG to control anti-donor memory responses was due to faster recovery of pre-existing memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.