are resistant to the antibiotic methicillin, termed MRSA (methicillin resistant Staphylococcus aureus). The calprotectin protein was detected to accumulate at the site of S. aureus infection, termed an abscess, in infected mice. The purified protein was shown to be an effective inhibitor of the growth of S. aureus in the test tube, and the mechanism of this inhibition was demonstrated to be through the tight coordination of manganese ions by the calprotectin complex. This in turn leads to manganese starvation of S. aureus cells when they are in the presence of calprotectin, whether in laboratory experiments or inside abscesses within the infected host. It was subsequently shown that manganese starvation resulted in a reduced ability of S. aureus to supply an essential enzyme, called superoxide dismutase, with its essential manganese cofactor. This enzyme is known to be crucial in defending the bacterium against a key weapon utilised by the mammalian immune system to kill invading bacteria.


Nutritional immunity in the intestine It is important to note that the calprotectin is known to play a number of important roles in the body, some of which are unrelated to its function in control of bacterial pathogens. This makes it an unreliable biomarker of inflammation. Nonetheless, calprotectin is also an important effector in the immune response to other pathogens, including those that cause infection through the colonisation of the mammalian gut. Cells of the intestinal epithelium also produce calprotectin, although notably the primary source of calprotectin in the intestine remains the neutrophil cells of the immune system, which infiltrate the gut lumen during the inflammatory response caused by the detection of pathogenic bacteria within the intestine. However, it’s notable that studies of the influence of calprotectin on bacterial infections within the mammalian intestine have found that zinc, rather than manganese, is the key metal whose availability is affected by the presence of calprotectin in this niche. Interestingly, this sequestration of intestinal zinc by secreted calprotectin can have dramatically different effects on the outcome of bacterial infection of the gut, depending on the bacterial pathogen that is encountered (Figure 2). Intestinal infection by the human pathogen Clostridium difficile


exhibited a classical nutritional immunity response to the presence of calprotectin. It was shown that the growth of C. difficile is strongly inhibited by calprotectin in laboratory experiments. This inhibition is caused by zinc starvation of the bacterium through tight coordination of the available zinc by the calprotectin complex. An analogous effect was observed during infection of mice with C. difficile; calprotectin secreted into the gut lumen inhibited bacterial growth and proliferation, whereas mice that were genetically modified to prevent them from being able to synthesise calprotectin were found to be especially vulnerable to succumbing to intestinal infection by C. difficile relative to wild type mice. Like C. difficile, growth of the Gram negative pathogenic


bacterium Salmonella enterica serovar Typhimurium was also found to be inhibited by the presence of calprotectin in the test tube, but the degree of growth inhibition was observed to be significantly less than


the inhibition of C. difficile. Crucially, however, the effect of calprotectin on Salmonella infections in mice were the opposite of that observed in the experiments with Clostridium infections; the presence of calprotectin appeared to increase the ability of Salmonella to establish infection in wild type mice. It was established that this difference was caused by the presence of a high affinity zinc acquisition system in Salmonella, which is able to compete with calprotectin for the available zinc. This zinc uptake system is absent from the genome of C. difficile. Mutant strains of Salmonella in which this zinc uptake system was abolished showed significantly decreased ability to colonise the mouse gut, and were hugely disfavoured relative to wild type Salmonella during competitive infection of wild type mice, but not in strains of mice that were unable to produce calprotectin. This indicates direct competition between the Salmonella zinc acquisition system and host calprotectin for zinc ions within the gut. Therefore, this high affinity zinc acquisition system enable Salmonella to compete against calprotectin, and thereby maintain its zinc supply to essential zinc-requiring enzymes under the calprotectin-mediated zinc limiting conditions in the lumen of the inflamed intestine. Furthermore,


Figure 2: Nutritional immunity has different effects on different pathogens inside the intestine. Schematic diagram illustrating how the nutritional immunity effector, calprotectin, affects the ability of the pathogenic bacteria (left) Clostridium difficile and (right) Salmonella enterica serovar Typhimurium to establish infection of the mammalian intestine. During inflammation, neutrophils invade the gut lumen and release calprotectin, which tightly coordinates metal ions in the lumen, most notably zinc. The very tight binding of zinc to calprotectin reduces the bioavailability of zinc, both to the resident bacteria of the microbiota and also to the pathogen C. difficile, which is unable to effectively compete against calprotectin. This results in C. difficile zinc starvation, making calprotectin an effective inhibitor of C. difficile infections. Conversely, Salmonella cells possess a high affinity zinc acquisition system that can more effectively compete against calprotectin for zinc, ensuring its continued access to this essential micronutrient. Calprotectin does, however, inhibit the ability of the microbiota to acquire zinc, thus calprotectin acts to inhibit their ability to protect the gut from colonisation by the pathogen.


FEED COMPOUNDER NOVEMBER/DECEMBER 2019 PAGE 43


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