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Heart Health Risks of Excess Dietary Mn2+

 

By: Carol A. Rouzer, VICB Communications
Published: October 20, 2017

 

High intake of Mn2+ increases the risk of bacterial endocarditis due to a complex interplay of factors.

 

Bacteria require nutrient metals for their survival, so one way host organisms protect themselves from pathogenic bacterial infections is to restrict metal access. This process, known as nutritional immunity, usually involves the production of chelating small molecules or proteins that bind nutrient metal ions and sequester them from invading pathogens. Nutritional immunity has its limitations, however, and can be thwarted if the dietary intake of metals exceeds the capacity of the chelating agents. Mn2+ is an important nutrient metal for many bacteria, including Staphylococcus aureus (Figure 1), the second most common cause of blood infections and the most common cause of bacterial endocarditis (infection of the heart). To control the availability of Mn2+ to potential pathogens, vertebrate hosts produce calprotectin, a protein that binds both Mn2+ and Zn2+ and is present at very high concentrations in blood neutrophils. The role of calprotectin in restricting S. aureus infection in the liver is well documented, but new evidence suggests that its protective effects may be tissue-specific. To further explore this possibility, Vanderbilt Institute of Chemical Biology (VICB) members Eric Skaar, Walter Chazin, and Richard Caprioli, along with their laboratories, investigated the interplay between dietary Mn2+ and calprotectin in S. aureus endocarditis [L. J. Juttukonda, et al. (2017) Cell Host & Microbe, http://dx.doi.org/10.1016/j.chom.2017.08.009].

 

 

FIGURE 1. Electron micrograph of S. aureus bacteria (gold) escaping from a white blood cell (blue). Figure is reproduced from NIAID and is in the public domain.

 

 

The investigators first tested the hypothesis that high levels of dietary Mn2+ could increase the susceptibility of mice to S. aureus infection. They fed mice one of four diets: a standard chow diet, a diet containing the minimum requirement of Mn2+, a diet containing no Mn2+, or a diet containing a high level of Mn2+. They used inductively coupled plasma mass spectrometry (ICP-MS) to confirm that the various diets correlated with expected changes in tissue Mn2+ levels in the heart, liver, kidney, and gastrointestinal tract. They then infected the mice with an intravenous infusion of S. aureus. Mice fed the high Mn2+ diet exhibited a significantly higher mortality rate than any of the other groups. This increased mortality was associated with a 10-fold higher bacterial burden in the heart but not in the liver or kidney. The low Mn2+ diet resulted in lower bacterial counts in the liver.


Levels of dietary Mn2+ had no effect on the recruitment of inflammatory cells to the heart. Exposure to excess dietary Zn2+ had no effect on S. aureus colonization of the heart. The high Mn2+ diet did not alter the normal gut microbiome, and it did not affect the outcome in models of Clostridium difficile infection of the gastrointestinal tract or Acinetobacter baumannii systemic or pulmonary infections. Thus, the effects of dietary Mn2+ appeared to be fairly specific for S. aureus endocarditis.


Further study demonstrated increased calprotectin levels in the S. aureus-infected hearts of mice on a high Mn2+ diet. However, the protein was concentrated around the abscesses rather than in them, suggesting that it was not reaching the bacteria. Mice lacking the gene for calprotectin (S100a9-/-) exhibited a lower bacterial burden in the heart and reduced mortality compared to wild-type mice when placed on the high Mn2+ diet. This unexpected finding suggested that calprotectin was actually deleterious rather than beneficial in the fight against S. aureus endocarditis.


The failure of calprotectin to help block S. aureus infection of the heart led the investigators to hypothesize that the protein was not effectively sequestering Mn2+ from the bacteria. To test this hypothesis, they used a strain of S. aureus (ΔmntH/C) lacking the genes for the two transport systems (MntH and MntABC) that the bacteria use to acquire Mn2+. These bacteria survive in environments rich in Mn2+ but do poorly if Mn2+ levels are low. The investigators found that this S. aureus strain grew equally well in the hearts of wild-type or S100a9-/-mice fed a diet containing control levels of Mn2+. This was not true in the liver, however, where growth of the ΔmntH/C strain was markedly reduced in wild-type mice as compared to S100a9-/-mice. These findings supported their hypothesis that calprotectin selectively fails to sequester Mn2+ from S. aureus in the heart. Note that the ΔmntH/C strain bacteria did not grow as well as parental strain bacteria in wild-type mice fed a control diet. However, prior exposure of the mice to a high Mn2+ diet enabled the ΔmntH/C bacteria to survive as well as the parental strain, confirming that excess dietary Mn2+ reaches the bacteria in the heart.


The finding that calprotectin does not sequester Mn2+ from S. aureus in the heart could explain why wild-type and S100a9-/-mice would exhibit equal bacterial burdens, but it did not explain why S100a9-/-mice actually exhibited lower bacterial burdens than wild-type mice. The researchers noted that S. aureus abscesses formed in the hearts of S100a9-/-mice were poorly defined and contained a lower number of neutrophils than those in the hearts of wild-type mice. This led the investigators to hypothesize that differences in neutrophil recruitment might exist between the two mouse genotypes. They tested this hypothesis by using an antibody to deplete neutrophils in both wild-type and S100a9-/-mice. The treatment eliminated the difference in S. aureus bacterial burden in the hearts of the mice. Furthermore, when they transplanted S100a9-/- bone marrow into irradiated wild-type mice and vice-versa, they found that the bacterial burdens in the hearts following subsequent S. aureus infection were consistent with those based on the genotype of the transplanted bone marrow. These findings suggest that there is an intrinsic difference between neutrophils of wild-type and S100a9-/- mice, such that calprotectin deficiency appears to correlate with a reduced ability to migrate to the heart in response to infection.


Mn2+ serves as a cofactor for a number of key bacterial enzymes, including SodA and SodM. Both of these enzymes are superoxide dismutases that protect S. aureus from damaging reactive oxygen species (ROS) produced by neutrophils as part of antibacterial host defenses. To investigate the role of these enzymes in S. aureus endocarditis, the researchers exposed mice to diets containing various Mn2+ levels and then infected them with bacteria lacking the genes for both superoxide dismutases (ΔsodA/M). They found that these bacteria grew very poorly in mice fed a control or low Mn2+ diet, but they survived as well as parental strain bacteria in mice fed a high Mn2+ diet. This suggested that Mn2+ alone could serve the same anti-oxidant function as superoxide dismutase. Additional support for this hypothesis came from studies that showed that incubation of ΔsodA/M bacteria in the presence of medium containing high levels of Mn2+ protected them from the toxicity of paraquat (a ROS-generating toxicant). High levels of environmental Mn2+ also protected S. aureus from killing by human neutrophils ex vivo.


Together the data suggest that calprotectin is not released by neutrophils in the heart, so it fails to provide protection against S. aureus infection in that tissue (Figure 2). This situation is worsened by the fact that calprotectin appears to promote neutrophil migration to the heart, which increases damaging inflammation without effectively limiting bacterial growth. High levels of Mn2+ further exacerbate the infection by enabling the bacteria to evade toxic ROS production. The investigators note that baseline levels of Mn2+ are higher in the liver than in the heart. Exposure to a high Mn2+ diet does not provide a survival advantage for S. aureus in the liver, where the levels of the metal are already high. However, the high Mn2+ diet increases heart levels to those found at baseline in the liver, apparently exceeding a critical threshold for bacterial survival.

 


FIGURE 2. Interaction between dietary Mn2+ and S. aureus survival in the heart. Neutrophils carrying calprotectin migrate to the infected heart, but an unknown tissue-specific factor prevents calprotectin release, so its beneficial effects are not realized. High levels of dietary Mn2+ both provide the needed cofactor for SodA and SodM production, enabling the bacteria to inactivate toxic superoxide. Mn2+ also serves as an antioxidant, inactivating toxic ROS produced by the neutrophil. All of these factors combine to promote S. aureus infection in heart tissue.

 

 

The clinical implications of these findings remain to be determined. However, the researchers note that some dietary supplements are particularly high in Mn2+, and perhaps these should be avoided. They also note that some conditions that increase the risk of bacterial endocarditis, such as intravenous drug use and chronic liver disease, are also correlated with high serum Mn2+ levels.

 

 

 

ViewCell Host Microbe article: Dietary Manganese Promotes Staphylococcal Infection of the Heart

 

 

 

 

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