The manganese transporter of the iron-free Lyme disease spirochete is essential for infection

Iron is a trace element essential for life. As such, living beings, including humans, must obtain iron from their diet. The same holds true for the bacteria that make us sick. Unfortunately for bacteria, iron is not readily available as it is tied up by various proteins in our body. Consequently, pathogenic bacteria have devised clever strategies to wrest iron away from these proteins.

The Lyme disease spirochete Borrelia burgdorferi lacks the machinery necessary for acquiring iron. In fact, B. burgdorferi can grow in the absence of iron. The reason that B. burgdorferi does not need iron is that the spirochete is missing most of the common metalloproteins that require iron to function. For the few metalloproteins that are present, manganese (Mn) may substitute for iron. This raises the question of how B. burgdorferi acquires Mn.

In the March 3 issue of PNAS, Ouyang and colleagues describe a potential Mn transporter BmtA (Borrelia metal transport protein A) encoded in the B. burgdorferi genome. What is their evidence that BmtA is a Mn transporter?

• Analysis of its amino acid sequence indicates that BmtA is a member of the ZIP family of metal transporters. The transporters sit in the membrane and transport metals such as iron, zinc, and manganese across the membrane. Most ZIP family members are predicted to have 8 transmembrane domains with potential metal-binding histidine residues within a "variable region" (see figure below taken from Guerinot 2000) and a signature sequence in the fourth transmembrane domain containing another metal binding histidine. BmtA lacks the his-X-his-X-his (X = any amino acid) metal binding site in the variable region, but it does have the signature sequence.
  
• When the bmtA gene was knocked out, the mutant B. burgdorferi was still able to grow in culture, but it was unable to accumulate Mn in its cytoplasm. The ability to accumulate Mn was restored when a plasmid expressing bmtA was introduced into the mutant.

To show conclusively that BmtA is a Mn transporter, the authors will need to incorporate purified BmtA into an artificial lipid bilayer (liposome) and demonstrate transport of Mn across the membrane.

The investigators next determined whether BmtA was required for infection of the mouse model of Lyme disease. They found that the bmtA mutant was unable to infect mice when injected into the skin. Infection was assessed by culturing heart, joint, and skin tissue removed 4 weeks after inoculation. The effect of the knockout was striking. None of the 66 organs sampled from the 22 mice inoculated with the bmtA mutant were culture positive. On the other hand, all 21 organs obtained from the 7 mice injected with the wild-type B. burgdorferi strain were culture positive. Infectivity was restored when the bmtA gene was introduced on a plasmid back into the bmtA mutant.

Because the bmtA knockout mutant grew in vitro yet was unable to grow in mice, BmtA must be essential for infectivity. Ouyang et al. presented two possible roles of BmtA in virulence in the Discussion of their paper. First, BmtA may be required for the activity of superoxide dismutase, which in B. burgdorferi is predicted to require Mn rather than iron. Superoxide dismutase detoxifies the reactive oxygen species (ROS) generated by phagocytic cells (neutrophils and macrophages) trying to ward off invading bacteria. Indeed, Ouyang et al. demonstrated that knocking out the bmtA gene rendered B. burgdorferi more sensitive to the oxidizing agent t-butyl hydroperoxide.

Second, Mn may contribute to the regulation of Borrelia genes encoding virulence determinants. The authors present as an example the transcriptional regulator BosR, which they state is a "Mn-dependent Fur homolog." This is incorrect as two different research groups have shown Mn to have no effect or even inhibit the activity of BosR. Nevertheless, there may be other Mn-dependent regulators of borrelial gene expression yet to be discovered.

The authors tout BmtA as a discovery that "may lead to new strategies for thwarting Lyme disease." That's probably true, but a word of caution should be expressed here. Any inhibitor of BmtA that's identified in future studies must have high specificity for the borrelial protein since ZIP family proteins are also found in humans.

Featured paper

Ouyang, Z., He, M., Oman, T., Yang, X., & Norgard, M. (2009). A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi Proceedings of the National Academy of Sciences, 106 (9), 3449-3454 DOI: 10.1073/pnas.0812999106

Other references

Boylan, J.A., Posey, J.E., and Gherardini, F.C. (2003). Borrelia oxidative stress response regulator, BosR: A distinctive Zn-dependent transcriptional activator. Proceedings of the National Academy of Sciences USA 100(20):11684-11689.

Guerinot M.L. (2000). The ZIP family of metal transporters. Biochimica et Biophysica Acta 1465(1-2):190-198.

Posey, J.E. and Gherardini, F.C. (2000). Lack of a role for iron in the Lyme disease pathogen. Science 288(5471):1651-1653.

Does male circumcision protect against syphilis?

Circumcision has been shown to reduce the risk of men contracting several sexually transmitted infections (STIs). Three randomized controlled trials (RCTs) published over the last few years have demonstrated that removing the foreskin of adult men diminished the risk of HIV infection by at least 50%. An article by Tobian and colleagues in last week's issue of the New England Journal of Medicine revealed a weak protective effect of circumcision against two other infections, herpes simplex virus 2 (HSV-2) and human papilloma virus (HPV), which cause genital herpes and penile warts, respectively. The same study showed no effect of circumcision on acquisition of Treponema pallidum, the agent of syphilis. You can find a nice critical analysis of the study here. Because I am interested in diseases caused by spirochetes, I will focus on the syphilis data.

Tobian et al. conducted two RCTs with similar designs. When the data were combined, they found that 50 of 2083 (2.4%) male adolescents and adults in Uganda who underwent circumcision became infected with T. pallidum over the following 24 month period. Similarly, 45 of 2143 (2.1%) control subjects became infected within the same time period, suggesting that circumcision had no effect on contracting T. pallidum. An editorial in the same journal issue points out that the study may have been underpowered to detect a protective effect (i.e., not enough subjects in the study).

Since the Tobian et al. study failed to give a simple answer to the question, I thought it would be illuminating to look at the older observational studies that examined the effects of male circumcision on syphilis transmission. Fortunately, I found a meta-analysis that compiled data from 14 research papers, most of which described cross-sectional studies.

The meta-analysis presented by Weiss and colleagues revealed a slight protective effect of male circumcision. The relative risks (RRs) along with the 95% confidence intervals (CI) are plotted in the graph. The RR in 11 of the 14 studies were adjusted for potential confounding factors such as age. The summary statistics listed at the bottom of the graph indicate a small protective effect (summary RR, 0.67; 95% CI, 0.54-0.83).

Ideally, all studies included in a meta-analysis would have similar RRs. However, if you look carefully at the graph, you will notice a wide variation in the RRs with some of the 95% confidence intervals failing to overlap. Using standard statistical calculations, the investigators determined that it was unlikely that the variation of the RR among the studies was due to chance (P = 0.01). In other words, differences in how the studies were designed and conducted led to significant variation in the outcomes. Consequently, the authors declared that there was significant heterogeneity among the studies and warned that the summary RR "should be interpreted cautiously."

The authors described one potential source of the heterogeneity. Looking at the plots again, you will note that the Cook and Parker studies demonstrated the largest statistically significant protective effect of male circumcision. Those two studies were conducted in the United States and Australia, respectively, where males are circumcised as infants. In contrast, the two largest studies, authored by Gray and Urassa, showed no effect of circumcision on the risk of becoming infected with T. pallidum. Those studies were conducted in Uganda and Tanzania, respectively, where males are not circumcised until they are adolescents or young adults. Many of the circumcised males examined in the two African studies, which were cross-sectional and case-control studies, could have contracted syphilis before being circumcised. Weiss et al. excluded subjects who were circumcised after their first sexual intercourse or after age eleven, but this information was not available for all studies. This would lead to an underestimate of the protective effects of circumcision.

You may look at the large protective effects of infant circumcision observed in the U.S. and Australian studies (RR = 0.25 and 0.19, respectively) and conclude that mass infant circumcision would be beneficial (at least for protection against syphilis). However, both studies involved men visiting STD clinics, and the results may not apply to the general population in those countries.

References

Tobian, A.A.R., Serwadda, D., Quinn, T.C., Kigozi, G., Gravitt, P.E., Laeyendecker, O., Charvat, B., Ssempijja, V., Riedesel, M., Oliver, A.E., Nowak, R.G., Moulton, L.H., Chen M.Z., Reynolds, S.J., Wawer, M.J., Gray, R.H. (2009). Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. The New England Journal of Medicine 360(13):1298-1309.

Golden M.R. and Wasserheit, J.N. (2009) Prevention of viral sexually transmitted infections--foreskin at the forefront (Editorial). The New England Journal of Medicine 360(13):1349-1350.

Weiss, H.A., Thomas, S.L., Munabi, S.K., and Hayes, R.J. (2006). Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis. Sexually Transmitted Infections 82(2):101-109.