Cholera and spirochetes: Introducing Brachyspira!

Cholera results in a severe form of diarrhea that can lead to dehydration, shock, and ultimately death without prompt treatment. The disease afflicts the poor in developing countries lacking clean water sources and sanitation infrastructure. Vibrio cholerae is the causative agent and can be viewed by microscopic examination of the so-called "rice-water" stool samples from cholera patients.

rice-water stool from a cholera patient (Figure 1 from Sack et al., 2004)

As reported in a recent issue of Emerging Infectious Diseases, Nelson and colleagues, while examining a cholera outbreak in Bangladesh back in 2006, found that stool samples in over a third of cholera patients contained spirochetes mingling with V. cholerae. Samples were fluorescently stained to aid identification of bacteria. One example is shown below. V. cholerae were visualized with a FITC-conjugated monoclonal antibody to its lipopolysaccharide (in red), and bacterial DNA was stained wtih DAP I (green). Only the merged image is shown below. V. cholerae are the rods with a slight bend and appear yellow (combination of red and green) with a red edge; the spirochetes are the W-shaped forms stained green.

bar = 10 µm

This wasn't the first time spirochetes were observed in rice-water stool. Over a century ago, Theodor Escherich (the discoverer of E. coli) was the first to witness spiral-shaped microbes in fecal samples from cholera victims.

What was the identity of these spirochetes? They were not any of the "Big 3" of Borrelia, Treponema, and Leptospira, which garner the most attention from spirochete researchers (and from the writer of this blog). Nelson and colleagues guessed that they were members of the genus Brachyspira as they are the only spirochetes known to live in the human intestine. They turned out to be correct. They successfully amplified the gene encoding the 16s rRNA with Brachyspira-specific PCR primers. The sequence of the PCR product revealed the spirochetes to be Brachyspira pilosicoli and Brachyspira aalborgi.

Brachyspira account for most cases of human intestinal spirochetosis, defined as the presence of spirochetes in the colon. Although colonization of the large intestine by spirochetes is uncommon in the Western world, up to half of those in developing nations may harbor intestinal spirochetes. A typical example is shown below (click on image for larger version).

Figure 3 from Esteve et al., 2006

The sectioned tissue, which was stained with H&E, was obtained by colonic biopsy. The left panel reveals a fuzzy layer covering the colonic epithelium. These are Brachyspira attached at one end to the lining of the colon. The density of spirochetes can reach up to 1,700 per square millimeter. The right panel shows a colonic biopsy from the same patient after successful treatment with the antimicrobial agent metronidazole. Note that the fuzzy layer has disappeared.

Whether intestinal spirochetes cause disease in humans is unclear. Many people with intestinal spirochetes do not suffer any ill effects, but others endure chronic diarrhea. The mode of transmission of Brachyspira is unknown, but scientists have surmised that ingestion of contaminated water is involved.

The role of Brachyspira in cholera, if any, is even more of a mystery. In the conclusion to their article, Nelson et al. present the hypothesis that intestinal spirochetes exacerbate the already devastating clinical course of cholera.

Featured paper

Nelson, E.J., Tanudra, A., Chowdhury, A., Kane, A.V., Qadri, F., Calderwood, S.B., Coburn, J., Camilli, A. (2009). High Prevalence of Spirochetosis in Cholera Patients, Bangladesh Emerging Infectious Diseases, 15 (4), 571-573 DOI: 10.3201/eid1504.081214

Other references

Esteve, M., Salas, A., Fernandez-Banares, F., Lloreta, J., Marine, M., Gonzalez, C.I., Forne, M., Casalots, J., Santaolalla, R., Espinos, J.C., Munshi, M.A., Hampson, D.J., and Viver, J.M. (2006). Intestinal spirochetosis and chronic watery diarrhea: Clinical and histological response to treatment and long-term follow up. Journal of Gastroenterology and Hepatology 21(8):1326-1333. DOI: 10.1111/j.1440-1746.2006.04150.x

Sack, D.A., Sack, R.B., Nair, G.B., and Siddique, A.K. (2004). Cholera. Lancet 363(9404):223-233. DOI: 10.1016/S0140-6736(03)15328-7

Leptospira heme oxygenase frees iron from heme

I mentioned in a recent post that iron is an essential trace metal that bacteria must acquire from its surroundings. (From that same post you will also recall that the Lyme disease spirochete B. burgdorferi is a rare exception that doesn't need iron.) Much of the iron in our body is trapped within the center of the heme molecule. Heme itself is not readily accessible as it is bound to host proteins such as hemoglobin. Pathogenic bacteria have evolved sophisticated systems to kidnap heme from host proteins and transport them into the cytoplasm. These complex systems, which include secreted degradative enzymes, heme capturing proteins, and transporter proteins that sit in the membrane, have been examined in numerous bacteria. However, the fate of heme after it is acquired by the bacteria is poorly understood. In some cases, the captured heme may be incorporated into bacterial proteins such as cytochromes, which participate in electron transport. In other cases, bacteria may need to extract the iron trapped in the middle of the heme molecule. Some bacteria possess the enzyme heme oxygenase, which extracts the iron caged within heme by the following reaction (adapted from Scheme 1 in Kikuchi et al., 2005):
Unlike its cousin that causes Lyme disease, the spirochete Leptospira requires iron for growth. Ben Adler's group at the Monash University in Australia isolated a Leptospira interrogans mutant with the transposon TnSC189 inserted into hemO, the gene encoding heme oxygenase. The properties of the hemO mutant is described in two papers in the journal Microbes and Infection. The graph below (figure 1B in Murray et al., 2008) shows that growth of the hemO mutant is impaired, although not completely, when hemoglobin is the sole source of iron in the culture medium. The residual growth of the mutant indicates that L. interrogans may possess another activity that extracts iron from heme.
In their follow-up study, Adler's group demonstrated that the hemO gene was necessary for L. interrogans to fully express its virulence in the hamster model of leptospirosis. For this study they used the hemO mutant and a control L. interrogans strain that had the TnSC189 element inserted in a noncoding region, presumably where gene expression would not be affected. The two strains were injected into the abdominal cavities of separate groups of hamsters, which were then monitored for 14 days. Only 8 of 24 hamsters (33%) survived the challenge with the control strain, whereas 20 of 24 (83%) injected with the hemO mutant survived. The difference in survival rates between the two groups was statistically significant (P = 0.001).

Although the hemO mutant was ineffective at killing hamsters, it was still able to colonize the kidneys of most of the animals. Colonization was assessed by culturing kidney or urine in Leptospira growth medium. The mutant was recovered by culturing of kidney or urine from 17 of 20 hamsters that survived the challenge with the hemO mutant and all 3 that died. These results were similar to what was obtained with hamsters inoculated with the control strain, which was recovered from all 8 animals that survived and all 12 that died. (Not all hamsters were examined for colonization.)

Why was the hemO mutant able to colonize the kidney when it was unable to extract iron from heme? Heme is not the only source of iron in the body. The mutant may have captured one of the other forms of iron present in the host. The genome of L. interrogans encodes several homologs of transporters that the spirochete may use to acquire non-heme sources of iron (Louvel et al., 2006). Since these other iron sources are less abundant than heme, the tissue burden (density of bacteria) of the mutant in the kidneys may have been lower than that of the control strain thereby allowing most of the hamsters challenged with the hemO mutant to survive.

One obvious limitation of the study is that the investigators did not attempt to complement the hemO mutation with a wild-type copy of the gene. However, I should point out that currently no plasmid is available that replicates in L. interrogans, rendering complementation of L. interrogans mutations difficult. The researchers did verify that the gene immediately downstream of hemO was still transcribed in the mutant.

This work is significant for the following reasons. First, although there have been two other studies that have examined the role of Leptospira genes in virulence, this study was the most satisfying to read because it was the first to show that a gene encoding a product of known function has a role in the virulence of Leptospira. Second and perhaps more importantly, it is the first to demonstrate the importance of a bacterial heme oxygenase in virulence.

Featured papers

Murray, G., Ellis, K., Lo, M., & Adler, B. (2008). Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin Microbes and Infection, 10 (7), 791-797 DOI: 10.1016/j.micinf.2008.04.010

Murray, G., Srikram, A., Henry, R., Puapairoj, A., Sermswan, R., & Adler, B. (2009). Leptospira interrogans requires heme oxygenase for disease pathogenesis Microbes and Infection, 11 (2), 311-314 DOI: 10.1016/j.micinf.2008.11.014

Other references

Kikuchi, G., Yoshida, T., and Noguchi, M. (2005). Heme oxygenase and heme degradation. Biochemical and Biophysical Research Communications 338(1):558-567. DOI: 10.1016/j.bbrc.2005.08.020

Louvel, H., Bommezzadri S., Zidane, N., Boursaux-Eude, C., Creno, S., Magnier, A., Rouy, Z., Médigue, C., Saint Girons, I., Bouchier, C., and Picardeau, M. (2006). Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp. Journal of Bacteriology 188(22):7893-7904. DOI: 10.1128/JB00711-06

The twentieth species of Leptospira

A new species of Leptospira was isolated from soil in Johor, Malaysia by researchers at the Universiti Putra Malaysia. The spirochete was dubbed Leptospira kmetyi to honor Emil Kmety, a Slovak bacteriologist who had made numerous contributions to the understanding of the genus Leptospira. L. kmetyi is the twentieth species of Leptospira to be validly published. The sequence of its 16S rRNA gene places L. kmetyi among the "pathogenic" species of Leptospira, as shown in the phylogenetic tree below (Figure 1 from Slack et al., 2009). (Click on the image for a larger version.) Further studies are needed to prove that L. kmetyi is truly pathogenic.

The tree shows that 19 of the species cluster into four major groupings or "clades" within the genus Leptospira as follows:

Pathogenic

• L. borgpetersenii
• L. weilii
• L. alexanderi
• L. santarosai
• L. noguchii
• L. interrogans
• L. kirschneri
• Leptospira genomospecies 1
• L. kmetyi

Novel

• L. wolffii
  

Intermediate

• L. fainei
• L. broomii
• L. inadai

Saprophytic

• Leptospira genomospecies 3
• L. biflexa
• L. wolbachii
• Leptospira genomospecies 4
• Leptospira genomospecies 5
• L. meyeri

The "novel" clade, which currently has L. wolffii as its only member, was first proposed last year in a paper by Slack and colleagues.

The species that was excluded from the phylogenetic analysis is L. licerasiae, which is a recently described member of the intermediate clade (Matthias et al., 2008).

By the way, I have never liked the designation "intermediate" because readers may assume an intermediate pathogenic potential between the pathogenic and saprophytic clades. At least one intermediate member, L. fainei, can cause severe disease, including Weil's syndrome and pulmonary hemorrhage (bleeding of the lungs).

Featured paper

Slack, A.T., Khairani-Bejo, S., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Bahaman, A.R., Craig, S., Harrower, B.J., and Smythe, L.D. (2009). Leptospira kmetyi sp. nov. isolated from an environmental source in Malaysia. International Journal of Systematic and Evolutionary Microbiology 59(4):705-708. DOI: 10.1099/ijs.0.002766-0

Other references

Matthias, M.A., Ricaldi, J.N., Cespedes, M., Diaz, M.M., Galloway, R.L., Saito, M., Steigerwalt, A.G., Patra, K.P., Vidal Ore, C., Gotuzzo, E., Gilman, R.H., Levett, P.N., and Vinetz, J.M. (2008). Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon. PLOS Neglected Tropical Diseases 2(4):e213. DOI: 10.1371/journal.pntd.0000213

Slack, A.T., Kalambaheti, T., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Chaicumpa, W., Bunyaraksyotin, G., Craig, S., Harrower, B.J., and Smythe, L.D. (2008). Leptospira wolffii sp nov., isolated from a human with suspected leptospirosis in Thailand. International Journal of Systematic and Evolutionary Microbiology 58(10):2305-2308. DOI: 10.1099/ijs.0.64947-0

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.