Zebrafish model of leptospirosis: Where's the relevance?

Scrutinized for the past several decades as a model of embryonic development, the zebrafish has recently been promoted as a vertebrate model for investigating the pathogenesis of infectious diseases. Zebrafish embryos are transparent, allowing microbiologists to readily view the course of infections in real time. Another advantage of the zebrafish model is that it's amenable, at least in theory, to large-scale genetic screens for mutated host or microbial genes that affect the infection process.


Davis and colleagues observed the early stages of zebrafish embryo infection by the spirochete Leptospira interrogans, as described in a recent issue of PLoS Neglected Tropical Diseases. They injected 10-100 spirochetes into the hindbrain of zebrafish embryos at 30 hours post-fertilization, when the innate immune response is fully functional. Macrophages rushed to the hindbrain and engulfed the invaders within the first 4 hours following inoculation of the spirochetes. L. interrogans was also rapidly phagocytosed when injected into the caudal vein.

Figure 1a from Lesley and Ramakrishnan, 2008. A zebrafish embryo 30 hours following fertilization. The hindbrain and caudal vein are indicated with the bracket and arrow, respectively.

Macrophages typically kill and destroy their prey following phagocytosis. However, spirochetes were still observed in the macrophages 24 hours following inoculation, suggesting that L. interrogans can survive inside macrophages.

Their most striking observation was the accumulation of infected macrophages near the dorsal aorta in a region known as the aorta-gonad-mesonephros (AGM), where hematopoietic stem cells are born. This was not a general phenomenon of bacterial infections as macrophages harboring Pseudomonas aeruginosa failed to accumlate at the AGM following its injection into zebrafish embryos.

Figures 2C and 2D from Davis et al., 2009. The embryo was infected with fluorescently stained L. interrogans. 24 hours following infection, most of the spirochetes were found near the dorsal aorta (brackets), with a few scattered around the ventral tail. Scale bar, 300 µm in panel C, 100 µm in panel D.

Here's what the authors concluded in the final sentence of the paper:

The strikingly specific delivery of leptospires to [the AGM] by phagocytes provides insights into pathogenesis by suggesting a novel mechanism for targeting of organs during leptospiral dissemination.

In other words, L. interrogans may be capable of steering macrophages towards specific organs. Once the macrophages arrive at their destination, the spirochetes may escape from the macrophage and colonize the organ.

The challenge for the authors in future studies will be to demonstrate the relevance of the zebrafish model to leptospirosis. Hamsters and guinea pigs are appropriate models for leptospirosis because the pathology and lethality of Leptospira infection in these rodents is similar to what's observed in human leptospirosis patients. The fate of Leptospira that macrophages capture in these rodents differs from what is seen in zebrafish embryos. Leptospira that are found inside macrophages in tissue sections from infected rodents often appear to be disintegrating. Nevertheless, it's possible that a few Leptospira survive phagocytosis and subsequently guide the macrophage towards the target organs.

Featured paper

Davis, J.M., Haake, D.A., & Ramakrishnan, L. (2009). Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues PLoS Neglected Tropical Diseases, 3 (6) DOI: 10.1371/journal.pntd.0000463

Other references

Lesley, R. and Ramakrishnan, L. (2008). Insights into early mycobacterial pathogenesis from the zebrafish. Current Opinions in Microbiology 11(3):277-283. DOI: 10.1016/j.mib.2008.05.013

The 14th species of the Lyme disease group of Borrelia

The cluster of genetically related Borrelia species that includes the Lyme disease spirochete B. burgdorferi is known in the scientific community as Borrelia burgdorferi sensu lato ("in the wider sense"). Three members of B. burgdorferi sensu lato account for most cases of Lyme disease worldwide. They are B. burgdorferi sensu stricto ("in the stricter sense"), B. garinii, and B. afzelii. Several other species of the cluster are suspected of causing a small number of Lyme disease cases in Europe and Asia.

The United States is home to at least four species of B. burgdorferi sensu lato. They are B. burgdorferi sensu stricto (the only species known to cause Lyme disease in the U.S.), B. bissettii, B. andersonii, and B. californiensis. The discovery of a fifth named U.S. species, christened Borrelia carolinensis, was published in the Journal of Clinical Microbiology earlier this year. The new species hails from South Carolina. Most of the isolates were cultured from cotton mice and eastern wood rats, but one isolate was obtained from an Ixodes minor tick feeding on an eastern wood rat. Whether B. carolinensis is capable of inducing Lyme disease is unknown.

The number of named species of B. burgdorferi sensu lato found worldwide now stands at 14:

• B. burgdorferi sensu stricto
• B. garinii
• B. afzelii
• B. andersonii
• B. bissettii
• B. californiensis
• B. carolinensis
• B. japonica
• B. lusitaniae
• B. sinica
• B. spielmanii
• B. tanukii
• B. turdi
• B. valaisiana

If you read my last post, you will notice that Borrelia lonestari, detected in one case of the Lyme-like illness STARI, is missing from the list. Although STARI clinically resembles a mild form of Lyme disease, genetically B. lonestari is more closely related to the set of Borrelia that causes relapsing fever.

Featured article

Rudenko, N., Golovchenko, M., Grubhoffer, L., and Oliver, J.H. (2009). Borrelia carolinensis sp. nov., a new (14th) member of the Borrelia burgdorferi sensu lato complex from the southeastern region of the United States. Journal of Clinical Microbiology 47(1):134-141. DOI: 10.1128/JCM.01183-08

STARI or Masters disease: More like Lyme than Lyme?

A tick-borne illness has been masquerading as Lyme disease in the southern United States over the past two decades. Victims first notice the expanding "bulls-eye" skin rash that is similar in appearance to the erythema migrans (EM) of Lyme disease. However, the tick that feeds on the victim is not the Ixodes tick that causes Lyme disease but the Lone Star tick Amblyomma americanum. Moreover, Borrelia burgdorferi, the Lyme disease spirochete, is not the infectious agent. B. burgdorferi has never been successfully cultured from a southern case of the EM-like rash, and sera from most of these patients test negative for Lyme disease by CDC criteria. Lyme disease itself is uncommon in the south as the resident Ixodes ticks rarely feed on humans; most ticks found attached to humans residing in the south are the Lone Star tick, which is unlikely to harbor or transmit B. burgdorferi.

The name bestowed upon the condition, "southern tick-associated rash illness" or STARI, is misleading because the territory of the Lone Star tick has been creeping into the northeastern and northern U.S., where Lyme disease is hyperendemic. The illness has also been dubbed "Masters disease" to honor Dr. Edwin Masters, who passed away last month. Dr. Masters' observations of skin rash patients in his private practice in Cape Girardeau, Missouri sparked the contentious CDC investigation that led to the first detailed description of STARI 14 years ago. You can read about his battles with the CDC in a series of blog posts by Pamela Weintraub, author of the book Cure Unknown, Inside the Lyme Epidemic (a book I hope to read some day).

Fig. 1 from Masters et al., 2008. Lone Star tick territory in green.

Contrary to popular belief, the erythema migrans of most Lyme disease patients does not present as a bull's-eye. In fact, in one study the EM-like rashes in Masters' STARI patients were much more likely to appear as a bull's-eye than the EM of Lyme disease patients from New York. In addition, the STARI patients were less likely than those with Lyme disease to suffer from accompanying symptoms such as joint and muscle aches, fatigue, headache, and stiff neck. A question that remains unresolved is whether arthritic, neurologic, or cardiac symptoms can crop up later, as they do in those afflicted with Lyme disease.

Figure 2 from Masters et al., 2008. EM-like skin lesions in Missouri STARI patients

The agent of STARI has eluded scientists. Borrelia lonestari was suspected at one time when it was detected by PCR in one patient and the Lone Star tick attached to his skin. (The spirochete could not be cultured since it does not grow in Borrelia culture medium.) However, B. lonestari could not be detected in a later study of a series of Masters' STARI patients. Thus, B. lonestari is unlikely to bring about most cases of STARI. The failure to identify the infectious agent of STARI has led some to question whether STARI has an infectious cause.

Masters was convinced that a spirochete, perhaps one closely related to Borrelia burgdorferi, was the agent of STARI. He has offered the following observations as evidence:

• Spirochetes have been observed in Lone Star ticks.
  
• Forms resembling spirochetes have been observed by silver staining of the EM-like skin lesions from Masters' STARI patients (see figure below).
• Extracts of B. burgdorferi reacted with sera from some STARI patients in ELISA tests, although the sera were Western blot negative according to CDC criteria. This observation indicates that antibodies were elicited against proteins closely related to those found in B. burgdorferi.
  Figure 8 from Masters et al., 1998. Silver stain of skin biopsy of an EM-like rash from a Missouri patient showing an apparent spirochete.
  

Of course these observations are far from definitive proof. Nevertheless, it is possible that a Borrelia spirochete is the culprit of EM-like lesions in the southern U.S. Like B. lonestari, some of these strains may not grow in the standard Borrelia medium used to cultivate B. burgdorferi from the skin rashes of Lyme disease patients. This would account for the inability of investigators to culture the spirochete from the skin rash of STARI patients.

Finally, how is STARI treated? Although the cause of STARI remains unknown, Edwin Masters declared that Lyme-like illness deserves Lyme-like treatment. That is, he recommended that antibiotics be administered to STARI patients according to Lyme treatment guidelines. Establishing whether antibiotics truly help will require a randomized placebo-controlled study.

Featured article

MASTERS, E.J., GRIGERY, C.N., & MASTERS, R.W. (2008). STARI, or Masters Disease: Lone Star Tick–Vectored Lyme-like Illness Infectious Disease Clinics of North America, 22 (2), 361-376 DOI: 10.1016/j.idc.2007.12.010

Other references

Masters, E., Granter, S., Duray, P., and Cordes P. (1998). Physician-diagnosed erythema migrans and erythema migrans-like rashes following Lone Star tick bites. Archives of Dermatology 134(8):955-960.

Wormser G.P., Masters, E., Liveris, D., Nowakowski, J., Nadelman, R. B., Holmgren, D., Bittker, S., Cooper, D., Wang, G., and Schwartz, I. (2005). Microbiologic evaluation of patients from Missouri with erythema migrans. Clinical Infectious Diseases 40(3):423-428. DOI: 10.1086/427289

Wormser G.P., Masters, E., Nowakowski, J., McKenna, D., Holmgren D., Ma, K., Ihde, L., Cavaliere, L.F., and Nadelman, R.B. (2005). Prospective clinical evaluation of patients from Missouri and New York with erythema migrans-like skin lesions. Clinical Infectious Diseases 41(7):958-965. DOI: 10.1086/432935

Role of the second messenger cyclic diguanylate (c-di-GMP) in the Lyme disease spirochete

Second messengers are the intracellular intermediaries that transmit the signals received from the environment (first messenger) to the cellular machinery that generates the appropriate response. Well known examples of second messengers in mammalian cells include cyclic AMP, cyclic GMP, calcium ion, and inositol triphosphate. A second messenger unique to bacteria is cyclic diguanylate, abbreviated c-di-GMP. First described in the 1980s, c-di-GMP is only now attracting wide interest among those who study signal transduction in bacteria.

Cyclic di-GMP is created from two GTP molecules by diguanylate cyclase and destroyed by phosphodiesterases. Genes encoding the opposing enzymatic activities can be identified by the conserved GGDEF motif in diguanylate cyclases and an EAL or HD-GYP motif in phosphodiesterases. Bacteria modulate the intracellular concentration of c-di-GMP by controlling the amounts and activities of the diguanylate cyclases and phosphodiesterases in response to changes in environmental conditions.

Cyclic di-GMP is best known for promoting the formation of biofilms. Biofilm assembly requires the synthesis and secretion of the special polysaccharides that make up the biofilm matrix and the down-regulation of motility. Both polysaccharide synthesis and the inhibition of motility are modulated by c-di-GMP. The molecule can also affect virulence functions. In many cases, the mechanistic details of how c-di-GMP exerts its effects remain unknown. The molecular target of c-di-GMP includes proteins with the "PilZ" domain (see figure). However, not all proteins bound by c-di-GMP possess the PilZ domain. In some bacteria, c-di-GMP can also bind specific sequences found within the 5' untranslated region of several mRNAs to modulate gene expression.

Figure 1 from Tamayo et al., 2007. DGC, diguanylate cyclase; PDEA, phosphodiesterase A; PDE, phosphodiesterase

The Borrelia burgdorferi gene rrp1 encodes the only protein in the Lyme disease spirochete containing the GGDEF motif. The Rrp1 protein consists of a receiver domain and the GGDEF domain, whose diguanylate cyclase activity requires phosphorylation of the receiver domain. A paper in the March issue of Molecular Microbiology revealed the genes whose expression is affected by Rrp1. The authors compared the transcript profiles (transcriptome) of a B. burgdorferi wild-type and an rrp1 deletion mutant by microarray analysis. It turned out that most of the genes affected by the mutation encode what the authors call the "core" cellular functions of Borrelia burgdorferi. The core functions allow the spirochete to seek out and capture nutrients from the environment, synthesize the building blocks necessary for assembling cellular parts, and extract energy from nutrients to fuel its activities. All bacteria, whether or not they cause disease, possess these core functions. Most transcripts from core genes were increased in the wild-type B. burgdorferi strain relative to the rrp1 mutant. The impaired growth of the rrp1 mutant compared to wild type is consistent with the importance of Rrp1 on the expression of the core functions of B. burgdorferi.

The investigators also found that the rrp1 transcript levels increased 6 fold when ticks harboring B. burgdorferi took a blood meal from mice. High levels of rrp1 mRNA were also maintained in B. burgdorferi growing in culture medium. These observations suggest that more rrp1 transcript is made when the spirochete is awash in nutrients, whether in blood or culture medium. Under these conditions, c-di-GMP signals B. burgdorferi to turn on genes necessary to acquire and metabolize the nutrients.

The protein that directly or indirectly senses changes in nutrient availability is likely to be the histidine kinase encoded by hpk1, the gene that lies immediately upstream of rrp1. The Hpk1 and Rrp1 proteins form a phosphorelay in which phosphate groups swiped from ATP molecules are transferred to the target Rrp1 protein in response to some signal in the environment.

In summary, Rrp1 diguanylate cyclase activity is enhanced at two levels when nutrients become abundant. First, rrp1 transcript levels are increased, leading to more Rrp1 protein being made. Second, the Rrp1 diguanylate cyclase activity is activated by phosphorylation. Increased c-di-GMP levels is the result. The c-di-GMP stimulates increased levels of transcripts emanating primarily from genes encoding the core cellular functions of B. burgdorferi. The question that remains unexplored is how c-di-GMP causes transcript levels to increase. One protein in B. burgdorferi harbors the PilZ domain, but as I mentioned earlier, PilZ is not the only protein domain capable of binding c-di-GMP.

Finally, what does this study reveal about the role of Rrp1 and c-di-GMP in Lyme disease? One possibility is that Rrp1 is involved in tick-to-human transmission and the early stages of the infection:

• As I already mentioned, Rrp1 upregulation in B. burgdorferi residing in a tick taking a blood meal may prepare the spirochete to metabolize the nutrients found in the blood as they are transmitted into the skin of the human victim.
• Transcripts expressed from several genes encoding factor H-binding proteins (some of the few non-core genes affected by the rrp1 knock out) were at higher levels when Rrp1 was present. Factor H is an inhibitor of the complement system found in our bloodstream. As such, binding of factor H by the spirochete may protect it from being killed by the host complement system. Indeed, the authors demonstrated that the rrp1 mutant was more sensitive to human serum than the wild-type B. burgdorferi strain. However, the significance of this observation is unclear as Borrelia garinii, another agent of Lyme disease, was just as sensitive as the B. burgdorferi rrp1 mutant to human serum. Additionally, earlier studies have shown that factor H is not necessary for successful B. burgdorferi infections (at least in the mouse model of Lyme disease).
• The ospC gene, which encodes another protein that may impair immune function during the early stages of infection, was also upregulated by Rrp1.
• Several transcripts expressing motility and chemotaxis functions are expressed at higher levels when Rrp1 is present. Motility and chemotaxis are considered to be core functions, but they may also be necessary for B. burgdorferi to establish infection in humans. Note that the proposed effect of c-di-GMP on B. burgdorferi motility is opposite of that found in other bacteria (see figure above).
  

The obvious experiment to perform is to test whether the rrp1 mutant can cause infection in the mouse model of Lyme disease. Unfortunately, the effect of rrp1 on virulence could not be tested as the authors were unable to knock out the rrp1 gene in an infectious strain of B. burgdorferi.

Featured paper

Rogers, E.A., Terekhova, D., Zhang, H.-M., Hovis, K.M., Schwartz, I., & Marconi, R.T. (2009). Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions Molecular Microbiology, 71 (6), 1551-1573 DOI: 10.1111/j.1365-2958.2009.06621.x

Image source

Tamayo R., Pratt J.T., and Camilli, A. (2007). Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annual Review of Microbiology 61:131-148. DOI: 10.1146/annurev.micro.61.080706.093426

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