Interruption of the Life Cycle of Schistosomiasis Parasite using Engineered Caulobacter Bio-films
Schistosomiasis is one of the world's most prevalent parasitic problems, affecting over 200 million people in over 75 countries. The problem is most acute in sub-Saharan Africa, Asia, and South America. Contact with parasite-infested water results in immediate infection from larvae that burrow into the skin and take up residence in blood vessels, primarily those around the urinary tract and intestine. Symptoms are visible in both the intestine and spleen and are most often the result of cellular, granulomatous inflammation around trapped eggs. Currently, methods of schistosomiasis control vary; widespread, short-term success has been shown with the chemotherapeutic agents oxamniquine and praziquantel. While a number of short-term treatments may have immediate effectiveness in tempering infection in the human host (in particular, drug treatment with oxamniquine and praziquantel), the long-term perspective both in terms of human treatment and environmental eradication shows little hope for parasite control. As a result, the goal of this prospectus is the introduction of an ecosystem friendly, genetically engineered bacterium that will interrupt and prevent parasite infection of the intermediate snail host. By affecting the parasite at a crucial stage in its lifecycle, not only will wild populations decrease but the incidence of human infection will also go down. Utilizing the natural anti-parasitical system of NO production, a strain of bio-film forming bacteria will be engineered to recognize and destroy the larval form of the parasite before it infects the intermediate snail host.
The parasitic infection schistosomiasis is a worldwide problem that affects over 75 countries and over 200 million individuals. While infection rates in some areas are decreasing and some control projects have had positive results, overall the number of infected individuals has "not been reduced and may well be increasing" (38). More specifically, some control programs have been successful in certain countries in Asia and the Americas and accordingly infection rates and risks in those areas are on the decline. Conversely, a predominant number of countries in Africa have been ineffective at controlling the parasite and as a result the number of infected individuals and, more importantly those at risk has risen sharply in recent years (38).
Schistosomiasis infection is caused by contact with the small free-swimming trematode flatworm of the genus Schistosoma. The parasite's lifecycle initially depends upon the infection of an intermediate hostthe freshwater snail Biomphalaria. Once the tiny larva, at this point called a miracidium, finds this temporary host, it will divide a number of times to produce thousands of new parasites or cercariae. These cercariae are then excreted from the snail into the water and now may infect humans, their final host (Figure 1) (15). Requiring only a few seconds of human contact, the cercariae penetrate an individual's skin and continue their life cycle in the blood stream of this definite host. Over a period of 30-45 days the parasite will mature into a long worm, pair with a mating partner, and settle in the blood vessels of the host (12, 37). Once mature, the female releases eggs, some of which are excreted through the host urine or feces while the remainder become trapped in various human tissues. Those eggs elicit the immune response that is the primary cause of the infection's symptomatology. Infection typically occurs in the liver, urinary tract and intestine and is rarely seen in other tissues. A urinary tract infection is usually characterized by painful urination, damage to the bladder, ureters, and kidneys, and in severe cases can even result in fatal bladder cancer (12). Intestinal infection is slower to develop and is usually characterized by bloody stools and an enlargement of the liver and spleen, as well as damage to intestinal tissue. Bleeding from damaged tissue seriously weakens the infected individual and can eventually cause death (12, 37).
One of the more complex aspects of infection is the apparent absence of both human and snail immune responses to the larvae and mature schistosome. In both the snail and human hosts the parasite somehow eludes host defense systems while maintaining the ability to reproduce. Interestingly enough, the schistosome has developed advanced systems to deal with host-specific immune responses (9). Upon infection of the intermediate host (Biomphalaria), the parasite ensures its survival by interfering with two necessary regulatory systems; the IDS (Internal Defense System) and the NES (neuroendocrine system) (14). For reasons which are currently unknown, the parasite secretes neuropeptide or neuropeptide-like elements that interfere with both the IDS and NES (14). This could occur through some modulation of gene expression, but the mechanism is currently unknown. In the case of the human host, the schistosome achieves concealment through "several unusual parasite adaptations," (32) among which are reduced surface antigenicity and the ability to combat and resist typical immune damage. As Fishelson has shown, there are four parasitic proteins that inhibit human proteasome, neutrophil, and antibody action, preventing the common immune response mechanisms from having any real effect both in terms of immediate elimination and immunity development. Further, some schistosomes have been shown to mask their surfaces with "adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatability complex class I, and beta(2)-microglobulin (beta(2)m), presumably as a means of avoiding host immune responses" (28).
As far as current treatments, a number of short-term solutions have shown positive effectsthe most attractive of which is the use of the chemotherapy drugs oxamniquine and praziquantel (12, 37). In recent years the price of these drugs, praziquantel in particular, has dropped significantly, making it a much more viable solution to third-world communities. Unfortunately, because of its consistently higher price over praziquantel, the use of oxamniquine has dropped, leading to the potentially "dangerous situation" of "praziquantel as the only available anti-schistosomal drug, with very serious consequences in the event that the parasite [develops] resistance to praziquantel" (37). Along these same lines, resistance to both drugs, albeit in low levels, has been shown in the field since 1973, further highlighting the potential threat of significant resistance. In addition to drug treatments, other methods of control such as mollusciciding and habitat destruction have been shown to have moderate short-term success. However, as was the case with drug treatment, these provide only acute solutions (2).
Schistosomiasis is found in over 75 countries around the globe, being most prevalent in Africa, South America and select areas in Asia. Generally speaking, infection is common in small, isolated, economically-stressed communities situated near rivers and/or major bodies of fresh water (12, 17, 37). In most of these communities, the nearby water is heavily relied upon for "personal or domestic purposes such as hygiene and recreation, [as well as] professional activities such as fishing, rice cultivation, irrigation etc." (37). As a result, contact with the water is an absolute necessity for the majority of residents, and infection can have seriously damaging effects socially and economically. Additionally, as mentioned earlier, initial infection does not convey immunity, and individuals are susceptible to repeated re-infections. In communities where water treatment and waste management essentially do not exist, this can result in nearly continuous outbreaks of schistosomiasis (26).
Drug treatment in these impoverished communities is very difficult. Despite the low cost ofpraziquantel and its relative accessibility, administering successful treatment proves to be much more taxing. The majority of individuals being unfamiliar with traditional Western medicine makes delivering and maintaining drug regimens on a case-by-case basis nearly impossible (17, 37). Further, lack of funding and infrastructure for proper education, water treatment and waste management combine to inhibit a stable solution. Additionally, a significant black market for pharmaceutical drugs adds further difficulties to the effective execution of drug treatment (2, 26).
Another problem facing schistosomiasis treatment is the HIV/AIDS epidemic. In many of the affected countries, African ones in particular, the HIV/AIDS epidemic has greatly overshadowed the urgency of the schistosomiasis problem. HIV/AIDS is a very pressing issue with shockingly high mortality and infection rates and a current number of infected individuals exceeding 28.5 million adults and children (6). Accordingly, huge amounts of funds and energy have been focused on education, prevention, and treatment of HIV, which has unfortunately resulted in reduced attention to areas such as schistosomiasis management.
In order to prevent schistosomiasis ifrom infecting the terminal human host and to maintain a more long-term solution for overall Schistosoma control, this research aims to interrupt the parasite at a crucial stage in its life cycle: prior to infection of its intermediate snail host. This interruption will be carried out by a bacterium genetically-engineered to recognize and eliminate Schistosoma parasites before they infect and colonize the snail. A strain of Caulobacter will be developed that is well suited to biofilm formation on the Schistosoma intermediate host Biomphalaria mansoni. Antibodies against parasite surface antigens will be introduced into the Caulobacter cell membrane, enabling the bacterium to recognize and bind to incoming parasites. Upon parasite recognition (i.e. antibody binding to the parasite surface antigen), cloned nitric oxide synthase (NOS) genes will be activated in the Caulobacter, resulting in self-destructing nitrous oxide (NO) production that will eliminate the parasite. In summary, the proposed strategy uses modified Caluobacter in the motile phase of their life cycle to recognize, bind to and eliminate Schistosoma parasites through antigen binding-induced, nitric oxide release.
The concept of nitric oxide as a parasiticidal, anti-microbial, anti-tumor agent is gaining strong worldwide support. Recently, a number of studies have shown that the use of oxygen and nitrogen radicals is a very effective means to regulate and prevent infection in a wide variety of systems (5, 22, 30). From humans to Drosophila to the Eastern Oyster, the use of nitric oxide as a fundamental immune defense mechanism is highly conserved and absolutely necessary for survival. As Nappi wrote in 2000, the "reactive intermediates of both oxygen and nitrogen constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and Eukaryotic parasites. These [intermediates] appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species" (30). Interestingly enough, current research is showing the role of NO to be more and more prominent in human as well as other organisms' immune defense systems. It has been shown to contribute to the immune response in "non-specific host defense" as well as to be a significant tool in the killing of tumor cells by cytotoxic effector cells (13).
In terms of the choice of bacteria, Caulobacter fulfills the requirements of being both a bio-film former as well as native to aquatic environments (35). Further, Smit et al. showed C. crescentus "is stable and amenable to high density monolayer growth and resists starvation" (35), which identifies it as an almost perfect candidate. Additionally, the proposed strategy takes advantage of the dimorphic life cycle of the bacterium. Caulobacter exists for almost half its life cycle as a swarmer cell (10). The primary purpose of these swarmer cells is to colonize new areas of potential bio-film growth, but in this case their motility will be used to seek out and destroy incoming Schistosome parasites.
Incorporating research from a variety of different fields, the experiment is projected to take place in a stepwise fashion over a number of years. While there are particular aspects of the project that will be conducted concurrently, several of the later trials are contingent upon the successful completion of earlier experiments. Additionally, it is important to note that while the particular goals of the project could be compromised by negative results, proposed experiments will contribute valuable knowledge to the field, whether or not these experiments are successful.
Stage IA: Isolation and Characterization of Surface Antigens of the Schistosome Miracidium
A number of experiments have previously been conducted to determine the existence of conserved surface antigens of the Schistosome parasite in its human host stage (or as cercariae) (1, 4, 14, 18, 21, 24, 28). A number of these studies have identified potential vaccine candidates with particular reference to Paramyosin (24, 28) as well as the Sm23, Sm28, and Sm37 surface antigen families.1 While research being conducted on parasite surface antigens primarily focuses on the cercariae or adult form, studies have been performed showing that conserved surface antigens do exist and can be tracked in the larval forms as well (16).2 A hybridoma technique followed by immunoblots will identify the relevent conserved larval surface antigens.3
IA1: Isolation of gene responsible for antibody to larval surface antigen
Once the antibody to the larval surface antigen has been identified, the genes coding for that antibody need to be isolated. This will be accomplished by creating a cDNA library of the selected hybridoma cell line, sequencing the variable domain of the antibody, and then cloning a peptidoglycan-associated lipoprotein gene into the same promoter as that of the antibody. This will promote binding of the antibody to the bacterial surface membrane.4
IB: Establishment of a strain of Caulobacter (most-likely C. crescentus) suited to healthy biofilm formation on the shell of the intermediate host Biomphalarai glabrata (11, 29)
Caulobacteria are "biofilm-forming members of the natural flora of soil and aquatic environments," (35) which makes them ideal candidates to be the aquatic host for the Schistosome-specific antibody. With the goal of attacking and eliminating the Schistosome parasite prior to its infection of the snail host, a Caulobacter biofilm will be engineered to display on its cellular surface the parasite-specific antibody --note Part II-- that is well suited to live on the shell of the intermediate snail host. The acclimation process will in essence be a forced evolution whereby, in a stepwise fashion, C. crescentus will be artificially selected to inhabit an environment more and more similar to the one they would encounter in the wild. Initial phases will include cultivating bio-films on sections of snail shell, then selecting for those individual bacteria which display higher reproductive success and greater longevity. Eventually, the hope is that an easily cultivatable, environmentally durable, snail shell-specific C. crescentus biofilm will evolve (11).5
IC: Isolation of genes involved in Nitric Oxide synthesis and characterization of effects on C. crescentus
A good deal of recent research has been conducted on the parasiticidal, anti-microbial, anti-pathogenic, and anti-tumor effects of nitric oxide (NO). Additionally, NO has been shown to operate in systems ranging from humans to bi-valves to Drosophila and even to single celled bacteria (3, 7, 19, 30, 31). In this particular situation, the parasiticidal effects of NO are being harnessed in order to eliminate the potentially infecting Schistosome parasite (miracidium). Genes for NO production already have been identified by Eizirik et al. (1996); the real task for our investigators is to isolate those genes and successfully insert them into C. crescentus under the control of an inducible promoter in (see Stage II) (20). Once cloned into the host bacterium, experiments will also be conducted to characterize the effects of NO induction and synthesis upon the C. crescentus.
Stage II: Microarray characterization of gene expression patterns during antibody-antigen binding of engineered swarmer cells
One of the major hurdles will be initiating NO synthesis specific to the binding of the antibody-expressing swarmer cell to the parasite surface antigen. A microarray will be used to determine a gene that is already expressed under these circumstances, and the promotor for this gene will be fused to the NO synthesis genes.6
Stage III: The Big Dance
After successfully engineering a biofilm-forming strain of C. crescentus that displays on its surface antibody to the miracidium Schistosome and contain s genes for NO synthesis that are induced only upon antibody-antigen binding, experiments will be conducted in a simulated natural environment to measure the effectiveness of the system. Efforts will be made within reason to replicate nutrient availability, natural flora and fauna and conditional changes i.e. temperature and tide.7
Legal and Ethical Issues
Concerns over the process of genetic modification and the introduction of such a highly engineered organism into a natural environment will be raised, but there are no consequences reasonably envisioned which would cause a significant problem. Additionally, issues surrounding the introduction of bacteria into the natural environment will most likely be noted, but as C. crescentus is a non-pathogenic bacterium, concerns about infection or adverse health effects should be minimal. Further, the introduced bacteria are effectively playing the role of kamikazes and will self-destruct upon binding to the Schistosome parasite.
Applicability to Other Countries
As mentioned earlier, Schistosomiasis is a worldwide problem occurring in over 75 countries and affecting approximately 200 million people, with another 400 million at high risk for infection. The proposed solution will be applicable to all of these problem areas. Success in different environments may require species-specific engineering to insure successful bio-film formation and antibody-antigen binding, both of which may differ on a case-to-case basis.
1 (4, 18, 21). As an aside, Dupre et al. showed effective immunization of murine hosts upon exposure to Sm28GST antigen followed by treatment with Praziquantel chemotherapeutic agent (18). The Praziquantel allowed for the "unmasking of the native GST enzyme at the surface of the worms, thus permitting its neutralization by the antibodies raised by DNA immunization" (4, 18)
2 In particular, there is evidence to show that the Schistosoma larvae constitutively express "several tegumental surface components" that contain recognizable carbohydrate epitopes (16).
3 Using parallel techniques to those outlined by Kurtis and Hernandez, monoclonal antibodies specific to larval parasite surface antigens will be obtained (24, 27). The screen for these larval antigens will be conducted using a number of previously known target antigens as described in the literature. More specifically, Schistosoma larval surface antigen will be prepared by techniques described by Hernandez, 1999. Female ICR mice (24) will be immunized with surface antigen adjuvant. Upon significant antibody production these mice will be sacrificed and their spleens harvested. Spleen cells will be isolated via a cell sorter and placed into the individual wells of 96-well plates where they will be fused with myeloma cells by the addition of polyethylene glycol. Purification on HAT medium will ensure successful hybridization (34, 36). At this point, immunoblots to potential surface antigens described by Abath, Argiro, Dunn, Fan, Hernandez, Kurtis and Loukas will identify the relevant conserved larval surface antigens.
4 Two methods could be employed to accomplish this: (1) A cDNA library of the selected hybridoma cell line would be created at which point cDNA's (in pairs of two to account for the gene for heavy chain and the gene for light chain) would be cloned into our Caulobacter host. Through a larval surface antigen screen, the cell expressing both the correct heavy and light chain (corresponding to the larval surface antigen) could be isolated in its bacterial host (2). To clone an antibody, the amino acid sequence of the variable domain needs to be determined. This can be accomplished by sequencing a purified sample of both the heavy and light chain proteins of the antibody to the larval surface antigen. Once accomplished, the same sequence will be cloned from hybridoma cDNA. This cDNA will be prepared by screening hybridoma mRNA with reverse transcriptase and probes specific to the constant regions of antibody heavy and light chains. Having isolated general antibody cDNA, the target genes for the specific antibodies will be cloned in the following manner: Hybridoma mRNA will be subjected to PCR twice, once using primers for the conserved regions and a second time using primers for the previously determined variable regions. In this way, the specific antibody cDNA will be generated and cloned into the Caulobacter (8, 25, 33, 36).
An additional step can be taken to further promote the transport and binding of the cloned antibody to the bacterial surface membrane. Further, this step may be taken to ensure that in the wild, the antibody is in fact being bound to the surface of the Caulobacter and not released into the surrounding mediumas the success of the strategy relies on the antibody being bound to the Caulobacter surface membrane. Fuchs et al. showed in 1991 that co-expression of the desired antibody with peptidoglycan associated lipoprotein (PAL) resulted in tight binding to the "murein layer of the cell envelope" with little to no effect "on cell growth and viability" (23). Thus, it is proposed that in the final Caulobacter strain the peptidoglycan associated lipoprotein gene be cloned into the same promoter as that of the antibody.
5 As an aside, there could be the potential to further engineer or select the strain of Caulobacter to be dependent on some aspect of the snail i.e. waste-products, essential nutrients, or something in the immediate environment to ensure/limit biofilm formation to the snail host only. This would most likely be similar to the use of selective media and plasmid cloning to ensure the cellular uptake of cloned genes.
6 The iNOS gene (NO synthetase gene) cannot be placed under the control of a promoter associated with quorum sensing proteins or surface binding proteins of the swarmer cell, as that would significantly hamper any chances of the biofilm survivingfor Caluobacter cells. In those cases, upon attaching to any surface or forming a biofilm, NO synthesis would be initiated resulting in the destruction of Caulobacter cells in the immediate vicinity. Thus, an RNA microarray experiment will be carried out in order to determine the differences in gene expression between a wild type swarmer cell colonizing or binding to a new surface and the engineered swarmer cell binding (via antibody-antigen bond) to the parasite surface antigen. In the latter case, a unique suite of genes will be activated which are not normally activated in the wild-type cells. Thus, an ideal place to situate the NO synthetase genes will be identified so that induction occurs only upon the binding of the antibody on the surface of the swarmer cell to the parasite antigen and not during any other time in the Caulobacter cell cycle.
7 Biomphalaria harvested from previously studied sites in sub-Saharan Africa will be seeded with both engineered and wild type C. crescentus and placed into an S. mansoni infested environment (with S. mansoni concentration similar to that of previously studied sites). Every two days snails will be removed, observed for biofilm characteristics, and examined for presence of parasite infection, using characteristics and criteria determined by earlier studies of snail response to parasite infection.
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Matthew Goldstein '04 is a biology major with an interest in putting his technical knowledge to work. He thanks Amy Vollmer, his roommate Dan Winkel, and the rest of his biology seminar for providing guidance, advice, and humor during his writing process. This paper was written for Professor Amy Vollmer's Biology 116: Microbial Processes and Biotechnology.