Characterization of Protein Profile of Filarial Parasite Brugia malayi after Depletion of Intracellular Endosymbiotic Bacteria Wolbachia through Antibiotics and Development of Antibiotic Delivery System for Antifilarial Targeting

Abstract

Lymphatic filariasis, a major tropical disease caused by mosquito borne filarial nematode worms W. bancrofti, B. malayi or B. timori is widespread in Asia, Africa and Latin America. Of these three species, W. bancrofti is responsible for more than 90% of the cases and is found throughout the tropics and in some sub-tropical areas including Asia, East Africa, Latin America, the Middle East, the Pacific Rim and Pacific Island. Brugia malayi is less prominent and constitutes remaining 10% incidence. India alone accounts for 40% of the 129 millions global prevalence of infection leading to loss of 1 billion US $ (~Rs. 5000 crores) each year in form of secondary treatment costs and loss of working days due to filariasis which highlights the considerable burden this disease places on individuals and on the community. The filarial nematodes are obligate parasites, characterized by their ability to persist in the lymphatics of infected host for many years (adult parasite life span 5-12 years). A striking feature of the filarial infection is the protracted and stable relationship reached between the host and the parasite. This balance serves to limit mortality of the host directly but fails to prevent substantial morbidity and disfigurement. In filaria endemic area, wide spectrum of clinical manifestations is observed. On one end are the asymptomatic amicrofilaraemic individuals commonly called as endemic normals (EN), to asymptomatic microfilaraemic (ASM) individuals called as ‘carriers’ to symptomatic chronic cases (CH) or those suffering from tropical pulmonary eosinophilia known as TPE cases. The pathology associated with the lymphatic filariasis results from a complex interplay of the pathogenic potential of the parasite and the consequent immune response of the host, and secondary bacterial and fungal infections. In its most obvious manifestations, enlargement of the legs, arm or breast and the genitals (vulva and scrotum) are quite common. In endemic province, 10-50% of men and up to 10% of women can be affected. In general, filarial nematode infection is less pathogenic and less likely to cause death than protozoan, bacterial or viral infections although their effects in terms of morbidity and economic loss are very significant. Filariasis is second only to malaria as the most important vector borne disease in India. The disease is endemic in 22 states and union territories. WHO has identified filariasis as one of six potentially eradicable infectious diseases and initiated ‘Global Programme to Eliminate Lymphatic Filariasis’ (GPELF) for its elimination as a public health problem by the year 2020. The current global filariasis control efforts (GPELF) advocate various strategies for the treatment and control which include (i) MDA with single-dose albendazole and ivermectin (Mectizan) tablets in areas, where lymphatic filariasis is co-endemic with onchocerciasis (river blindness) caused by Onchocerca volvulus, annually for 4–6 years, (ii) single dose albendazole and diethylcarbamazine (DEC) in areas, where onchocerciasis is not endemic, annually for 4–6 years, (iii) exclusive use of DEC-fortified tablet or cooking salt for 1–2 years in some settings, (iv) vector control measures, especially the use of insecticide-treated materials, where appropriate and cost-effective, (v) home-based self-management of lymphoedema and elephantiasis for affected individuals and (vi) improved access to surgical intervention for men with hydrocoele. Although, the MDA programme has provided encouraging resulting with development of newer control strategies, availability of sensitive and specific immunodiagnostic assays nevertheless, lack of public awareness and literacy specially in developing nation’s elimination of lymphatic filariasis in a very near future seems to be a difficult task. Apart from this, diethylcarbamazine (DEC) or ivermectin principally effective against circulating microfilariae (mf) resulting into recurrence of microfilaraemia after withdrawal of the drug, moreover both the drugs have low adulticidal/embryostatic effect. Recently, the evidence of emergence of resistance against mainstay drugs like albendazole and ivermectin have started to show up which also makes this challenge tougher than past. Starting from the mid-1970s, electron microscopy studies showed the presence of intracellular bacteria in the body of various species of filarial nematodes, including O. volvulus, W. bancrofti, B. malayi, Difilaria. immitis and Litomosoides sigmodontis. Wolbachial presence have been observed in the lateral chords of both male and female filariids, in the reproductive apparatus of females (i.e. in the oogonia, oocytes, embryos and microfilariae) and also in the larvae present in the mosquito vector. The endosymbionts are also reported to be necessary for filarial worm fertility and survival, thus making them a promising target for the development of a potential long-term sterilizing or macrofilaricidal (adulticidal) drug. Certain antialpha proteobacterial agents, most notably tetracycline and doxycycline apart from rifampicin and azithromycin, have exhibited detrimental effects on parasitic nematode development and fertility, through Wolbachia depletion. The main features of tetracycline treatment are interruption of embryogenesis and inhibition of larval development and adult worm viability following elimination of Wolbachia. Although, the present combination chemotherapy has offered encouraging results, however, lack of adequate action on adult worms, an alternative strategy is still required. Therefore, from the chemotherapeutic perspective against filarial nematode infections, the anti-wolbachial targeting strategy is of paramount research interest. This approach has presented a superior and comprehensive antifilarial efficacy over the conventional treatment strategy (DEC/ivermectin with albendazole). The motive behind the use of antibiotics in filariasis is the attrition of endosymbiont Wolbachia, which eventually inversely affects the fitness and survival of filarial parasites, thereby indicating an intricate reciprocal dependence between the two. In past, several studies employing tetracycline have emphasized the role of wolbachial depletion in its antifilarial efficacy, although few others did not deny a possible direct action of this drug as well. Taking into consideration of the above reports, the proposed study was sought to evaluate the antifilarial profile of doxycycline and rifampicin in our model system i.e. B. malayi-Mastomys coucha and B. malayi-Meriones unguiculatus (jirds model). Since B. malayi and B. pahangi intraperitoneal (i.p.) jird models have invariably been used in the past to evaluate antifilarial efficacy of potential agents by various groups owing to the convenience and suitability, the identical animal model system was opted. To draw a comparative antifilarial profile of these antibiotics with tetracycline, various treatment regimens of these anti-wolbachial antibiotics were used i.e. intraperitoneally in jird model infected through the same i.p. route and orally in mastomys infected subcutaneously. The jird i.p. model harbouring adult parasites in the peritoneal cavity has the convenience of quantitatively monitoring the degree of micro- and macrofilaricidal efficacy and parasite death or calcification can be directly observed for a better comparison with controls, unlike s.c. infections where only a certain percentage of inoculated larvae (L3) reach adulthood leading to animal to animal variation in worm establishment. The results of these experiments demonstrated that doxycycline possess a beforehand antifilarial (micro- and macrofilaricidal) efficacy in comparison to both tetracycline and rifampicin. It is a general belief that Wolbachia is an obligatory requirement for the survival and fecundity, however several studies carried out earlier employing tetracycline or oxytetracycline concluded that wolbachial depletion is a slow process and requires a prolonged duration of treatment. In contrast to tetracycline and rifampicin, the present study demonstrated that doxycycline imparted very quick and superior antifilarial efficacy. In order to find out the reason underlying the superior efficacy doxycycline another experiments were designed in which this antibiotic was administered at different dose concentrations and at various treatment schedules through intraperitoneal route in jirds having a patent infection in their peritoneal cavity itself. These experiments also revealed similar results as obtained in previous trial. In these experiments, as the antibiotic was directly administered in the close vicinity of parasites, therefore, to rule out the possibility of toxicity for the reason of quick efficacy of doxycycline in i.p./jird model, a comparative cytotoxicity assessment of the three antibiotics along with the standard filaricide DEC was carried out. The results obtained from this experiment revealed that among the three antibiotics and standard filaricidal drug DEC tested, doxycycline was the safest as it was found to be imparting least toxicity to metabolically active human kidney fibroblast cell line. Nevertheless, the reason for the beforehand efficacy of doxycycline remained unanswered. To substantiate whether action of doxycycline on Wolbachia resulted into the death of filarial worms PCR (primers specific for 16S rDNA of B. malayi Wolbachia) and immunoblotting (specific antibodies against Wolbachia surface protein) studies were performed using adult B. malayi recovered from jirds treated with doxycycline or tetracycline for comparison with untreated worms to observe Wolbachia depletion, if any. These experiments demonstrated that prolonged treatment schedule employed in the present study were able to deplete Wolbachia completely irrespective of the two antibiotics used. In contrast, the shorter treatment regimens of doxycycline failed to deplete bacteria, in spite of exerting remarkable adulticidal and microfilaricidal efficacy. These results apparently indicate that wolbachial depletion is not the sole factor responsible for killing the filarial parasite moreover some other mechanism might be responsible for the early antifilarial efficacy of doxycycline. In the present study, the results of our earlier experiments also demonstrated that anti-wolbachial treatment strategy resulted into a comprehensive antifilarial efficacy, however to achieve better efficacy through Wolbachia depletion required a prolonged duration of treatment. Though, this approach demonstrated a comprehensive antifilarial efficacy, instead of this, prolonged treatment apparently seems not feasible, if employed for mass drug administration. Therefore, proposition for liposomized drug delivery system appears worthwhile, as it is proven to be effective as a targeting/delivery vehicle for intracellular as well as deep-seated pathogens. This delivery system results into a slow release of drug, which remain, in circulation for long time. Moreover, the dose of drug can be reduced several times as compared to that administered as free drug, leading to reduction in its toxic side effects. Earlier findings from our lab have also demonstrated that drugs (DEC and tetracycline) in liposomized form proved much more effective against filarial worms in rodent model as compared to free drug. A combination of anti-rickettsial and antifilarial drug could possibly be used as a suitable tool to deplete Wolbachia in parasitized host with effective micro and macrofilaricidal effect with reduced adverse reactions in human filarial infections. Therefore, in order to increase efficacy of these antibiotics with minimal toxic manifestations and to reduce the dose amount and its frequency, we prepared liposomized formulation of doxycycline and rifampicin. Both of these antibiotics were encapsulated in neutral liposomes and entrapment efficacy of these antibiotics was found to be comparatively very high over tetracycline. Besides this, the in vivo release of these antibiotics demonstrated that the serum concentration of both of these anti-wolbachial antibiotics remained above their MICs up to 48 hours post injection of a single dose of the formulation. Based on these observations, the dose of both the antibiotics was reduced to 10mg/kg in the present study, which was administered at an interval of every 72 hours. As it was observed in in vivo study that the drugs remained in circulation for more than two days, the total numbers of doses were reduced to five in contrast to the total twelve doses of tetracycline administered in our previous study. The results obtained by employing these formulations alone or in combination with DEC demonstrated that only five doses of these liposomized antibiotics were found to be very effective against microfilaraemia as well as adult parasites. The liposomization of these drugs augmented the antifilarial efficacy significantly over free drug administration. The single combination of these drug formulations (doxycycline or rifampicin) with standard antifilarial drug DEC although enhanced the microfilaricidal efficacy, however, no remarkable enhancement in adulticidal/embryostatic efficacy could be observed. Whilst in another experiment combination of both the liposomized antibiotics with standard drug DEC acted synergistically in providing significantly higher antifilarial efficacy. It is well documented that tetracycline and other antibiotics imparted lethal effects on microfilariae as well as adult parasites, resulting into complete blocking of embryogenesis, production of microfilariae and significant reduction in recovery of live adult worms. Several reports have emphasized on the anti-wolbachial activity of tetracycline leading to antifilarial efficacy indicating an obligatory relationship of Wolbachia with the parasite. More recently, the complete genome of Wolbachia from B. malayi has been fully sequenced and it is postulated that B. malayi cannot synthesize riboflavin or heme endogenously and therefore relies solely on Wolbachia for these co-factors necessary to make glutathione and all nucleotides. Thus it would be imperative to conclude an obligatory mutualistic relationship between the two. Our findings also support the reciprocal interdependency between endosymbiont and nematode host. Since Wolbachia are present intracellularly within the filarial nematodes, their components are likely to be present in the filarial proteins. Therefore, we sought to investigate the contribution of the endosymbiont Wolbachia in the protein profile of its nematode host B. malayi. Consequently, proteomic approach was employed to make out the difference between the protein profile of Wolbachia intact and depleted B. malayi worms. For this Wolbachia depletion was achieved by treating infected jirds with tetracycline where the infection and subsequent drug treatments were given through i.p route. It also took 90 consecutive day treatment for complete elimination of Wolbachia as reported earlier in Mastomys coucha carrying L3 induced B. malayi infection. The depletion of endosymbiont was confirmed at DNA level through PCR using Wolbachia (16S rDNA) specific primers and at protein level by immunoblotting and reacting with anti-wsp antibody. An initial comparative SDS-PAGE profile of tetracycline treated and untreated adult B. malayi parasites showed that many protein bands were either down-regulated (Molecular weights 180, 95, 42, 40 and 36kDa) or totally disappeared (Molecular weights 90, 87, 65, 46, 39 and 27kDa) from Wolbachia depleted B. malayi parasite thereby further establishing the intricate relationship between the two. The differences in both the protein extracts were found consistent when these were employed on 2DE gel electrophoresis. The analysis of adult B. malayi whole worm extract with proteomic software recognized approximately 100 protein spots following Coomassie staining of 2D gel and out of theses 56 showed differential expressions amongst the treated and untreated worms. These proteins were further subjected to MALDI-TOF for their identification using NCBI, MSDB and Swiss-Prot databases. A total of 14 protein spots completely disappeared from treated worms. Of these 6 antigens could be identified viz. i) hypothetical proteins from C. briggsae, ii) thioredoxin peroxidase from Brugia pahangi, iii) hypothetical protein from Brugia malayi, iv) elongation factor 1α from Stator furcatus, v) ENSANGO00000017163 and vi) ENSANGP00000026642 from Anopheles gambiae. The tetracycline treatment resulted into the down-regulation of 30 proteins of B. malayi. Of these 6 could be analyzed as two isoforms of enolases, superoxide dismutase, small heat shock protein, 60S ribosomal protein L15 and one unidentified protein. In contrast to down-regulation of expression of protective anti-stress proteins, tetracycline significantly up-regulated the expression of 9 other proteins. Interestingly, of these 6 could be identified as major structural proteins except one known as B. malayi antigen, whose function is yet to be determined. Nevertheless, the over-expression of these proteins is quite interesting and warrants further investigation. In addition to these up-regulated proteins, the emergence of a new protein resembling oxidoreductase of Sulpholobus solfataricus is difficult to explain, however, it has recently been reported to be a multifunctional enzyme showing high efficiency in catalyzing the reduction, oxidization and isomerization of disulfide bridges and might play a special role in adaptation to extreme conditions. Proteomic analysis highlights two important findings; firstly, few proteins with unknown functions disappeared after treatment and showed homology with earlier identified protein/s in other non-filarial species especially arthropods. These may possibly be of wolbachial origin and therefore can be further exploited as targets for new antifilarial drugs. Secondly, some of the proteins that are being down-regulated were identified as stress proteins which provide protection to the parasite against oxidative stress generated by host immune cells and thus may be important for parasite survival and are possibly regulated by bacterial presence. These proteins may also serve as possible antifilarial protein target/s. The present study is the first proteomic study on a human lymphatic filarial parasite B. malayi providing some functional proteomic data that unravels the complex and interdependent relationship between Wolbachia and its host B. malayi. Apart from providing important nutrient supplements, Wolbachia also appears crucial for host immune evasion by filariids. Several studies have confirmed that wolbachial components generate severe inflammatory reactions (Th1 immune response) by inducing the cells of innate immunity such as dendritic cells, macrophages and monocytes through different toll like receptors (TLR’s). Wolbachia is also involved in phenotypic dysregulation and antigen presenting function of monocytes and macrophages, which play as an important link between the innate and acquired immunity. These are the major target cells for immunomodulation caused by parasite derived or Wolbachia derived molecules leading to the host’s cellular hyporesponsiveness. Besides these, multiple mechanisms of dysregulation have been identified in humans and model systems, including parasite-mediated apoptosis of dendritic cells (DC) and T cells, down-regulation of MHC class I and class II on filarial exposed DC, the generation of T cell suppressing alternatively activated macrophages. Several groups recently working on elucidation of the mechanistic basis of cellular hypo-responsiveness established the role of regulatory T cells (Treg) and induction of CTLA-4 for the same. In this context, it was sought to appraise wolbachial involvement in induction of Treg cells or CTLA-4. Thus an experiment was planed in Balb/c mice which were sensitized with somatic adult B. malayi antigen (Wolbachia intact and Wolbachia depleted) either once or repeatedly and these immune parameters were recorded using FACS. Adult A. viteae antigen (naturally deprived of Wolbachia) was used as negative control. The present study demonstrates that Wolbachia as well as filarial parasite derived molecules led to the clonal expansion of regulatory T cells repertoire and CD4 expressing CTLA-4 molecules. It has earlier been suggested that induction in these cell population resulted into the emergence of cellular hyporesponsiveness through IL-10 dependent manner. The results obtained in this study also corroborated these findings since following a single sensitization with either of Wolbachia intact or depleted B. malayi antigen reduced the T cell responsiveness. However, presence of Wolbachia in filarial antigen caused predominant cellular un-responsiveness. Multiple sensitizations with Wolbachia intact B. malayi antigen reversed the immunological scenario characterized by a remarkable down regulation of Treg and CD4 expressing CTLA-4 populations associated with dramatic increase in the total CD4 and CD8 populations. Down regulation in regulatory T and CD4 cells expressing CTLA-4 population indicates selective clonal anergy apparently due to wolbachial occurrence. Another important aspect of this study was to investigate the effects of wolbachial and filarial antigen sensitizations on the macrophage function. It was very interesting to observe that macrophages were also affected in the same fashion as T cells. Besides these, similar type of tolerance was also observed in these peritoneal macrophages. Sensitization of peritoneal macrophages with Wolbachia intact B. malayi antigen also induced nitric oxide release which was down regulated after multiple sensitizations. This phenomenon suggests the generation of tolerance in macrophages which did not even respond to bacterial LPS. Conclusively, the work embodied in this thesis provides few new insights as: • Amongst the three anti-wolbachial antibiotics, doxycycline experimentally exhibited beforehand antifilarial efficacy over tetracycline and rifampicin • Liposomal delivery system enhanced the antifilarial efficacy of both doxycycline and rifampicin at a much lower dose and frequency and both the antibiotics combinedly demonstrated synergistic efficacy. • The proteomic approach was used to identify alteration in protein profile after tetracycline treatment that led to identification of various proteins which can be exploited as protein target. • Wolbachia is importantly involved in induction of cellular un-responsiveness (a hallmark of filarial infections) and apparently causes T cells anergy as well as macrophage tolerance.