Tissue localization of collagenase and leucine aminopeptidase in the bovine filarial parasite Setaria cervi

BACKGROUND: Like other helminth proteases, filarial proteases have also been shown to require for parasite survival inside the host and mediate various physiologic processes such as tissue invasion, feeding, embryogenesis and host immune evasion. Many of these proteases have shown potential for vaccines and chemotherapeutic agents against active filarial infections. Setaria cervi is a bovine filarial parasite and serves as a good parasite model for the studies in lymphatic filariasis. Recently, a 175 kDa collagenase and leucine aminopeptidase (LAP) have been purified and characterized from the bovine filarial parasite S. cervi and shown to be potential vaccine candidate and diagnostic marker, respectively for human lymphatic filariasis. However, their tissue localizations and putative roles in the parasite biology have not yet been examined and thus remain unclear. Therefore, the current study attempts to localize and explore the putative roles of these two enzymes in S. cervi. METHODS: The tissue distributions of 175 kDa collagenase and leucine aminopeptidase in S. cervi were examined by immunohistochemical and histochemical methods, respectively. Immune sera obtained from the jirds immunized with collagenase served as primary antibody, rabbit anti-mouse IgG-HRP conjugate as secondary antibody and DAB as the substrate for the immunostaining of collagenase. Leu-βNA was used as the substrate for the histochemical staining of LAP. RESULTS: Both the collagenase and LAP were present in the body wall; however, they differ in their distribution pattern in different layers of body wall. Collagenase was mainly localized in epicuticle, cuticle, syncytial hypodermis and the nerve cord region whereas LAP was more concentrated in epicuticle, longitudinal muscle layers and almost absent or very faintly stained in syncytial hypodermis and nerve cord region. Both collagenase and LAP showed their common distributions in intestine, uterus and mature eggs, growing embryos and mf. Very strong immunostaining of collagenase in the outer body surface of the parasite indicates its major role in host-parasite relationship whereas the presence of LAP in muscular region suggests its role in tissue remodeling. The common presences of collagenase and LAP in the S. cervi intestine, ovary, uterus, eggs and mf suggest that they also have collaborative roles in molting, nutrition and embryogenesis. The data obtained on their immunological characterizations and their presence in important parasite organs give strong indication that they are critical for the survival of filarial parasite and thus can be good vaccine candidates and/or diagnostic markers for human lymphatic filariasis. CONCLUSION: The manuscript reports for the first time the tissue distribution of collagenase and LAP in the bovine filarial parasite S. cervi and discuss their putative roles in vivo. Our findings also open the avenue to examine the roles of these two proteases in vivo, which will require further experiments like using their natural substrates and/or specific inhibitors in each tissues

Evaluation of Wuchereria bancrofti GST as a Vaccine Candidate for Lymphatic Filariasis

Lymphatic parasites survive for years in a complex immune environment by adopting various strategies of immune modulation, which includes counteracting the oxidative free radical damage caused by the host. We now know that the filarial parasites secrete antioxidant enzymes. Among these, the glutathione-S-transferases (GSTs) have the potent ability to effectively neutralize cytotoxic products arising from reactive oxygen species (ROS) that attack cell membranes. Thus, GSTs have the potential to protect the parasite against host oxidative stress. GSTs of several helminthes, including schistosomes, fasciola and the filarial parasite Seteria cervi, are also involved in inducing protective immunity in the host. The schistosome 28 kDa GST has been successfully developed into a vaccine and is currently in Phase II clinical trials. Thus, GST appears to be a potential target for vaccine development. Therefore, in the present study, we cloned W. bancrofti GST, and expressed and purified the recombinant protein. Immunization and challenge experiments showed that 61% of protection could be achieved against B. malayi infections in a jird model. In vitro studies confirm that the anti-WbGST antibodies participate in the killing of B. malayi L3 through an ADCC mechanism and enzymatic activity of WbGST appears to be critical for this larvicidal function

Comparative Genomics of the Anopheline Glutathione S-Transferase Epsilon Cluster

Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated, gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (ψAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, ψAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera, though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site within GSTE5 may represent adaptations to new habitats

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

Helminth immunoregulation: The role of parasite secreted proteins in modulating host immunity

Helminths are masterful immunoregulators. A characteristic feature of helminth infection is a Th2-dominated immune response, but stimulation of immunoregulatory cell populations, such as regulatory T cells and alternatively activated macrophages, is equally common. Typically, Th1/17 immunity is blocked and productive effector responses are muted, allowing survival of the parasite in a “modified Th2” environment. Drug treatment to clear the worms reverses the immunoregulatory effects, indicating that a state of active suppression is maintained by the parasite. Hence, research has focussed on “excretory–secretory” products released by live parasites, which can interfere with every aspect of host immunity from initial recognition to end-stage effector mechanisms. In this review, we survey our knowledge of helminth secreted molecules, and summarise current understanding of the growing number of individual helminth mediators that have been shown to target key receptors or pathways in the mammalian immune system

<span style="font-size: 20.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">Antibody responses of <i>Wuchereria bancrofti </i>patients to recombinant <i>Brugia pahangi </i>superoxide dismutase </span>

35-40<span style="font-size: 14.5pt;mso-bidi-font-size:8.5pt;font-family:&quot;Times New Roman&quot;,&quot;serif&quot;">Lymphatic filarial parasite Brugia malayi contains significant amount of Cu/Zn superoxide dismutase (SOD) activity in the extract of different life stages and in the excretory-secretory product of adults. In the present study recombinant SOD from B. pahangi has been used to see the antibody response in Wuchereria bancrofti infected patients. The recombinant SOD from B. pahangi reacted specifically with W. bancrofti infected sera in ELISA and immunoblotting. The reactivity of IgM subclass was more as compared to IgG subclass both in the asymptomatic microfilaraemic and symptomatic amicrofilaraemic when tested by ELISA. Serum from other helminthic infection was very low and found to be insignificant. The antibody response to rec SOD was directly proportional to the number of microfilariae in infected patients. The circulating filarial SOD was detected in filarial patients using polyclonal antibodies raised against recombinant Cu/Zn SOD in rabbits. The apparent molecular masses as determined by immunoblotting were 29 and 22 kDa. The specificity of recombinant SOD could be explored for its use in immunodiagnosis of lymphatic filariasis. </span