Microsporidia are obligate intracellular pathogens that cause severe disease in immunocompromised humans. While albendazole is the treatment of choice, no therapy exists that effectively treats all forms or causes of human microsporidiosis. Recent studies show that the microsporidian Encephalitozoon intestinalis binds glycosaminoglycans (GAGs) associated with the host cell surface, and that the divalent cations manganese (Mn2+) and magnesium (Mg2+) augment spore adherence to host cells by activating a constituent on the spore surface. These studies also illustrate a direct relationship between spore adherence and host cell infection; inhibition of spore adherence leads to reduced host cell infection while augmentation of spore adherence increases host cell infection. In light of recent studies, microsporidia spore adherence has become a promising target for the development of novel therapeutics to treat or even prevent human microsporidiosis. The goal of this study was to further characterize the molecular mechanisms governing spore adherence by identifying specific constituents on microsporidia spores that participate in spore adherence with host cells. A 40 kDa Encephalitozoon cuniculi host cell binding protein was discovered and identified as ECU01_0820, hereafter known as Encephalitozoon cuniculi microsporidia spore adherence protein (EcMsAP). The gene encoding EcMsAP has multiple heparin-binding motifs and an integrin-binding domain, which are characteristic of proteins that interact with constituents on the cell surface. Immuno-transmission electron microscopy reveals that native EcMsAP is located on the plasma membrane, endospore, exospore, and the anchoring disk of microsporidia spores. Recombinant EcMsAP and antibodies to recombinant EcMsAP both inhibit spore adherence and host cell infection. However, the deletion of heparin-binding motif #1 from the EcMsAP gene results in the loss of ability to inhibit spore adherence and infection. Host cell-binding assays reveal that recombinant EcMsAP binds Vero and CHO cell lines, but exhibits attenuated binding to glycan-deficient CHO cell lines. Finally, biomolecular interactions analysis provides direct evidence that EcMsAP is a glycan binding protein. This study not only identifies a potential microsporidial vaccine candidate, it further supports the assertion that microsporidia spore adherence is a critical step in the host cell infection process.

Melanosome biogenesis is a highly regulated endosomal maturation process wherein structural fibers harbouring immature melanosomes acquires its biosynthetic proteins through the secretory pathway and finally matures into a functional organelle. These processes were shown to be dependent on several cytosolic protein complexes such as AP (adaptor protein)-1, AP-3, BLOC (biogenesis of lysosome-related organelles complex)-1, -2 and -3; in addition to kinesin motor KIF13A and Rab GTPases 7, 32 or 38. Mutations in the subunits of these complexes or Rab38 result into defective melanosome maturation leading to occulocutaneous albinism, a clinical phenotype commonly observed in Hermansky-Pudlak syndrome (HPS). Moreover, molecular function of these complexes in regulating the biogenesis of melanosome is partially known. The delivery of cargo to maturing melanosomal membranes requires fusion machinery that includes Rab GTPases, tethering factors and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. However, the SNAREs involved in the transport of cargo to melanosomes is poorly understood. In this study entitled as “understanding the SNARE dynamics during melanosome biogenesis” we focus on functional role of endosomal Qa-SNARE protein, Syntaxin 13 (formally called STX12, herein referred to as STX13) in the organelle biogenesis and its transport in and out of melanosome. Moreover, these studies show that STX13-mediated cargo transport require a melanosomal membrane localized R-SNARE VAMP7 and these SNAREs are interdependent on each other in regulating their steady state distribution. In addition, this study illustrated the possible mechanism of SNARE recycling which occurs indirectly through AP-3 complex. Thus, these studies underscore the STX13‟s role in cargo transport to maturating melanosomes and its trafficking routes to and from the melanosomes. Chapter-I describes the literature review on melanosome biogenesis; Chapter-II lists the experimental procedures used in this study and Chapter-III to V focuses on results and discussion, segregated into three sections. Chapter-III: Screening and identification of endosomal SNAREs involved in the trafficking of melanosomal proteins. Our preliminary RNAi screen for SNAREs involved in melanosome biogenesis revealed STX13 as one of the Qa-SNARE affecting pigmentation and cargo transport. STX13, a recycling endosomal SNARE has been reported to interact with pallidin, a subunit of BLOC-1; however the functional role of this interaction in pigment formation is unknown. In addition, previous studies from our lab have shown that STX13 colocalize with endosomal Rab11 and partially with EEA1- or Rab5-positive organelles in melanocytes. Together, these observations insinuated us to characterize the functional role of STX13 in melanosome biogenesis. Upon STX13 inactivation, wild type mouse melanocytes showed hypopigmentation due to mistargeting of cargo such as TYRP1 and TYR to lysosomes. Knockdown of STX13 dramatically decrease the population of immature and mature melanosomes. Moreover, STX13 associate with the melanosome cargo on endosomal tubular structures. In addition, deletion of regulatory domain in STX13 increases the cargo transport to melanosomes due to its increased SNARE activity. This is possibly due to loss in intracellular regulation of SNARE occur through multiple factors such as SM (Sec1p/Munc18) proteins. Together this data suggests that STX13 mediates cargo transport to melanosomes from recycling endosomes. Chapter-IV: Functional characterization of the SNAREs involved in melanosomal maturation. Several in vitro studies have shown that a set of four SNAREs such as Qa, Qb, Qc (or Qbc) and R control the membrane fusion event duing the cargo transport. Additionally, this process is further regulated by SM proteins in in vivo. Electron microscopic studies in melanocytes have shown that melanosomal proteins were delivered to the melanosomal membrane through recycling endosomal tubular domains. Moreover, our RNAi screen show that STX13 possibly acts as Qa-SNARE in mediating the fusion events between melanosomal membranes and the endosomal tubular or vesicular intermediates. However, the role of other SNAREs for this membrane transport is unknown. It has been shown that the expression of VAMP family SNAREs such as VAMP3, VAMP7 and VAMP8 increased with melanogenesis upon differentiation of melanoma cells. VAMPs belong to the class of R-SNAREs, in which VAMP7 is known to interact with VARP (abbreviation) and AP-3 (mediates the trafficking of TYR) separately, and these molecules are known to regulate the cargo transport to melanosomes. However, the precise role of VAMP7 in pigment granule maturation is unknown. Therefore, we set out to characterize the functional role of VAMP7 in melanosome biogenesis. VAMP7 has been shown to localizes to multiple sub-cellular compartments and regulate the several transport steps in other cell types. Our study found that GFP-epitope tagged either human or rat VAMP7 localize to melanosomes at steady state in wild type mouse melanocytes. Knockdown of VAMP7 causes hypopigmentation of melanocytes and misroutes the cargo to lysosomes. Further, the inactivation of VAMP7 in melanocytes phenocopies the STX13 depletion, suggesting both the SNAREs are required for the melanosome biogenesis. In addition, knockdown of STX13 target the VAMP7 to lysosomes; while inactivation of VAMP7 affect the localization of STX13 to recycling tubular structures. Subsequently, the dominant active mutants of STX13 were not able to rescue the pigmentation or cargo transport defects in VAMP7 knockdown melanocytes. Together, the data suggests that STX13 functions from recycling endosomes and VAMP7 on melanosome membrane for the transport of cargo to melanosomes Chapter-V: Understanding the mechanism of STX13 recycling during melanosome biogenesis. At steady state, SNAREs are localized to the membranes of specific organelles where they mediate or regulate the membrane fusion. During this process, three or two Q-SNAREs on one membrane (in a trans-SNARE complex, possibly formed by Qa, Qb, Qc or Qbc) interact with a R-SNARE on another member to form a SNAREpin complex. Post-fusion, SNAREs are disassembled by SNAP and NSF proteins and then recycled back to the original compartment for next round of fusion. Here, we address the mechanism of post-fusion recycling of STX13 from melanosomes to endosomes. Previous studies have shown that STX13 mislocalize to melanosomes in AP-3-deficient melanocytes, suggesting a role for AP-3 in recycling the SNARE from melanosomes. Bioinformatic analysis of the N-terminal region of STX13 revealed the presence of two canonical adaptor binding motifs 3YGP6L and KETNE80L81L, resembling the tyrosine-based (YXXø) and dileucine-based motif [DE]XXXL[LI], recognized by several adaptor proteins. Point mutagenesis of these motifs in STX13 had no effect on their steady state distribution indicating that STX13 possibly uses non-canonical residues for its recycling. Further, deletion of the N-terminal region (either 1-129 or 14-129 aa) in STX13 redistributes the SNARE to melanosomes. Moreover, the activity and the trafficking of recycling defective STX13 mutants are dependent on another HPS complex, BLOC-2 and the SNARE, VAMP7. Absence of 1-129 region in STX13 or mutations in the subunits of AP-3 perturbs the steady state localization of STX13 suggesting an indirect role for AP-3 in recycling of STX13 to endosome via non canonical motifs present in its 1-129 aa region.

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