Studies on Erp (Exported Repetitive Protein, Rv3810) of M. tuberculosis H37Rv

Abstract

The Erp (Exported Repetitive Protein, Rv3810) protein of Mycobacterium tuberculosis (Mtb) is a highly virulent secretory protein with a canonical signal sequence. It has three domains: a highly conserved amino terminal domain, a central variable region consisting of P(G/A)LTS repeat motif and a conserved carboxy terminus domain rich in proline and alanine. The number and the fidelity of PGLTS repeats vary considerably among mycobacterial species. Disruption of the Mtb erp gene has been reported to impair Mtb multiplication in cultured macrophages and mice. It has been found to be most highly upregulated cell envelop gene under nutrient starvation condition. There has been sufficient information on the role of Erp in maintaining cell wall integrity and colony morphology of mycobacterium. However, the response of Erp to the host/macrophage is not explored. Not much work has been done to study the protein-protein interaction of host with Mtb. These studies might give us critical insights on disease establishment. Among all the mycobacterial proteins, there is a very high likelihood of secretory proteins of Mtb being involved in interaction(s) with macrophage proteins. The interactions of Erp with mouse macrophage proteins have been attempted, keeping in mind the identification, functional characterization and discovery of new drug targets against TB, if possible. The work carried out here is the first of its kind where the secreted virulence determinant “Erp” of Mtb has been used as a “bait” to fish out the “prey” proteins encoded by Mouse macrophage cDNA library using yeast two hybrid (Y2H) assay. A total of nine putative host protein partners of ΔssErp (Erp devoid of signal sequence) were found by Y2H assay. Out of the nine macrophage genes, amplification of eight were successfully achieved. ΔssErp was found to interact directly with Serine Palmitoyl Transferase 2 (SPT2), as confirmed by in vitro GST-Pull down assay. Co-Immunoprecipitation studies in BHK21 fibroblast cell line confirmed that both Erp and ΔssErp interacted with SPT2 under in vivo conditions. SPT2 is the enzymatic subunit of SPT enzyme involved in rate limiting and first step of de novo ceramide production in the eukaryotic cell. Further more, coimmunoprecipitation studies of the deletion mutants have revealed that the amino terminus domain of Erp may interact with the carboxyl terminus domain of SPT2. Erp undergoes post-translational modification as is evident by the doublet obtained when Erp protein of Mtb H37Rv was over expressed in mammalian cell. Interestingly ΔssErp from non pathogenic M. smegmatis failed to interact with SPT2. Confocal studies revealed that both Erp-CFP, ΔssErp-CFP localized in endoplasmic reticulum (ER) while ΔNErp-CFP localized mostly in the nucleus. Also Erp-CFP and ΔssErp-CFP co-localized with SPT2-YFP, but ΔNErp-CFP failed to do so. SPT2 is present in all the mammalian cells and SPT2 mutants are known to be embryonic lethal, thus, making it extremely difficult to study the biological significance of interaction of ΔssErp with SPT2 under in vivo conditions. Therefore, we attempted to explore the significance of interaction by treating the cell lysate with purified ΔssErp or ΔNErp protein. Treatment of BHK21 cell lysate with purified recombinant ΔssErp protein exhibited a dose dependent increase in SPT activity which is responsible for the condensation of Palmitoyl CoA with L-Serine leading to the generation of 3- Ketosphinganine. This increase was abolished when same amount of ΔNErp protein was used for the treatment. Once 3-Ketosphinganine is formed it is committed to be converted into ceramide by de novo pathway. Thus it can be concluded that an increase in SPT enzyme activity by ΔssErp leads to an increase in ceramide production. Ceramide or its other sphingolipid intermediates like Ceramide-1-phosphate, Sphingosine, Sphingomyelin, S1P, etc have been shown to modulate the innate immune response of macrophage towards mycobacteria. Ceramide acts as a second messenger in a number of signaling pathways and it also causes apaotosis. We propose that Erp protein of Mtb interacts with mouse macrophage SPT2 leading to an increase in SPT enzyme activity which further increases the de novo biosynthesis of ceramide. Ceramide being a known inducer of apoptosis might assist in the spread of the mycobacteria. The precise mechanism by which Erp-SPT2 interaction allows survival of Mtb in macrophage needs to be further elucidated.