The mycobacterial tlyA gene product, Rv1694 (MtbTlyA), has been annotated as “hemolysin” which was re-annotated as 2′-O rRNA methyl transferase. In order to function as a hemolysin, it must reach the extracellular milieu with the help of signal sequence(s) and/or transmembrane segment(s). However, the MtbTlyA neither has classical signals sequences that signify general/Sec/Tat pathways nor transmembrane segments. Interestingly, the tlyA gene appears to be restricted to pathogenic strains such as H37Rv, M. marinum, M. leprae, than M. smegmatis, M. vaccae, M. kansasii etc., which highlights the need for a detailed investigation to understand its functions. In this study, we have provided several evidences which highlight the presence of TlyA on the surface of M. marinum (native host) and upon expression in M. smegmatis (surrogate host) and E. coli (heterologous host). The TlyA was visualized at the bacterial-surface by confocal microscopy and accessible to Proteinase K. In addition, sub-cellular fractionation has revealed the presence of TlyA in the membrane fractions and this sequestration is not dependent on TatA, TatC or SecA2 pathways. As a consequence of expression, the recombinant bacteria exhibit distinct hemolysis. Interestingly, the MtbTlyA was also detected in both membrane vesicles secreted by M. smegmatis and outer membrane vesicles secreted by E. coli. Our experimental evidences unambiguously confirm that the mycobacterial TlyA can reach the extra cellular milieu without any signal sequence. Hence, the localization of TlyA class of proteins at the bacterial surface may highlight the existence of non-classical bacterial secretion mechanisms.
Traditional radiation therapy is often associated with significant toxicity to normal cells that could limit cancer therapy's success. The greater understanding of the molecular differences between cancer cells and normal cells has led to targeted therapies in cancer treatment. We discovered the radiation-inducible expression of Glucose regulated protein 78kDa (GRP78) in cancer using phage-display peptide libraries. In this study, we screened a peptide library and discovered novel peptides targeting GRP78. The top ten peptides were further evaluated for their affinity of binding to recombinant GRP78 using surface plasmon resonance technology. Since GRP78 is induced on cancer cells' surface, we irradiated the lung cancer cell lines A549 and H460 to evaluate peptide binding. Flow cytometry analysis of Dylight 488 conjugated peptides led to identifying peptides A and K with the highest binding level to irradiated A549 and H460. Peptides A and K were compared for their efficacy to deliver liposomal doxorubicin specifically to tumors. Copper-free click-chemistry was used to conjugate peptides to the liposome surface. Whole-body near-infrared imaging was performed to evaluate the biodistribution of the peptide conjugated liposomes in nude mice bearing A549 and H460 tumors. Both peptides A and K specifically delivered liposomes to the tumors compared to the non-targeted liposomes. In pilot tumor growth experiments, we found that GRP78 targeting peptide conjugated liposomes delayed the growth of tumors in nude mice compared to non-targeted liposomes. The inhibition of tumor growth was further enhanced in combination with radiation, indicating efficient delivery of the radiation-sensitizer doxorubicin specifically to tumors. Currently, studies are underway to evaluate the microscopic biodistribution of doxorubicin in tumor sections. Mass spectrometry analysis of various organs will be performed to determine the pharmacokinetics of liposomal doxorubicin delivered by GRP78 targeting peptides. In conclusion, our novel GRP78 targeting peptides specifically deliver liposomal doxorubicin to tumors. Doxorubicin encapsulated in GRP78 targeting peptide conjugated liposomes enhances the tumor growth inhibitory activity of radiation therapy. Specific delivery of radiation-sensitizing drugs encapsulated in a liposome by peptides that target radiation-inducible GRP78 is a promising therapeutic strategy for various cancers. Citation Format: Abhay K. Singh, Chandresh Shyam, Calvin Lewis, Wendy Zhang, Sapna Deore, Vaishali Kapoor, Dennis E. Hallahan. GRP78 targeting peptides deliver liposomal doxorubicin specifically to cancer and enhance the efficacy of radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1446.
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