Publicação
Mycobacteriophage Ms6 : exploring the involvement of Gp1 on LysA export
| Resumo: | Mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) phage that infects the non-pathogenic Mycobacterium smegmatis. Similarly to what happens with all other dsDNA phages studied so far, Ms6 must compromise host cell integrity in order to release its progeny at the end of the lytic cycle. Ms6 lytic operon is organized into five genes. In addition to the endolysin (lysA) and holin-like genes (gp4 and gp5), two accessory lysis genes are found, gp1 and gp3 (lysB), which reflects a novel mechanism of phage-mediated lysis. lysB encodes an enzyme with lipolytic activity whereas gp1 encodes a chaperone-like protein. Gp1 interacts with the N-terminal region of LysA and enables its access to the peptidoglycan layer in a holin-independent manner. However, some aspects concerning Gp1 role in the lytic process are not completely clear. In this work we present data obtained using a recombinant Ms6 carrying gp1 and lysA fused to tag sequences. Subcellular fractionation of M. smegmatis infected cells revealed that Gp1 is present on the cell wall and cell membrane fractions, while LysA seems to be restricted to the cell wall. Despite the association of Gp1 with the cell envelope, translational fusions with the E. coli alkaline phosphatase gene have shown that Gp1 is not endowed with a signal sequence. These results together with the observation that Gp1 is not able to promote the export of the first 60 amino acids of LysA fused to PhoA’ suggest that Gp1 and LysA are exported as a complex. The association between the two proteins may be important to keep LysA inactive until the proper time of lysis. The study of bacteriophages opens new perspectives regarding the treatment of bacterial infections and, in this case, it may also contribute to a better understanding of the diverse mechanisms employed by bacteriophages to lyse their hosts. |
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| Autores principais: | Martins, Francisco André de Lemos |
| Assunto: | Mycobacteriophage Ms6 Mycobacteria Lysis Ms6 Gp1 Secreted endolysins Teses de mestrado - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) phage that infects the non-pathogenic Mycobacterium smegmatis. Similarly to what happens with all other dsDNA phages studied so far, Ms6 must compromise host cell integrity in order to release its progeny at the end of the lytic cycle. Ms6 lytic operon is organized into five genes. In addition to the endolysin (lysA) and holin-like genes (gp4 and gp5), two accessory lysis genes are found, gp1 and gp3 (lysB), which reflects a novel mechanism of phage-mediated lysis. lysB encodes an enzyme with lipolytic activity whereas gp1 encodes a chaperone-like protein. Gp1 interacts with the N-terminal region of LysA and enables its access to the peptidoglycan layer in a holin-independent manner. However, some aspects concerning Gp1 role in the lytic process are not completely clear. In this work we present data obtained using a recombinant Ms6 carrying gp1 and lysA fused to tag sequences. Subcellular fractionation of M. smegmatis infected cells revealed that Gp1 is present on the cell wall and cell membrane fractions, while LysA seems to be restricted to the cell wall. Despite the association of Gp1 with the cell envelope, translational fusions with the E. coli alkaline phosphatase gene have shown that Gp1 is not endowed with a signal sequence. These results together with the observation that Gp1 is not able to promote the export of the first 60 amino acids of LysA fused to PhoA’ suggest that Gp1 and LysA are exported as a complex. The association between the two proteins may be important to keep LysA inactive until the proper time of lysis. The study of bacteriophages opens new perspectives regarding the treatment of bacterial infections and, in this case, it may also contribute to a better understanding of the diverse mechanisms employed by bacteriophages to lyse their hosts. |
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