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Mycobacteriophage-mediated lysis : the role of LysB proteins
Publication . Gigante, Adriano Marcelo Dos Santos; Pimentel, Madalena Maria Vilela, 1961-; Moniz-Pereira, José António Frazão
Most bacteriophages, or simply phages, lyse bacterial hosts by producing at least two proteins, holins and endolysins, which allow the release of the viron progeny at the end of a lytic cycle. Holins are membrane proteins responsible for the lysis timing and endolysins are enzymes that disrupt the peptidoglycan mesh. In addition to these two essential lysis proteins, phages infecting Gram-negative hosts synthesize a third class of lysis proteins, the spanins, necessary to overcome the bacterial outer membrane. Mycobacteriophages, the phages that infect mycobacteria, also have to face an outer membrane for phage release, although with a completely different composition from that of Gram-negative bacteria. Most mycobacteriophages encode a protein, named LysB, an enzyme with mycolyl-arabinogalactan esterase activity that hydrolyzes the link between the outer membrane and the cell wall. In this work the mycobacteriophage Ms6 was used as a model to investigate the role that LysB plays in lysis of Mycobacterium smegmatis. It is shown that Ms6 LysB is an essential lysis protein for an efficient lysis of mycobacteria. LysB proteins are highly diverse and some, including Ms6 LysB, have an extended N-terminus. The presented results demonstrate the ability of this region to bind to peptidoglycan, with a higher affinity for the M. smegmatis peptidoglycan, and absence of this region affect the lysis progression and results in a faster rise period. Based on comparative modeling and site directed mutagenesis the catalytic triad involved in the esterase activity showed the catalytic triad formed by Ser168, Asp249 and His318 to be essential for the esterase activity. A comparison with LysBs from other mycobacteriophages shows that Ms6 LysB has the highest catalytic activity. Overall, the results presented in this thesis define the importance of LysB in phagemediated lysis and further supports the notion that a complex host cell envelope can only be overcome by an appropriate phage lytic toolkit.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/87685/2012