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Projeto de investigação
Role of the peptidoglycan hydrolases in the interaction of Streptococcus pneumoniae with an infected host
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The mycobacteriophage ms6 lysb n-terminus displays peptidoglycan binding affinity
Publication . Gigante, Adriano M.; Olivença, Francisco; Catalão, Maria João; Leandro, Paula; Moniz-Pereira, José; Filipe, Sérgio R.; Pimentel, Madalena; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); MDPI - Multidisciplinary Digital Publishing Institute
Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.
Study of penicillin resistance in Streptococcus pneumoniae
Publication . Martins, Jorge Miguel Alves; Filipe, Sérgio
Bacterial antibiotic resistance constitutes a major and current threat worldwide and the
development of new antibacterial strategies is considered a priority for organisms such as
Streptococcus pneumoniae.
The murMN operon in S. pneumoniae has long been associated with an elevated β-lactam
resistance. Its expression is responsible for the presence of branched muropeptides on the
peptidoglycan produced by multiple strains resistant to penicillin.
Recent work from our lab demonstrated that it was possible to restore penicillin resistance
to mutants without the murMN operon. Three mutants, called suppressors, were selected through
several rounds of population analysis profiles in increasing penicillin concentrations, in two
separate occasions. The suppressor mutants had a decreased susceptibility to penicillin and
contained several mutations in regions of the bacterial chromosome, which did not include the
gene groups usually associated to β-lactam resistance. Some of these mutations could be
associated with the TA synthesis pathway or the cell wall synthesis regulating factors.
The purpose of this work was to understand how the suppressor mutants, which express
PBP with low affinity to penicillin, but lack the murMN operon, managed to decrease their
susceptibility to penicillin.
To characterize the phenotypical effects of each mutation, the effects of two mutations
present in Pen6ΔmurMN suppressor 1.1, mutations lytR(R76G) e htrA(A361P), were first
analyzed. It was observed that the combination of both lytR(R76G) and htrA(A361P) is necessary
and sufficient to decrease penicillin susceptibility in the strain Pen6ΔmurMN, but not each
mutation individually. In fact, the mutant that received htrA(A361P) does not show any changes
in susceptibility, whereas the one that received lytR(R76G) showed only a slight decrease in
susceptibility to this antibiotic.
To characterize the effect of mutations in LytR protein, and to produce specific antibodies
for this protein, derivatives of LytR were expressed, in Escherichia coli, whose purification was
successful.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-MIC/30746/2017