SBE-33 Prize 2015
Dr. Cecilia Artola Recolons
Instituto Química Física "Rocasolano", CSIC. Department of Crystallography and Structural Biology
Structural biology of macromolecular machines involved in Peptidoglycan recycling and their implications in antibiotic resistance
I have worked as a Postdoctoral fellow in the Spanish National Research Council, and I did my Ph.D. with Prof. Dr. Juan A. Hermoso, in the Department of Crystallography and Structural Biology. My Ph.D project was in collaboration with Prof. Dr. Shahriar Mobashery from the University of Notre Dame (Indiana, USA), where I had the opportunity to learn science during my research stay in 2012.
I have a B.S. in Chemistry, by the University of Oviedo (2009) and in 2008 I worked as a research assistant with Dr. Jason Halfen (University of Wisconsin, USA).
The relationship between cell wall recycling and antibiotic resistance has been proved essential to look for new antibiotic targets in bacteria. My Ph.D. thesis research is focused on understanding the mechanism of key proteins involved in the recycling process of the bacterial cell wall, analyzing their structural biology and different functions, using X-ray crystallography and bioinformatics techniques.
In Gram-negative bacteria, the recycling process serves to control induction of the expression of beta-lactamases. Although there are many enzymes involved in this process, there are some key steps that need of certain proteins. The first step is the cleavage of the saccharide bonds that conform the PG. This work is done by the lytic transglycosylases (LTs).
This work presents the crystallographic structure of two essential Lytic transglycosylases. MltE (Artola-Recolons et al. Biochemistry, 2011) was the first endoactive lytic transglycosylase ever discovered, and we have proposed it to be the enzyme starting the recycling process. The crystallographic structure shows how this protein is attached to the inner leaflet of the outer membrane and how that MltE can accommodate up to eight sugars unlike the rest of LTs.
The other LT studied, MltC (Artola-Recolons et al. ACS Chemical Biology, 2014), is able to react, in a processive way, over both cross-linked and non-cross-linked peptidoglycan chains due to its additional module, described for the first time and highly conserved in bacteria. MltC is attached to the inner leaflet of the outer membrane and has a mobile region that allows the enzyme to move around the peptidoglycan chains, which could have crucial implications in flagellar mobility and biofilm formation.
Pseudomonas aeruginosa is a human pathogen that causes infection and generalized inflammation and sepsis. It has been shown that the penicillin resistance mechanism of P. aeruginosa is substantially different from the rest of Gram-negative organisms. Analysis of the genes of revealed P. aeruginosa to posses three paralogous amidases: AmpD, AmpDh2 and AmpDh3.
As in the rest of bacteria, AmpD is placed in the cytoplasm. However, AmpDh2 and AmpDh3 only appear in P. aeruginosa, and have a periplasmic localization. Both enzymes appear to be involved in virulence of this organism. This work has shown a whole mechanism of action for both AmpDh2 (Martínez-caballero et al. JACS, 2013) and AmpDh3 (Lee et al. JACS, 2013), explaining their specificity for the soluble or insoluble fraction of the Peptidoglycan.
The crystallographic structure and the complexes with cell-wall analogs were a key factor for determining the mechanism of these two enzymes. Although the structure of the monomer is very similar between AmpDh2 and AmpDh3, their quaternary structure has been proved essential to understand their role in recycling. What is more, both proteins AmpDh2 and AmpDh3 complement each other in the turnover and maturation of the cell wall.
This work tries to correlate the activity of lytic transglycosylases, with that of AmpDh2 and AmpDh3 working altogether in the periplasm of the bacteria, and performing unique activities in the PG recycling process.