Saturday, 23rd June 2018
Virology and Microbiology

       Bacterial Cell Division and Antibiotic Resistance







Juan Alfonso Ayala









Research summary:

Bacteria are protected from environmental offenses by an external cell wall. This structure consists of a strong yet elastic peptidoglycan polymer called the murein sacculus. Integrity of the sacculus is essential for bacterial viability and morphogenesis. Because the sacculus is both essential and exclusive for the bacterial cell, the enzymes involved in peptidoglycan metabolism (PBPs (penicillin-binding proteins), transglycosylases, transpeptidases, racemases, carboxypeptidases, etc) have become preferred targets for antibiotic development. During cell division, assembly of proteins at a division ring has the effect of constricting the membrane and producing a cell wall septum. The synthesis of a rigid peptidoglycan septum involves a particular set of these dedicated PBPs enzymes.


The objectives of our group are the analysis of the bacterial growth and cell division under diverse molecular approaches, and to study the mechanisms of resistance to the beta-lactam antibiotics, that have been developed by pathogens of clinical origin. Under this general scheme, we have developed two main aspects. One is the molecular characterization of the CTX-M beta-lactamases family, their mobilization mechanisms and the ancestral origin of the CTX-M-1 subfamily. And also the identification of PBPs and their role on the resistance mechanisms of anaerobic strains and Gram-negative enteric bacteria was analyzed. A new family of PBPs, belonging to the COG1680, (PBP4B, AmpH) with high homology with class C beta-lactamases (AmpC) and been involved in morphogenesis are going to be analyzed in the new project. An also a PBP (PBP4 from Pseudomonas), involved in the mechanism of expression of ampC will be characterized both at molecular and functional level.

We have achieved the purification of several of these enzymes (PBP1B, PBP3, and inactive variants) together with a good number of substrates (including whole sacculi, as the largest available structure, fragmented peptidoglycan and smaller-sized precursors as lipid II and UDP-muramyl pentapeptide) to assist in the set up of "in vitro" screening assays. This "in vitro" peptidoglycan synthesis assay will we used for search of new antibacterial compounds

Contrary to traditional ideas, recent investigations showed that the cell wall is a highly variable and dynamic structure. Previous research from our group demonstrated the induction of structural changes in the sacculus in response to antibiotic challenge as a key step to trigger defence mechanisms. Furthermore, bacterial secondary metabolites secreted as effector molecules in intercellular signalling are a previously unrecognized important source of adaptive changes in bacterial cell walls. All these advancements mean that traditional ideas on peptidoglycan metabolism need to be deeply revisited and reassessed pondering the ecological niches of microorganisms. Our current investigation aims to improve our understanding of the molecular mechanisms underlying the adaptive changes exhibited by the cell wall in response to antibiotics and other environmental challenges. To do so, we will study peptidoglycan enzymology in response to stress conditions, regulation of beta-lactam resistance factors, and identification of the extra/intra-cellular signals that trigger these responses. Particular emphasis will be made on the research of low molecular weight PBPs and inducible beta-lactamase systems as sensors of cell wall damage. The results should lead to the discovery of new pathways in the cell wall metabolism, which should provide a closer to real vision of peptidoglycan diversity in nature. The results from these studies will be of substantial help to better understand fundamental questions about bacterial social behaviour in poly-microbial communities and adaptability against environmental challenges. This is an ambitious project that relies significantly in collaborative relations with an important number of domestic and foreign laboratories, and is planned to promote a serious inter disciplinary effort including expertise areas as diverse as microbiology, crystallography, chemistry and bio-informatics.

Main achievements:

1. - Characterization of a new extended spectrum b-lactamase KLUA-9 of a Kluyvera ascorbata susceptible to cephalosporins and the genetic organization of the environments of blaKLUA9. In addition, we have carried out in collaboration with the project COBRA of the European Union a study on prevalence of ESBLs in Europe with the definition of a family CTX-M, as an emergent problem of change in the expression of the resistance in Europe. Also it has been identified and characterized the genetic surroundings of the b-lactamase of extended spectrum, PER2, of little diffusion in Spain, but emergent in the South cone of Latin America.

2. – L forms have been characterized in Escherichia coli and it has been demonstrated that they need to have peptidoglycan to carry out the bacterial division, and this fact has supposed a landmark in the identification of the division mechanisms. This work has been remarked in the Journal of Bacteriology and Microbe journals

3. - The analysis of resistant and virulent mutants of Pseudomonas aeruginosa in the model of Caenorhabditis elegans, has allowed to reconfirm in real time the theory on the influence of the infection in the evolution processes. This work has been remarked in the WEB page of the CSIC.

4. - We have carried out in collaboration with the University of Leon the characterization of the HMW-PBPs of the actinomicetes Corynebacterium glutamicum  that present the peculiarity to show growth of apical peptidoglycan and lacks homologous to the cytoskeleton of actin of other Gram negative bacteria.

5. - We have put in evidence for the first time in the South cone of Latin America the appearance of the genetic variant cr of the gene aac6'-Ib, that confers resistance to fluoroquinolones, in enterobacteria resistant to ciprofloxacin. 

6. - Based on analysis of peptidoglycan structure of Aeromonas PBP4 mutants, we have proposed a model for induction of the expression of b-lactamase mediated by a two component regulatory system.


Relevant publications:

1.- D. M. Livermore, R. Canton, M. Gniadkowski, P. Nordmann, G. M. Rossolini, G. Arlet, J. A. Ayala, T. M. Coque, I. Kern-Zdanowicz, F. Luzzaro, L. Poirel and N. Woodford. (2007). CTX-M: changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 59: 165–174. (IF: 5,068) (266 citations).

2.- Sauvage E., Kerff F., Terrak M., Ayala J.A., and Charlier P. (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev. 32(2):234-58. (IF: 11,796) (126 citations).

3.- van Heijenoort, Y., M. J. Gómez, M. Derrien, J. A. Ayala, and J. van Heijenoort. (1992). Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP1B and PBP3. J. Bacteriol. 174:3549-3557 (IF: 4,299) (99 citations).

4.- Terrak, M., T. K. Ghosh, J. van Heijenoort, J. van Beeumen, M. Lampilas, J. Aszodi, J. A. Ayala, J. M. Ghuysen, and M. Nguyen-Disteche. (1999). The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli. Mol. Microbiol. 34:350-364. (IF: 6,906) (88 citations).

5.- Fernandez-Cuenca, F., L. Martinez-Martinez, M. Conejo, J. A. Ayala, E. J. Perea, and A. Pascual. (2003). Relationship between b-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems against clinical isolates of Acinetobacter baumannii. J. Antimicrob. Chemother. 51:565-574. (IF: 5,068) (80 citations).