The secret of bacterial armour

T

he better you know your enemy, the better you can fight him. This could have been Jean-Marie Ghuysen's motto. Not to go to the front to fight a flesh-and-blood enemy, but to fight tiny but merciless invaders: bacteria " In the '50s, after working for a pharmaceutical company, Jean-Marie Ghuysen returned to the University of Liège, working in the Bacteriology Department of the Faculty of Medicine ", explains Jean-Marie Frère, Professor Emeritus at the University of Liège, and a specialist in bacterial resistance. " At that time, we knew that bacteria needed armour to protect themselves from their own osmotic pressure ", continues Jean-Marie Frère. Bacterial membranes are highly permeable to water, and without a rigid structure - the bacterial wall - these microorganisms would burst under the pressure of incoming water, diluting their contents. " Scientists therefore knew of the existence of this armor, responsible for the mechanical stability of the bacterial cell and which can be compared to a three-dimensional lattice. They also knew that penicillin prevented the formation of this lattice . "On the other hand, Jean-Marie Ghuysen's predecessor had discovered a series of bacterial extracts capable of lysing other bacteria ", continues the Professor. "We're talking about actinomycetin, a mixture of enzymes secreted by bacteria of the genus Streptomyces", adds the scientist.

Back in the world of research, Jean-Marie Ghuysen was determined to make progress in the fight against bacteria. His ambition was to elucidate the structure of the bacterial wall, in order to identify the elements that needed to be targeted in order to pulverize this armor. " He wanted to use the enzymes secreted by Streptomyces and so set about isolating them, characterizing their activity and the consequences of their interactions with the bacterial wall ", explains Jean-Marie Frère. Thanks to the resources available at the time, he was able to propose a model for the structure of this wall, made up of a polymer he called "peptidoglycan". He also identified the action of bacteriolytic enzymes to destroy it. " In those days, the main method used was column chromatography to isolate the largest possible quantities of pieces of the wall," explains Jean-Marie Frère. " We can isolate the peptidoclycan from bacteria by boiling them with detergent. Everything is then dissolved except the peptidoglycan . From there, Jean-Marie Ghuysen activated the various bacteriolytic enzymes and tried to separate the pieces of peptidoglycan on the basis of their size or charge. " These pieces were then re-cut using chemical processes to obtain very small pieces of the bacterial wall which could be identified after chromatography on paper ", continues the Professor. This is how Jean-Marie Ghuysen discovered that the bacterial wall is made up of sugars and amino acids. 

In the course of his analyses, Jean-Marie Ghuysen was able to confirm that it is indeed sugars and amino acids that make up the walls of all bacteria. " With a few minor variations depending on the type of bacteria," says Jean-Marie Frère. " The bacterial wall is made up of linear chains of sugars, between which are formed bridges composed of peptides of 6 to 14 amino acids. The main differences observed from one bacterium to another are at the peptide level, in the way the sugar chains are linked together ". This major work was published in 1967 in the prestigious journal Science(1). Following this momentous discovery, Jean-Marie Ghuysen set up a multidisciplinary team whose ambition was to understand, on an atomic scale, how penicillin kills bacteria. "It was from the creation of this team, after Jean-Marie Ghuysen's retirement, that the Centre d'Ingénierie des Protéines (Protein Engineering Center) was created ", emphasizes Jean-Marie Frère. Made up of specialists in molecular biology, biochemistry, genetic engineering, protein chemistry, enzymology, radiocrystallography and other specific disciplines, this team will focus on gaining a fundamental understanding of the structure and catalytic mechanism of the enzymes responsible for bacterial wall synthesis. This should enable the scientific world to understand how penicillin acts as a grain of sand in this construction process. In today's context, where bacteria have responded to the massive use of antibiotics with resistance, Jean-Marie Ghyusen's team's approach opens up new prospects for the design of molecules effective against bacteria.

" In 1968-69, Jean-Marie Ghuysen's team thought they had identified two enzymes involved in bacterial wall construction, but one was sensitive to penicillin and the other not ," explains Jean-Marie Frère. Thinking he had uncovered a mechanism of resistance to penicillin, Jean-Marie Ghuysen decided to take a closer look and see how these enzymes actually interacted with penicillin. The idea behind this was to find new molecules capable of interfering with the penicillin-resistant enzyme to prevent it from playing its role in bacterial wall formation. "It was at this point that I joined Jean-Marie Ghuysen's team, after completing a doctorate in Canada and a post-doctorate in the USA on enzymes ", says Jean-Marie Frère. " It later turned out that the two enzymes we had identified had nothing to do with each other, and therefore did not represent a penicillin-resistant or a non-penicillin-resistant version ," adds Jean-Marie Frère. Research is all about trial and error, and not all leads to major discoveries. However, it's worth pointing out that the initial intuition was correct. "Later, it was shown that certain resistant strains (including the infamous methicillin-resistant Staphylococcus aureus, also known as MRSA) owed this resistance to the acquisition of a gene coding for a modified protein that penicillin inactivates very slowly, despite an undeniable structural similarity with sensitive enzymes. That said, several other "world firsts" were subsequently achieved in Jean-Marie Ghuysen's laboratory. We showed that penicillin-sensitive enzymes bind to penicillin via a serine residue ", says Jean-Marie Frère. " Our team has also proposed a whole reaction pathway for penicillin's mechanism of action. And we have obtained the first results on the three-dimensional structure of a penicillin-sensitive enzyme ", adds the scientist. " Finally, Jean-Marie Ghuysen's team also determined the structure of enzymes that were not sensitive to penicillin, and which functioned according to a different mechanism to that of enzymes that can be inhibited by penicillin ".

Professor Jean-Marie Ghuysen's work has been universally recognized, earning him prestigious awards. He has chaired numerous symposia at international conferences, and is a sought-after speaker at universities and meetings around the world. He received honorary doctorates from the universities of Nancy, Debrecen and Montreal. "Jean-Marie leaves behind him a thriving center with over seventy scientists and technicians. He left a lasting imprint on Belgian and international science, and his numerous publications (over three hundred and fifty) will long be remembered not only by scientists around the world, but also by his three children and eight grandchildren, one of whom is ... a pharmacist!" writes Jean-Marie Frère in his academic eulogy for Jean-Marie Ghuysen at the Royal Academy of Belgium.

Scientific reference

(1) Jack L. Strominger & Jean-Marie Ghuysen. Mechanisms of Enzymatic Bacteriolysis. Cell walls of bacteri are solubilized by action of either specific carbohydrases or specific peptidases. Science 14 Apr 1967:Vol. 156, Issue 3772, pp. 213-221 DOI: 10.1126/science.156.3772.213