Study Elucidates Mechanism That Makes Bacteria More Resistant to Antibiotics

Process that makes bacteria stop behaving like single-cell organisms swimming freely in a medium and assemble into a colony to form a biofilm is described at the molecular level (image: Pseudomonas aeruginosa culture / Wikimedia Commons)

Bacterial biofilm formation is one of the main problems caused by infections. It occurs when bacteria radically change lifestyle in response to adverse environmental conditions, such as excessively high temperatures, lack of nutrients, alterations in pH, or the presence of antibiotics. They stop behaving like single-cell organisms swimming freely in a medium and assemble into a large colony, in which individuals adhere to a surface and to each other to produce a protective extracellular matrix made up mostly of sugars.

This biofilm, which tends to grow, is highly resistant to antibiotics, increases the toxicity of pathogens, and boosts the infection to a chronic stage. Understanding the mechanism whereby bacteria switch from free-swimming mode to biofilm mode is therefore one of the top priorities in microbiology. A great deal of research has focused on this problem in the past decade.

A study performed by an international collaboration of specialists coordinated by Marcos Vicente de Albuquerque Salles Navarro, a Brazilian researcher, has just made an important contribution to the effort to elucidate this mechanism. The current issue of Proceedings of the National Academy of Sciences of the United States of America (PNAS) carries an article describing their findings, entitled “Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa”.

Navarro is a professor in the Department of Physics & Interdisciplinary Science at the University of São Paulo’s São Carlos Physics Institute (IFSC-USP) and an awardee of a Young Investigator grant from FAPESP for the research project “Structural and functional studies of proteins involved in c-di-GMP-mediated signaling pathways”.

In many pathogens, including the bacterium Pseudomonas aeruginosa, on which the study focuses, the transition from free-swimming form to biofilm is orchestrated by the nucleotide cyclic diguanylate monophosphate (c-di-GMP). This nucleotide functions as an intracellular signaling molecule that emerges inside bacteria in response to some external stimulus and is involved in several physiological processes, including the control of gene expression in the transition between lifestyles.

“Fluctuations in intracellular levels of c-di-GMP make bacteria switch off the genes responsible for flagella, the filamentous protein structures that enable these single-cell organisms to swim in a liquid medium, and switch on the genes responsible for producing the extracellular matrix of polysaccharides that envelopes and protects the bacterial colony,” Navarro told Agência FAPESP.

This process was already well known in the scientific community and reported in the literature. The novelty of this study is its clarification of the role of a specific protein called FleQ in the functioning of c-di-GMP.

“FleQ is a key protein in the transition between the bacterium’s two life forms,” Navarro said. “It acts as a receptor of c-di-GMP. On interacting with the nucleotide, FleQ inhibits the expression of flagellar biosynthesis and promotes the expression of polysaccharide biosynthesis. What we did was describe exactly how this happens.”

FleQ is known as a “transcription factor” because it mediates the transcription of DNA into messenger RNA. Transcription factors bind to specific DNA sequences and recruit to the binding site all the machinery responsible for transcription. At the site, the transcription machinery reads the genes in DNA that will be transcribed into RNA and later translated into proteins.

“When the level of c-di-GMP is low, FleQ presents as a hexamer. In other words, the thousands of atoms in the molecule interact with each other to form a hexagonal assembly. This complex structure binds to two specific DNA regions, promoting transcription of the genes responsible for formation of the flagellum and inhibiting transcription of the genes responsible for formation of the polysaccharide matrix. This gives rise to the free-swimming lifestyle in the bacteria,” Navarro said.

“However, when it interacts with c-di-GMP, FleQ changes shape completely to form a pair of trimers instead of a hexamer. In this new shape, it’s unable to activate transcription of the flagellar genes and instead activates matrix polysaccharide transcription genes. We demonstrate this process rigorously at the molecular level.”

To discover all of this, the researchers used various physical methods, such as X-ray crystallography, which enabled them to determine the spatial structure of FleQ and the huge change undergone by this protein in the presence of c-di-GMP. In addition, they produced mutants to identify how specific alterations influence FleQ’s activity. “We performed a complete in vitro study,” Navarro said, “and supplemented it with a functional in vivo investigation to see how these variants of the protein acted on the bacterium.”

For Navarro, the study is basic science, but the potential applications are self-evident. Understanding how c-di-GMP works is an emerging topic in microbiology owing to the importance of finding new ways to combat infectious processes using new antibiotics and new adjuvants, especially at a time when multi-drug-resistant strains of bacteria are so widely disseminated. With proof that bacteria can be made more susceptible by interfering in the signaling pathways that promote biofilm formation, research is booming in the area.