Discovery of a new bacterial cell wall recycling vector

The vector discovered by Professor Felipe Cava and PhD student Michael Gilmour at Umeå University is critical to the integrity of the cell wall of the plant pathogen Agrobacterium tumefaciens, and is a suitable lab model for some human pathogens.

Bacteria are surrounded by a protective exoskeleton, the cell wall. The cell wall is important to bacteria because it determines their shape and enables them to protect themselves. Many of our best antibiotics target the proteins that build and remodel this structure. As bacteria grow and divide, they remodel their cell wall, which causes the release of cell wall fragments called myropeptides. The released neuropeptides can end up in the environment, where they can have a far-reaching effect on interactions between species, or they can be transported back into the cell for recycling. To recycle murine peptides, some bacteria use a transmembrane transporter called AmpG. However, many bacteria do not possess this transporter, and thus it is not known how or if they recycle their cell wall.

When bacteria encounter an antibiotic that targets cell walls, such as penicillin, excess peptides are released. Some bacteria detect these excess peptides during recycling and use this as a signal to produce enzymes called β-lactamases that break down the antibiotic. However, not all bacteria do this, and neuropeptide recycling is not properly explored.

– We were really interested in studying the recycling of cell walls because its purpose is not well understood. For example, E. coli bacteria produce several proteins to recycle their cell wall, but preventing recycling has no effect on their ability to thrive. To better understand this process, we wanted to study alternative bacteria that do things differently, says Michael Gilmore, first author of the study.

By studying Agrobacterium tumefaciens, a bacterium that causes coronary gall disease in plants, researchers have identified a new vector that takes on the role of AmpG in this and related bacteria. By searching for genes required for growth when the synthesis of new cell walls is reduced by the antibiotic Fosfomycin, they found that a transporter belonging to the ABC family (ATP-binding cassette) appears to be essential for bacteria to survive. When they removed the vector, they found that myopeptides accumulated in the growth medium of the cells, while there were no myopeptides in the cells. The newly discovered transporter has been named YejBEF-YepA.

When the researchers tested the carrier mutant for resistance to β-lactam antibiotics such as ampicillin, they found that it became extremely sensitive. As expected, this is consistent with a decrease in β-lactamase enzyme activity. Remarkably, however, the vector mutant became more sensitive to ampicillin than a strain that completely lacked β-lactamase, meaning that more was bound to happen. To further investigate this, the researchers attempted to stress the strain defective in recycling by culturing it in a low-osmolarity medium where the support provided by the cell wall becomes very important. The bacteria grew very poorly and showed swelling and decomposition, which meant that the integrity of the cell wall was greatly reduced.

This transporter appeared in our assay and was an ideal candidate for a novel transporter of muropeptides. We had expected to see a decrease in antibiotic resistance due to decreased expression of the beta-lactamase enzyme, but how sick the bacteria became when the vector was removed was very surprising, says Michael Gilmore.

When the researchers studied the chemistry of the cell wall in more detail, they found that the thickness of the cell wall in the vector mutant was much less than normal, which corresponded to a corresponding decrease in cell wall particles. In addition, the cell wall was less bound than normal and was more dependent on an unusual type of cross-linking enzyme. They concluded that the loss of recycled cell wall material must be an important cause of the antibiotic sensitivity and cell wall defects seen in bacteria lacking the vector.

Interestingly, the vector has already been reported previously, which is important for Sinorhizobium meliloti to form a plant symbiosis and for the mammalian pathogen Brucella melitensis to be able to resist antimicrobial peptides. However, its function as a cell wall recycling transporter has not yet been revealed. It could therefore represent a promising target for antibiotics or adjuvants in some human pathogens, but also suitable for cultivation in plant pathogens or symbionts.

The universal peptide transporter, YejBEF, appears to have been selected to transport polypeptides through the evolution of a novel subunit, YepA. Interestingly, the vector is present in many other bacteria, including human pathogens such as Brucella and Ochrobactrum, and this work is just the beginning of characterizing its potential role in infection, for example, says Professor Kava, lead author of the study.

The results are published in the scientific journal Nature Communications. Professor Felipe Cava and PhD student Michael Gilmour belong to the Department of Molecular Biology at Umeå University and are also affiliated with the Laboratory for Molecular Infectious Medicine in Sweden (MIMS), the Umeå Center for Microbial Research (UCMR) and the Laboratory for Integrated Science (ISILAB).