The transporter that researchers have found is vital for cell wall integrity in the plant pathogen Agrobacterium tumefaciens, a convenient lab model of some human pathogens.
Bacteria
are enclosed by a protective exoskeleton, the cell wall. The cell wall
is essential for bacteria since it determines their shape and allows
them to protect themselves, and 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 results in the release of
cell wall fragments known as muropeptides. The released muropeptides can
go to the environment, where they can have a far-reaching impact in
interspecies interactions, or be transported back into the cell for
recycling. To recycle muropeptides, some bacteria use a membrane
transporter called AmpG. However, many bacteria don’t have this
transporter, and so it is not known how or if they recycle their cell
wall.
When
bacteria encounter a cell-well targeting antibiotic such as penicillin,
excess muropeptides are released. Some bacteria detect these excess
muropeptides during recycling, and use this as a signal to produce
enzymes called β-lactamases which break down the antibiotic. However,
not all bacteria do this, and muropeptide recycling is not well
understood.
“We were really interested in studying cell wall recycling because its purpose is poorly understood. The bacterium E. coli for
example produces multiple proteins to recycle its cell wall, but
blocking recycling has no effect on its ability to thrive. To understand
this process better, we wanted to study alternative bacteria which do
things differently,” says the first author on the study.
Studying the bacterium Agrobacterium tumefaciens,
a bacteria which causes Crown Gall disease in plants, the researchers
identified a new transporter which takes over the role of AmpG in this
and related bacteria. Screening for genes which are required for growth
when new cell wall synthesis is reduced by the antibiotic Fosfomycin,
they found that a transporter belonging to the ATP-binding cassette,
ABC, family seemed to be become essential for the bacteria to survive.
When they deleted the transporter, they found that muropeptides
accumulated in the cells’ growth medium, while no muropeptides were
present inside the cells. The newly found transporter is named
YejBEF-YepA.
When
the researchers tested the resistance of the transporter mutant to
β-lactam antibiotics such as ampicillin, they found that it became
extremely sensitive. As expected, this corresponded with a decrease in
the activity of a β-lactamase enzyme. Remarkably however, the
transporter mutant became even more ampicillin sensitive than a strain
which completely lacked the β-lactamase, meaning that there must be more
going on. To investigate this further, the researchers tried stressing
the recycling defective strain by growing it in a low-osmolarity medium
where the support provided by the cell wall becomes very important. The
bacteria grew very poorly, and displayed swelling and lysis, meaning
that the integrity of the cell wall was greatly reduced.
“This
transporter showed up in our screen and represented an ideal candidate
for a new transporter of muropeptides. We expected to see a drop in
antibiotic resistance due to lower β-lactamase expression, but how sick
the bacteria became when deleted the transporter was very surprising,”
says the author.
Studying
the cell wall chemistry in more detail, the researchers found that the
thickness of the cell wall in the transporter mutant was much lower than
normal, which corresponded with a similar decrease in cell wall
precursor molecules. Also, the cell wall was less crosslinked that
normal, and relied more on an unusual type of crosslinking enzyme. They
concluded that the loss of recycled cell wall material must be a major
cause of the antibiotic sensitivity and cell wall defects seen in
bacteria missing the transporter.
Interestingly, the transporter has actually been reported before, as being important for Sinorhizobium meliloti to form a plant symbiosis, and mammalian pathogen Brucella melitensis to
resist antimicrobial peptides. However, it is not until now that its
function as a cell wall recycling transporter has been revealed. It
could therefore represent a promising target for antibiotics or
adjuvants in certain human pathogens, but is also relevant to
agriculture in either plant pathogens or symbionts.
“It
seems that a general peptide transporter YejBEF has been co-opted to
transport muropeptides by the evolution of a new subunit, YepA.
Interestingly, the transporter is present in many other bacteria
including human pathogens like Brucella and Ochrobactrum, and this work
is just the start in characterising its potential role in, for example,
infection,” concludes the senior author of the study.
https://www.nature.com/articles/s41467-022-35607-5
http://sciencemission.com/site/index.php?page=news&type=view&id=publications%2Fpeptidoglycan-recycling&filter=22