Scientists find new bacterial cell wall proteins

E. coli are a type of Gram-negative bacteria, so they have two membranes, inner and outer ones. Without the protection of their outer membrane, Gram-negative bacteria die.

Creating new antibiotics is especially important because bacteria are constantly developing resistance to current drugs, said Thomas Silhavy, a professor in the molecular biology department.

“In the last 10 years, I think only one new drug for Gram-negative bacteria has been put on the market, so they are becoming resistant to all the ones out there,” Silhavy said. “There aren’t as many new drugs in the pipeline for [Gram-negative bacteria] as there are for the Gram-positive ones.”

Gram-negative bacteria like E. coli, salmonella and neisseria can cause serious illnesses including typhoid, plague, gonorrhea and meningitis. E. coli has been in the media spotlight recently after the bacteria were found in commercially available meat and spinach.

“We’re interested in understanding how bacteria make the outer membranes,” molecular biology lecturer Natividad Ruiz said. “The outer membrane has several components, including one called LPS [lipopolysaccharide] which is made in the inner membrane and somehow gets to the outer membrane, and we were trying to figure out the parts of that LPS pathway.”

Though some of the components of that pathway have previously been discovered, Ruiz said, it has been difficult to find the missing pieces because there are roughly 4,500 proteins encoded in the genes of the E. coli bacteria. Scientists currently do not know the function of about 40 percent of these proteins.

There were too many unknown proteins in the E. coli to test each one and find its role in outer-membrane formation, Ruiz said, so the research team instead decided to study a similar species of bacteria, Blochmannia floridanis, which evolved from the same ancestor as E. coli but lives only inside the cells of carpenter ants.

“Since E. coli live in the environment, and Blochmannia only live inside ants’ bodies, Blochmannia has deleted a lot of genes which it no longer needs to survive anymore,” Ruiz said. “Blochmannia only have 583 of the 4,500 E. coli genes.”

Blochmannia share all the core components of the E. coli bacteria, like the crucial outer membrane, but because the Blochmannia bacteria have so many fewer genes than the E. coli, they were much easier to study, Ruiz said.

This reductionist approach allowed the researchers to identify which of the 583 proteins were in the right location to be involved in LPS transport and which ones the bacteria need to survive, since the bacteria die without LPS. These elimination processes reduced the number of proteins possibly involved in LPS transport from 583 to two, Ruiz said.

The researchers then examined these two proteins in the E. coli bacteria.

“I made a mutant of the E. coli where I could tell them when to turn on the genes that code for these proteins by adding a particular sugar, and then I could say, ‘Make no more of this protein,’ and see what happened,” Ruiz said. When she stopped production of either of the two proteins, Ruiz found that the LPS could not get to the outer membrane and the bacteria died.

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These two proteins represent crucial missing components of the LPS transport pathway, said Luisa Gronenberg, a Harvard graduate student who helped verify that the new genes were actually involved in the LPS assembly pathway.

“Researchers in the field knew that we were missing important pieces of the machines that transport LPS from the inside of the cell to the outside,” Gronenberg said in an e-mail. “Now we have proteins at each step of the pathway from inside to outside, there are no more obvious missing pieces. The next step is to figure out what each protein does and how exactly they work together.”

Learning how the LPS assembly pathway works may also help scientists find methods for shutting down the pathway and develop new antibiotics, Gronenberg said. Ruiz also explained that LPS in the outer membrane of Gram-negative bacteria is a strong barrier against a lot of antibiotics currently on the market and also elicits very strong reactions from the immune system. People can even die of septic shock when their immune systems overreact to the LPS in Gram-negative bacteria, she said.

“Since [the LPS] pathway is essential for the survival of most Gram-negative bacterial species, being able to shut it down would give us a new method of killing bacteria,” Gronenberg said. “This is very important because as bacteria are becoming increasingly resistant to our old antibiotics we are running out of ways to kill Gram-negative bacteria.”

In the future, it might be possible to find drugs that completely inhibit the LPS pathway, killing Gram-negative bacteria, Ruiz said. Scientists could also develop drugs that would weaken the LPS pathway so that the outer membrane would not have the proper amount of LPS at all times; this would then allow current antibiotics to penetrate the crippled membrane.