Live Legionella pneumophila induces MUC5AC production by airway epithelial cells independently of intracellular invasion

Yoshitomo Morinaga,a Katsunori Yanagihara,a Nobuko Araki,a Yohei Migiyama,a,b Kentaro Nagaoka,a,b Yosuke Harada,a,b Koichi Yamada,a,b Hiroo Hasegawa,a Tomoya Nishino,b Koichi Izumikawa,b Hiroshi Kakeya,b Yoshihiro Yamamoto,b Shigeru Kohno,b,c Shimeru Kamihiraa aDepartment of Laboratory Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1, Sakamoto, Nagasaki, 852-8501, Japan.

bSecond Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

cGlobal COE Program, Nagasaki University, Nagasaki, Japan.

Published on the web 20 January 2012.

Canadian Journal of Microbiology, 2012, 58:(2) 151-157, 10.1139/w11-123

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Purdue scientists reveal how bacteria build homes inside healthy cells

Public release date: 20-Dec-2011
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Contact: Elizabeth K. Gardner
ekgardner@purdue.edu
765-494-2081
Purdue University

WEST LAFAYETTE, Ind. - Bacteria are able to build camouflaged homes for themselves inside healthy cells - and cause disease - by manipulating a natural cellular process.

Purdue University biologists led a team that revealed how a pair of proteins from the bacteria Legionella pneumophila, which causes Legionnaires disease, alters a host protein in order to divert raw materials within the cell for use in building and disguising a large structure that houses the bacteria as it replicates.

Zhao-Qing Luo, the associate professor of biological sciences who headed the study, said the modification of the host protein creates a dam, blocking proteins that would be used as bricks in cellular construction from reaching their destination. The protein "bricks" are then diverted and incorporated into a bacterial structure called a vacuole that houses bacteria as it replicates within the cell. Because the vacuole contains materials natural to the cell, it goes unrecognized as a foreign structure.

"The bacterial proteins use the cellular membrane proteins to build their house, which is sort of like a balloon," Luo said. "It needs to stretch and grow bigger as more bacterial replication occurs. The membrane material helps the vacuole be more rubbery and stretchy, and it also camouflages the structure. The bacteria is stealing material from the cell to build their own house and then disguising it so it blends in with the neighborhood."

The method by which the bacteria achieve this theft is what was most surprising to Luo.

The bacterial proteins, named AnkX and Lem3, modify the host protein through a biochemical process called phosphorylcholination that is used by healthy cells to regulate immune response. Phosphorylcholination is known to happen in many organisms and involves adding a small chemical group, called the phosphorylcholine moiety, to a target molecule, he said.

The team discovered that AnkX adds the phosphorylcholine moiety to a host protein involved in moving proteins from the cell's endoplasmic reticulum to their cellular destinations. The modification effectively shuts down this process and creates a dam that blocks the proteins from reaching their destination.

The bacterial protein Lem3 is positioned outside the vacuole and reverses the modification of the host protein to ensure that the protein "bricks" are free to be used in creation of the bacterial structure.

This study was the first to identify proteins that directly add and remove the phosphorylcholine moiety, Luo said.

"We were surprised to find that the bacterial proteins use the phosphorylcholination process and to discover that this process is reversible," he said. "This is evidence of a new way signals are relayed within cells, and we are eager to investigate it."

The team also found that the phosphorylcholination reaction is carried out at a specific site on the protein called the Fic domain. Previous studies had shown this site induced a different reaction called AMPylation.

It is rare for a domain to catalyze more than one reaction, and it was thought this site's only responsibility was to transfer the chemical group necessary for AMPylation, Luo said.

"Revealing that this domain has dual roles is very important to identify or screen for compounds to inhibit its activity and fight disease," he said. "This domain has a much broader involvement in biochemical reactions than we thought and may be a promising target for effective treatments."

During infection bacteria deliver hundreds of proteins into healthy cells that alter cellular processes to turn the hostile environment into one hospitable to bacterial replication, but the specific roles of only about 20 proteins are known, Luo said.

"In order to pinpoint proteins that would be good targets for new antibiotics, we need to determine their roles and importance to the success of infection," he said. "We need to understand at the biochemical level exactly what these proteins do and how they take over natural cellular processes. Then we can work on finding ways to block these activities, stop the infection and save lives."

A paper detailing their National Institutes of Health-funded work is published in the current issue of the Proceedings of National Academy of Sciences. In addition to Luo, Purdue graduate student Yunhao Tan and Randy Ronald of Indiana University co-authored the paper. Luo next plans to use the bacterial proteins as a tool to learn more about the complex cellular processes controlled by phosphorylcholination and to determine the biochemical processes role in cell signaling.

Writer: Elizabeth K. Gardner, 765-494-2081, ekgardner@purdue.edu Source: Zhao-Wing Luo, 765-496-6697, luoz@purdue.edu

Related website:
Luo lab: http://bilbo.bio.purdue.edu/luolab/

Related news release:
Purdue biologists identify new strategy used by bacteria during infection: http://www.purdue.edu/newsroom/research/2011/110712LuoNature.html

PHOTO CAPTION:
Purdue associate professor of biological sciences Zhao-Qing Luo, at right, and graduate student Yunhao Tan look at the growth of Legionella pneumophila bacteria in a petri dish. (Purdue University photo provided by Laurie Iten and Rodney McPhail)

A publication-quality photo is available at http://news.uns.purdue.edu/images/2011/luo-legionella.jpg

Abstract on the research in this release can be found at: http://www.purdue.edu/newsroom/research/2011/111220LuoPNAS.html

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Biologists discover new way in which bacteria hijack healthy cells during infection

Purdue University biologists identified a new way in which bacteria hijack healthy cells during infection, which could provide a target for new antibiotics.

Zhao-Qing Luo, the associate professor of biological sciences who led the study, said the team discovered a new enzyme used by the bacterium Legionella pneumophila - which causes Legionnaires' disease - to control its host cell in order to take up residence.

"Legionnaires' disease is a severe form of pneumonia, and this finding could lead to the design of a new therapy that saves lives," Luo said. "At the same time it also provides great insight into a general mechanism of both bacterial infection and cell signaling events in higher organisms including humans."

Successful infection by Legionella pneumophila requires the delivery of hundreds of proteins into the host cells that alter various functions to turn the naturally hostile environment into one tailor-made for bacterial replication. These proteins tap into existing communication processes within the cells in which an external signal, such as a hormone, triggers a cascade of slight modifications to proteins that eventually turns on a gene that changes the cell's behavior, he said.

"Pathogens are successful because they know how information in our cells is relayed and they amplify some signals and block others in order to evade the immune system and keep the cell from defending itself," Luo said. "Despite our understanding of this, we do not know much about how the proteins delivered by the bacteria accomplish this - how they work. This time we were able to pinpoint an enzyme and see how it disrupted and manipulated a specific signaling pathway in order to create a better environment for itself."

The signaling pathway involved was only recently identified, and the discovery by Luo and graduate student Yunhao Tan also provides a key insight into its process. A paper detailing their National Institutes of Health-funded work is published online in the current issue of the journal Nature.

The signaling pathway involves a new form of protein modification called AMPylation in order to relay instructions to change cell behavior and has been found to be used by almost all organisms, Luo said.

The bacterial enzyme discovered by the Purdue team, named SidD, reverses or stops the AMPylation process, he said.

"It had not been known before if the AMPylation signaling process was reversible or if it was regulated by specific enzymes," Luo said. "Now we know that it is, and we have a more complete picture that will allow us to use it as a scientific tool to learn more about complex cellular processes. By being able to turn the signaling on and off, we can control different activities and detect mechanisms we wouldn't see under normal physiological conditions."

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