Scientists identify bacterial protein that inhibits inflammatory response

AGÊNCIA FAPESP/DICYT An international group of scientists led by Dario Zamboni, a professor at the University of São Paulo’s Ribeirão Preto School of Medicine (FMRP-USP) in Brazil, have discovered the strategy used by Coxiella burnetii, a potentially dangerous bacterium, to trick the immune system and inhibit the inflammatory process triggered by defense cells when they come into contact with pathogens of this kind.

This discovery represents a significant step forward in combating C. burnetii, considered highly pathogenic, as well as in paving the way for new treatments against sepsis, a type of systemic inflammation that is the main cause of death in intensive care units (ICUs) and a major cause of late hospital mortality in Brazil.

The group performed experiments as part of a Thematic Project supported by FAPESP and coordinated by Zamboni. The results have recently been published in an article in the journal Nature Communications.

“We’re particularly interested in C. burnetii because it’s a very subversive bacterium,” Zamboni said. “It inhibits several stages of immune system activation, and this makes it highly virulent. A single bacterium is thought to be capable of causing infection and sickness in a healthy individual.”

Usually found in secretions (vaginal mucus, milk, feces, urine and semen) produced by cattle, goats, pigs and other livestock, C. burnetii infects humans through inhalation, causing an atypical pneumonia known as Q fever. In some cases, infection can damage the liver or heart sufficiently enough to cause endocarditis (inflammation of the heart’s inner lining) and alterations in the cardiac valves.

“We already knew of this bacterium’s ability to inhibit the macrophages it infects, so the cells that should phagocytize it aren’t activated and the inflammatory response can’t begin. Our aim was to find out how, by discovering the molecular mechanisms involved,” Zamboni said.

Macrophages are normally the first line of defense cells to go into action when a pathogen infects an organism and are a key element of the immune response. In macrophages, caspases are a group of proteins that agglomerate to form the inflammasome, a complex of immune system receptors and sensors, including neutrophils as well as other phagocytes and defense cells, triggering a protective inflammatory process outside the infected cells.

In the case of Gram-negative bacteria such as C. burnetii, inflammation is triggered when cell receptors recognize bacterial lipopolysaccharide (LPS), and this in turn activates a type of inflammasome mediated by caspase-11, the protein studied by Zamboni’s group.

Other major human pathogens that are also Gram-negative bacteria include Escherichia coli, Shigella, Salmonella, Pseudomonas and Legionella pneumophila. All of these microorganisms contain LPS and activate the caspase-11-mediated inflammasome.

In vitro assays

Through laboratory experiments and in vitro assays with macrophages from mice, the scientists tested two hypotheses: the first was that LPS from C. burnetii was not capable of inflammasome activation, and the second was that the bacterium must secrete some other type of inflammation-inhibiting substance in the presence of inflammasome activation.

“To find out which was right, we used a co-infection approach,” Zamboni said. “We infected a cell with C. burnetii, and one day later we infected the same cell with L. pneumophila, a bacterium that’s known to induce the caspase-11-mediated inflammasome.”

The group observed that even with L. pneumophila inside the cell, the inflammasome was not activated, proving that C. burnetii was actively inhibiting inflammation via the secretion of some substance. What that substance was remained to be determined.

“We knew C. burnetii secreted tens of proteins inside the cell, so we expressed several mutants of L. pneumophila, each of which expressed a different protein from C. burnetii,” Zamboni said.

When they infected murine macrophages with the mutants of L. pneumophila, the scientists found that the bacterium that expressed the gene annotated as CBU1823, later labeled IcaA (for inhibition of caspase activation), was the only one capable of inhibiting the formation of the caspase-11-mediated inflammasome.

According to Zamboni, the discovery contributes to a better understanding of why C. burnetii is so virulent. “This microorganism uses several proteins to evade the immune system. We show that IcaA is one of them,” he said.

This discovery also opens up the possibility of using molecules with a similar action to IcaA to inhibit caspase-11, he added, helping to combat inflammatory diseases such as sepsis. Many cases of sepsis, especially those caused by Gram-negative bacteria, are associated with the activation of caspase-11.