Under Pressure: Complement-Sensitivity of Gram-Negative Bacteria

Abstract

Klebsiella pneumoniae is a bacterium commonly found in the environment but also on humans. It usually causes no harm to a healthy person. However, in people with a weak immune system Klebsiella can lead to severe disease and even death. These infections can be difficult to treat, as Klebsiella can become very resistant to antibiotics. Because these factors are often found in hospitals, where people have weakened immune systems and are generally more vulnerable, this bacterium is also known as a hospital bug. In order to prevent infections with Klebsiella via vaccinations or help the body to fight an ongoing infection, antibodies are considered as a promising tool. These very specific proteins of the human immune system guide the human immune reaction. One of the first systems in the human immune system to react to these bound antibodies is the complement system. The complement system labels bacteria for immune cells to destroy them, but it can also directly kill some bacteria by forming a hole in the bacterial cell wall. However, bacteria such as Klebsiella can prevent the immune system from killing them. Those mechanisms are not always well understood. The goal of this PhD thesis was to study the different ways that bacteria have evolved to prevent being killed by the complement system. First, we looked at the surface of the bacterial cell wall. In Klebsiella, O-antigen side chains stick out from the surface and coat the whole bacterium. Bacteria can express a variety of different O-antigens. We noticed that if Klebsiella had a specific O-antigen on their surface, they would better survive the attack of the immune system. We then looked more into the details, and could show that the O1-antigen prevents the complement system to form a hole in the bacterial cell wall. Second, we looked at how the bacterium changes its surface when evolving to resist an antibiotic. We found that in Klebsiella, mutations in a specific regulatory gene were responsible for resistance colistin. At the same time, that very mutation caused a change in the bacterial surface composition. This change made binding of antibodies possible and allowed the activation of the complement system. The complement system was then able kill the bacteria via membrane attack complexes. Third, we looked more closely at the mechanism we found in the first paper. The MAC pore that is formed in bacterial cell walls is the result of a line of reactions that happen one after the other. These reactions are very tightly regulated in the human body, because they can otherwise lead to diseases. We found that O-antigen of Klebsiella would overactivate the complement at a critical step. This overactivation would disbalance the protein ratio needed to form a MAC. By interfering with MAC formation the bacteria could survive. We could also show that in this case, to our surprise, inhibiting complement – something you usually don’t want to do during an infection – would increase killing of Klebsiella. Lastly, we looked at how the equally dangerous bacterium Pseudomonas aeruginosa prevents being killed by the immune system. By looking at all the genes that Pseudomonas expresses when exposed to the complement system, we found a couple of genes to be expressed more often in bacteria that were more resistant. One set of genes was particularly interesting, because it was previously undescribed. Among other things, we found that one of these genes lead to the expression of a new undescribed protein. This protein prevented that Pseudomonas was being killed by the complement system even though complement formed a hole in the cell wall.