Francisella tularensis: How The Dangerous ‘Rabbit Fever’ Bacterium Evades Your Immune System

By: Laura Ross

For decades, the bacterium Francisella tularensis has been considered remarkably dangerous. It is one of only 5 bacteria classified as a Tier-1 Select Agent by the US government. These are bio-agents that are believed to “pose a severe threat to public health and safety.” This is in part because the bacterium has a startlingly low infectious dose, which is the number of bacteria that must enter the host in order for an infection to occur. Only 5 to 50 organisms are required for a full-blown F. tularensis infection; the resulting disease is called tularemia. In contrast, Bacillus anthracis, the causative agent of anthrax and a fellow Tier-1 Select Agent, has an infectious dose of 10,000 spores; Vibrio cholerae, which causes tens of thousands of deaths every year, requires between 10,000 and 1,000,000 organisms to establish an infection. F. tularensis can be grown in large quantities in the laboratory, and it is easily aerosolized. For this reason, scientists have long recognized the potential of F. tularensis as a powerful biological weapon. If F. tularensis was deployed as a biological weapon, we would expect to see many people suddenly develop an acute, febrile illness about 3-5 days after exposure to the bacteria. The complications of tularemia can be severe, and, if left untreated, the mortality rate of an F. tularensis infection can be as high as 30-60%. In the case of a biological attack, rapid medical response would be required to prevent deaths among the exposed. The United States Center for Disease Control has prepared for a scenario like this by stockpiling antibiotics that can be used to treat tularemia.

This picture, taken using scanning electron microscopy, shows a mouse immune cell infected with F. tularensis.

F. tularensis infects a wide range of animals, and humans generally become infected through contact with an infected animal. Ticks, mosquitoes, and deer flies can all transmit tularemia to human hosts; infections can also be caused by exposure to the carcasses of infected animals. Hunters can develop tularemia after skinning infected rabbits, which is why tularemia is sometimes called “Rabbit Fever.” Luckily, tularemia infections are rare; while there was a spike in tularemia infections in 2015, scientists are not currently concerned about a natural tularemia outbreak. Additionally, tularemia cannot be passed directly from person to person and, with proper diagnosis, it responds well to antibiotic treatment. However, tularemia remains incredibly dangerous because it can be spread quickly and easily and is often difficult to accurately diagnose. Researchers believe that the aerosol release of 50kg of F. tularensis over a large city would lead to as many as 250,000 infections, which would likely include around 19,000 deaths.

So, how does this unique bacteria evade your immune system and cause such a dangerous infection? In 2016, researchers Maj Brodmann, Roland F. Dreier, Petr Broz, and Marek Basler set out to investigate this question in their paper “Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape.” To understand the intriguing findings of this paper, it is important to understand how F. tularensis interacts with the host organism that it infects.

When F. tularensis cells enter a host organism, the bacteria are targeted by macrophages, a type of white blood cell that engulfs and digests foreign particles in a process called phagocytosis. When the macrophage engulfs an F. tularensis cell, it isolates the pathogen in a vesicle called a phagosome. In order for the invading bacterium to be digested, the phagosome must fuse with a lysozyme, an antimicrobial enzyme that is a fundamental part of the host’s immune system. In order to survive, it is essential for the bacterium to avoid digestion at all costs. To survive, the bacterium must escape the phagosome before it fuses with the lysozyme. Once the bacterium escapes, it can replicate within the cytosol of the macrophage until it eventually causes the cell to lyse, or explode, allowing the bacteria to spread throughout the host. Escape from the phagosome is crucial for F. tularensis to establish an infection. This escape process is tricky, and requires unique, specialized molecular machinery.  

It turns out that a specialized nanomachine called a Type VI Secretion System (T6SS) plays a key role in phagosomal escape of F. tularensis.  An excellent, detailed breakdown of the canonical, traditional T6SS can be found here. The following figure, excerpted from Basler (2015), shows the basic assembly, structure, and mechanics of a typical T6SS, using V. cholerae as a model.           

The T6SS has four main components: the membrane complex, the baseplate complex, the Hcp tube, and the contractile sheath. The sheath in particular is key to the function of the T6SS. It assembles around the Hcp tube and then rapidly contracts, allowing the spike at the end of the T6SS to pierce through the membrane of the target structure and inject special proteins called effector proteins. In the case of F. tularensis, these effector proteins help the bacterium to escape the phagosome. After this process is finished, the sheath is recycled; it is repeatedly assembled and disassembled in a cycle. To see a great claymation depiction of a T6SS secretion system in action, check out this video made by Robin VanHouten at the University of Puget Sound, which shows how V. cholerae uses a T6SS to deliver toxins to other cells.     

However, the T6SS of F. tularensis appears to have some significant differences from the canonical T6SS previously studied in bacteria like V. cholerae. While it has the same basic structure and mechanism, several key proteins involved in the assembly of the canonical T6SS have no known equivalent in F. tularensis. In particular, F. tularensis seems to be missing an equivalent of ClpV, an unfoldase that is a crucial part of the canonical T6SS. An unfoldase is an enzyme involved in unfolding proteins; ClpV, the T6SS unfoldase in bacteria like V. cholerae, plays an important role in disassembling and recycling the sheath. Additionally, F. tularensis possesses many genes with unknown functions. In this paper, Brodmann et al. (2016) explored the role of these unknown genes, and determined which gene was involved in the recycling and disassembly of the sheath. The scientists carried out their research using Francisella novicida, a close relative of F. tularensis. F. novicida is often used in place of F. tularensis for laboratory experiments because it is rarely pathogenic in humans, but highly pathogenic in mice. It is so closely related to F. tularensis that findings about its T6SS can be used to understand the T6SS of F. tularensis.

First, the researchers tested the role of the protein ClpB. Previous research had suggested that ClpB was involved in heat shock survival and immune response. To find out if ClpB also played a role in sheath disassembly, Brodmann et al. (2016) created mutants without the clpB gene. This allowed them to determine how the bacteria behaved without the ClpB protein. Time-lapse imaging showed that bacteria without ClpB never disassembled their sheaths. Brodmann et al. (2016) went on to repeat this experimental design for a whole host of other F. novicida genes and proteins. Then, in the laboratory, researchers infected macrophages with the bacteria with these mutant bacteria and found that, in several cases, the bacteria struggled to escape the phagosome.

The results of this experiment are shown in the figure below. In this graph, wt represents unaltered, wild-type bacteria; ΔpdpB is a mutant bacteria with a fundamental structural component called PdpB removed; and ΔclpB is the mutant without ClpB. Panel D shows the number of bacteria that were able to escape the phagosome into the cytosol. It is clear that, in comparison to the wild-type, only a tiny fraction of bacteria without clpB were able to escape into the cytosol, and none of the bacteria without pdpB could escape. The two graphs in Panel E demonstrate bacterial virulence by mapping the amount of cell death triggered and the number of cytokines released. Again, bacteria without clpB cause only a tiny fraction of the immune response elicited by wild-type F. novicida, and bacteria without pdpB show no immune response at all.

These data support the idea that ClpB is an important player in the sheath disassembly process. This was an interesting finding, as it means that ClpB has a dual role: it both helps disassemble the sheath and helps the bacterium survive heat shock. This is in stark contrast to the ClpV unfoldase of the canonical T6SS, which has no second function. The data also confirmed the researchers’ hypothesis that PdpB is a fundamental structural component of the T6SS. These mutant experiments also allowed the researchers to identify PdpC and PdpD as effector proteins secreted by the T6SS. These two proteins are crucial for Francisella to escape the phagosome, and researchers confirmed that F. tularensis and its close relative F. holoarctica cannot escape the phagosome without pdpC. In addition, the researchers identified several other key structural proteins for T6SS assembly; these findings were confirmed with live cell imaging.

At first, it might seem that this paper is focused on small, insignificant details about F. tularensis. However, understanding the mechanics of the F. tularensis T6SS is incredibly important for understanding its virulence and developing new treatment techniques. In the case of a mass outbreak or a bioterrorist attack, this data could be useful in creating therapies that specifically target the very system that allows F. tularensis to be so effective at infecting its hosts. Additionally, this study demonstrates that there are multiple ways to run a Type VI Secretion System, and the data collected by Brodmann et al. (2016) could help future researchers understand the ways these secretory systems function in other bacteria. There are still many unanswered questions about the F. tularensis T6SS; in particular, future research may help us understand the specific roles of the effector proteins, and give us a better picture of how the F. tularensis T6SS relates to T6SSs in other bacteria. This paper represents an important contribution to a dynamic field of research, and the authors have brought us one step closer to fully understanding this dangerous pathogen.

About the Author:


Laura Ross '18
Laura Ross is a senior at Mount Holyoke studying astronomy with a concentration in astrobiology. In her free time, she enjoys playing guitar, bass, and drums. In the future, she hopes to become a certified nurse-midwife.