Molecular Basis of the Disease state

Current information at the cellular level of the disease state focuses on the effect of the bacterial pathogen on cell surface receptors and the downstream effects within the host; however, it seems that the field is lacking a lot of knowledge on many of these specific interactions. Additionally, most of the information obtained regarding the disease mechanism for R. rickettsii is based on experiments using a very similar bacteria R. conorii. Regardless, because of the major similarity between the two species, it is viable to consider the mechanism of infection of R. conorii as being generally identical to that of R. rickettsii. This statement is supported by the fact that OmpB is potentially conserved functionally across all rickettsia species due to the large degree of shared sequence identity and homology of the specific protein (Chan et al. 2009).

Due to the fact that Rocky Mountain spotted fever is characterized by the effects of R. rickettsii on endothelial cells, it is important to consider the mechanism of binding between the host cells and bacteria cells as it is what controls for the disease state. The most important surface protein for the mechanism of infection of R. rickettsii is OmpB, as it is what allows for the bacteria to bind and enter endothelial cells through the Ku70 receptor (Chan et al. 2009). This study indicates that although very little is known about the specific interactions of the complex, OmpB is sufficient to mediate invasion of host endothelial cells via attachment to Ku70 on the surface (Chan et al. 2009). OmpA also has a role in this process, but what it binds and how it helps to mediate invasion of the host cell is not clear. Additionally, a recent study shows that knockdown of OmpA does not have a significant effect on the virulence of R. rickettsii because OmpB is what is paramount (Noriea et al 2015).

ku heterodimer
Figure 1: Crystal structure of Ku heterodimer (PDB number: 1JEQ).

The landmark experiment that established Ku70 as the receptor for OmpB was carried out approximately ten years ago and was the first study that showed the receptor-ligand interaction allowing for the entry of rickettsial species into the cells (Martinez et al. 2005). Studies done by this group showed that upon the binding of OmpB to Ku70, the E3 ubiquitin ligase, Cbl, ubiquitinates Ku70 and the rickettsia present is phagocytosed as a result of a different signal-transduction pathway that uses Cdc42 (Martinez et al. 2005 and Chan et al 2009). However, the exact mechanism of how the structure of Ku70 facilitates the invasion of the rickettsial species using OmpB is still unclear. This is surprising due to the fact that the crystal structure of Ku70 has been uncovered; however, because OmpB is present on the outside of the bacteria along with numerous other unknown strucutres, it is very difficult to isolate a crystal strucutre of the protein by itself (Walker and Ismail 2008).After phagocytosis, R. rickettsii is shown to cause the lysis of the phagosome, which is then followed by the use of the Arp2/3 complex to polymerize actin monomers that function to push the bacteria through the cytoplasm and into the filopodia (Martinez and Cossart 2004). After this process, R. rickettsii are able to leave the cell and enter adjacent cells by pushing through the filopodia and into an adjacent endothelial cell to continue to replicate and infect more and more epithelial cells.

Screen Shot 2016-05-06 at 5.03.12 PM
Figure 2: Mechanism of interaction of host endothelial cell with R. Rickettsii (Walker and Ismail 2008).

The binding of OmpB to Ku70 produces downstream effects that can be used to explain some of the negative symptoms of Rocky Mountain spotted fever. In addition to the fact that endothelial cells are disrupted upon invasion of the bacteria, the expression of two major enzymes involved in inflammatory response are affected. One of these enzymes, p38 MAP kinase, is activated by the binding of OmpB to Ku70 and causes for an increased inflammatory response in the body (Rydkina et al. 2005). The next enzyme, cyclooxygenase-2, is activated at the transcriptional level by signaling of p38 MAP kinase, which in turn causes for the release of prostaglandins (Rydkina et al 2009). The activation and stimulation of these enzymes via infection serve to alter the inflammatory response in infected humans to contribute to the symptoms of the disease. Pro-inflammatory response is also induced by infection of the bacteria as it causes increased expression of cytokines and chemokines (Rydkina et al. 2005). The increased production of chemokines is intriguing as to whether it benefits or hinders the disease due to the fact that they stimulate T cell movement to the site of infection (Walker and Ismail 2008). Additionally, R. rickettsii plays a role in altering cell apoptosis, which allows for the bacteria to manipulate cell death in order to be able to survive for longer periods of time in infected cells (Bechelli et al. 2009). While there is still a lot that is unclear regarding the infection with this bacteria, studies are continuously done to uncover more about the mechanisms of R. rickettsii as they pertain to Rocky Mountain spotted fever.

6 Replies to “Molecular Basis of the Disease state”

  1. Tyler, you did such a great job explaining this disease in a clear and concise way. I can tell that it must have been hard to research this since there is still a lot we do not know about Rocky Mountain Spotted Fever. Something that I am a little confused about on this page is the role of OmpA in mediating invasion of host endothelial cells via attachment to Ku70. You say that it does indeed have a role but the mechanism of this role is not yet well understood. However, right after you explain that a recent experiment showed that knockdowns of OmpA does not have a significant effect on the virulence of R. rickettsii, which indicates OmpA does not really have role in this process. I felt these two claims were a bit contradictory. It is possible that two separate studies found different results, because authors tend to indirectly talk to each other and argue through their research. Do you happen to have an idea of what the overall attitude of OmpA’s role is in the literature?

    1. Hey Steph thanks for the great questions! In terms of the role of OmpA, it is shown to bind to a receptor on the outside of endothelial cells during the process of adhesion and eventual entry into the cell; however, although it is involved in adhesion, its function is not critical to allow entry. This essentially means that although OmpA is shown to bind, deleting it would still result in entry into the cell due to the binding of OmpB to Ku70 being the critical complex (Noirea et al. 2015). I hope this clears up any of the confusion you may have. The paper I cited in my response should give you any additional information you need regarding the role of OmpA if you are curious for further research!

  2. Hi Tyler. Nicely written description on rickettsii infection and what is know about its entry into the cell. I know you mentioned that there is little known about how OmpB induces endocytosis of the Ku70/OmpB complex, but I was wondering if anything was known about requirements for endocytosis of the receptor. For example, is the protein phosphorylated on the cytosolic side and this serves a s a signal for the ubiquitin ligase to bind after Cdc42 signaling? Does the ubiquitin ligase Cbl bind directly or is it mediate through some adaptor protein that is recruited due to a conformational change after OmpB binding? Is anything known abut the oligomerization of the receptor and any roles that may play in internalization? (No need to answer all of these questions, and many of them might be unknown, was just curious if you had any hypothesis on how you think OmpB is triggering endocytosis or what the downstream effect is on the receptor that triggers endocytosis).

    1. Hey Christopher thank you for all the questions! While this area of the bacterial entry into the cell seems to be difficult to understand in the literature, it seems that the process of OmpB binding and triggering endocytosis is dependent on a cascade of downstream effects. The studies done by Chan et al. 2009 provide the most insight into the exact events at the molecular level. It seems that the exact process here depends on the binding complex shown in the first block Figure 2 on this page . How exactly these signaling events result in the internalization of the bacteria seems to be difficult to determine due to the amount of things involved .However, I have seen information explaining that Cbl is recruited to the site of entry by the OmpB binding complex and it interacts directly with the receptor. Additionally, I have not come across anything specifically that gives any sort of hypothesis of exactly how these events cause for the bacteria to enter the cell, which means that a lot of research needs to be done. I am very sorry that I am unable to answer some of your questions with specific studies in the literature, but it just seems that not a lot is known about the exact process that you are curious about.

  3. Hello Tyler!
    I really enjoyed reading your blog on Rocky Mountain Spotted Fever. The picture of the arm with the rash on the title page appears freaky and almost like chicken pox spots. I enjoyed your use of hyperlinks to key articles and thought they were helpful.
    You noted how the bacteria binds to OmpB which results in downstream affects with Ku70 and endothelial cell. With the crystal structure for OmpB and Ku70, are there predictions or structural models which may explain this observation? Also, if such model where to exist, do you feel developing inhibitors to target this interaction or target the active site of Ku70 would be more effective?
    Great work, Tyler! I think you did a through job with your research and summary. That Figure you created (Figure 2 on the title page) is very well done!
    Tyler Florio

    1. Hey Tyler thank you for all the very nice words about my project, as well as some very insightful questions. While the crystal structure for Ku70 is very clear, it is difficult to make predictions regarding OmpB due to the fact that crystal structures of the it are normally shown in complex with other proteins on the outer membrane. If a structural model was to exist, I think it would be extremely beneficial to develop inhibitors of this reaction by somehow inactivating OmpB. I think it would be difficult to target the active site of Ku70 due to the fact that it plays a large role in a number of nuclear functions as described in the literature by Martinez et al. 2005. However, if more information is uncovered regarding the structure of OmpB, I think that an inhibitor binding to it could be very beneficial to diminishing the virulence of R. rickettsii so that it is unable to bind to Ku70.

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