"PhD Defense: Nanomechanics of pathogenic attachment: Uropathogenic Escherichia coli and Human Immunodeficiency Virus"
Who: Alvaro Alonso, Nanobiomechanics Group
Place: CFM Auditorium
Date: Friday, 15 December 2017, 11:00
The role of mechanical force in physiology is becoming increasingly recognized as an important factor in the human health. Mechanical force affects the function, the organization and the remodeling of organs and tissues in the human body, which are processes triggered by events occurring at the single-molecule level. In the same way mechanical forces are actively present during disease, and their involvement is calling the attention of the scientific and medical communities. Among diseases, the pathogenic infections are not exempt of mechanical forces, especially during the pathogen attachment to human tissues where surface proteins from both the pathogen and the host interact with each other. Currently, the traditional treatments against infections are facing the challenge of the bacterial antibiotic resistance and the lack of a cure for some pathogenic diseases of worldwide spread. In this sense, the pharmacological targeting of the mechanical attachment of the pathogen at the single-molecule level is seen to be a new approach to fight infections. This new point of view enables the design and discovery of new drugs, and the new fields of Mechanomedicine and Mechanopharmacology provide a suitable framework for the development of new treatments against pathogenic attachment. Although the last years have witnessed an increasing knowledge regarding single-molecule mechanics, little is known about the mechanical features of the proteins used by pathogens for attachment. Hence, basic research is needed to unravel the mechanics of these molecules and to provide a basis from where a pharmacological treatment can be developed.
With this goal, my thesis has focused on the study of the nanomechanics of cell-surface proteins involved in viral and bacterial adhesion. Specifically I have studied the proteins used by uropathogenic Escherichia coli to attach to the urinary tract, and the human protein receptor CD4 used by Human Immunodeficiency Virus (HIV) for recognizing and infecting immune cells.
We hypothesized that bacterial proteins display a high mechanical resistance based on their adhesion function in the urinary tract. For the CD4 protein it was hypothesized that its mechanical extension is related with the Human Immunodeficiency Virus infection since this process requires the spatial rearrangement of many elements for bringing the virus closer to the cell membrane. Many disciplines and techniques are involved in this thesis but the most important technique applied for the direct manipulation of single protein molecules is single-molecule force spectroscopy.
In the first part of this thesis the mechanical resistance of the proteins that build the attachment organelle of uropathogenic Escherichia coli was studied. This extracellular structure is called type I pilus, a filamentous appendage composed of hundreds of proteins used during urinary tract infection. We found that pilus proteins show not only a remarkable resistance to mechanical unfolding but also that they follow an unfolding hierarchy pattern tightly connected to their position in the pilus structure. Our results reveal that conserved disulfide bonds in each of the pilus proteins are a crucial feature for their mechanical stability. Besides unraveling its nanomechanical architecture, the biogenesis of the pilus was studied wherein we recreated in vitro the same events that take place during the maturation and folding of the proteins previous to their incorporation into the pilus. During our investigation we found that the enzyme DsbA involved in disulfide bond creation during the first step of pilus proteins maturation shows an additional pronounced chaperone effect, which helps to fold to pilus proteins in a greater extent than the putative real chaperone of this system. With these findings we provide for the first time a detailed description at the single-molecule level of the type I pilus mechanical architecture and biogenesis.
In the second part of this work the nanomechanics of the lymphocyte T cell-surface protein CD4 were studied. The CD4 protein is used by the HIV particle to attach to these cells. The subsequent events for viral infection require the approaching of the particle to the cell membrane to interact with other proteins meanwhile attached to CD4. We have discovered that the first two domains of CD4, D1D2, are able to unfold at low forces. We suggest that this is the range of forces that a viral particle could produce due to thermal motion. In this scenario CD4 would act as a shock absorber in which its domains can unfold at low forces by the virus, therefore increasing the length of the tether and allowing the virus to explore the cell membrane looking for the other proteins required for viral fusion. Also the regulation by oxidoreductase enzymes of the disulfide bonds of CD4 during HIV infection is important for the infectivity of the virus. We demonstrated that only the mechanical unfolding of CD4D1D2 makes possible the access of the oxidoreductase enzyme to the buried disulfide bonds, supporting the idea that the viral particle could unfold CD4 in vivo. A mathematical model was developed based on the infectivity of cells expressing engineered variants of CD4 showing different tether lengths. The infectivity increases with the length of the tether, and the model allows interpreting that the in vivo infection would require the extension of CD4. Finally the effect of Ibalizumab, an antibody known to be a good neutralizer of HIV infection was tested. This antibody?s epitope is placed between CD4D1 and CD4D2 domains and the exact mechanism for its potent neutralizing effect is unknown. We have found that the mechanical stability of CD4D1D2 is increased in the presence of Ibalizumab, suggesting that the binding of the antibody hinders the extension of CD4 by the virus and this avoids the subsequent events of the infection. This supports again that the mechanical extension of CD4 is mandatory for successful virus infection. Altogether our results suggest that CD4 mechanochemical extension may play a critical role during HIV infection.
In the light of these findings, we think that our results could help to develop new therapeutic strategies oriented to alter the nanomechanics of proteins involved in pathogenic attachment, in the context of the new fields of Mechanopharmacology and Mechanomedicine. In the case of bacteria, the use of molecules which disrupt the strength of the pilus proteins would circumvent the current limitations of traditional antibiotic treatments. For HIV, we suggest that an increased mechanical stabilization of CD4 receptor induced by antibody binding could prevent the infection or diminish its chances of success.
Supervisor: R. Perez-Jimenez