First person – Damián Lobato-Márquez and José Javier Conesa

Damián Lobato-Márquez

How would you explain the main findings of your paper in lay terms?

D.L.-M. & J.J.C.: The bacterial pathogen Shigella flexneri is one of the major causes of human diarrhoeal disease worldwide. S. flexneri infects the epithelial cells in our intestine, where bacteria highjacks a host protein called actin to propel itself and disseminate from cell-to-cell. To counteract S. flexneri invasion, epithelial cells employ cell-autonomous immune responses, including septins and anti-bacterial autophagy. Septins are host proteins that play key roles in cell homeostasis and can recognize and entrap actin-polymerizing S. flexneri into cage like structures. The entrapment of S. flexneri stop actin-based motility, and these bacteria are targeted for autophagy (an evolutionarily conserved mechanism that recycles host components and eliminates invading pathogens). In this research, we employed state-of-the-art correlative cryo-light and cryo-soft X-ray tomography to study the poorly understood connection between septin cages and autophagy. We discovered that septins interact with autophagic vesicles (in ways that are still under investigation), and entrapped bacteria get labeled with the signaling molecule ubiquitin to recruit autophagy. Ubiquitin can assemble into different forms, and we discovered that the specific lysine 63 (K63) linkage is the one involved in targeting entrapped S. flexneri to autophagy. Strikingly, we discovered that septins and K63-linked ubiquitin are present in separate microdomains (or patches) on the bacterial cell surface.

Were there any specific challenges associated with this project? If so, how did you overcome them?

D.L.-M.: The most challenging part of this work was the optimization of the cryo-light and cryo-SXT pipeline. This is a technique especially suited to visualizing membrane-based organelles (including autophagosomes). However, this technology can only be applied to biological samples in a handful of research facilities worldwide and the access to this infrastructure is highly restricted, especially when working with human pathogens. After a successful grant application, the ALBA synchrotron (Barcelona, Spain) granted me 5 days of beam time to perform all experiments. However, it was a challenge to optimize such a correlative pipeline and acquire all required data in just 5 days! We were lucky that, at the time, Javier Conesa (co-first author in this work) was working at ALBA. This provided us extra time to optimize the conditions required to develop the imaging pipeline. In addition, the combination of my infection biology and fluorescence microscopy skills with Javi's cryo-SXT expertise was key to successfully imaging Shigella–septin cages in situ.

J.J.C.: I completely agree with Damián; the optimization of the correlative pipeline was the most tricky part. Nevertheless, working with a pathogen that it is considered as a potential bioterrorism threat in Damián's home Institution was not easy, because we worked in close-to-native conditions; that implies, we had to inactivate the pathogen (chemically fixing it) while preserving the subcellular structure.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

We would say we have not one but two ‘eureka’ moments:

D.L.-M.: To me the surprise came during the study of the ubiquitin linkages decorating entrapped S. flexneri. We found that septins and K63-linked ubiquitin are present in different microdomains or patches on the bacterial cell surface.

J.J.C.: For me the ‘eureka’ moment was when we discovered that under cryo-SXT, septin cages are visualized as X-ray-dense structures (∼30 nm resolution), suggesting that these anti-bacterial structures are enriched in additional host cell components (including lipids).

These two discoveries opened new research avenues in the lab investigating the actual composition of septin cages and the septin-ubiquitin interplay.

Why did you choose Journal of Cell Science for your paper?

D.L.-M.: The Journal of Cell Science is a respected journal that publishes highly quality research. Some of the JCS manuscripts I read during completion of this project inspired the experimental approaches and discoveries reflected in our research work.

J.J.C.: Journal of Cell Science is a peer-reviewed journal with high-quality research that fits well with the line of our work.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

D.L.-M.: Ramón Díaz Orejas. He was Professor of Microbiology at CIB-CSIC (Madrid, Spain). Beyond supervising my PhD work, Ramón is a good friend who taught me that all scientific discoveries are interesting and important in their own way. This transformed the way I see other people's research and helped me connect with other scientists.

J.J.C.: Jose L. Carrascosa. He was Professor and Director of the Structure of Macromolecular Department at CNB-CSIC and my PhD supervisor. He was always helping to clarify my mind when I had to make a difficult decision.

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

D.L.-M.: Even though he never worked as a chemist, my father studied chemistry. This was probably my first connection to science. But what really motivates me to pursue a career in science is the curiosity of seeing what nobody else has seen.

An interesting moment of my career comes from my PhD. At the end of my BSc studies, I was offered a position to do my PhD at CNIC (Madrid, Spain). At the same time, my wife Laura had started her PhD work at the CIB-CSIC. She heard the lab next door was looking for a PhD candidate and put me in touch with it. I ended up doing my PhD at CIB-CSIC funded by a competitive fellowship that allowed me to meet scientists that shaped the breadth of my science. I will always be grateful to Laura.

J.J.C.: I think I am curious by nature. I always wanted to know how things work inside. I like machines, but what I like the most is biological systems. An interesting moment of my career was when I started my postdoc at ALBA synchrotron; being a biologist by training, I discovered a new world where very different disciplines where integrated to answer complex biological questions. I fell in love with multidisciplinary approaches, so I keep applying them to my research.

Who are your role models in science? Why?

D.L.-M.: Personally, I try to focus on the science rather than on who does it. That said, I find inspiring those group leaders (especially women), who have a family and a healthy work-life balance.

J.J.C.: I am not currently following anyone's model but I admire hard workers like the people awarded with the Nobel Prize of Chemistry in 2017, Jacques Dubochet, Joachim Frank and Richard Henderson.

What's next for you?

D.L.-M.: I was recently awarded a starting grant to initiate my independent research line as Principal Investigator at the CNB-CSIC (Madrid, Spain). Here, I will focus on the study of the fascinating lifestyle of emerging Rickettsia infections, obligate intracellular bacteria that cause life-threatening infections.

J.J.C.: I am currently working as permanent staff, so for the moment I will settle for some time in my new position and keep thinking in the next adventure.

Tell us something interesting about yourself that wouldn't be on your CV

D.L.-M.: I am a calisthenics aficionado [enthusiast].

J.J.C.: I love to spend my free time with the family and doing artistic projects.