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Symbiont-mediated defense of amoebae against Legionella pneumophila - molecular mechanisms and pathogen ecology

Periodic Reporting for period 1 - SymPathInfect (Symbiont-mediated defense of amoebae against Legionella pneumophila - molecular mechanisms and pathogen ecology)

Reporting period: 2015-10-01 to 2017-09-30

Bacterial pathogens are generally investigated in the context of diseases. Their environmental lifestyle and reservoirs are often neglected. This project aims to fill this gap by studying the interaction of free-living amoebae with their bacterial symbionts and the bacterial pathogen Legionella pneumophila.
Free-living amoebae are ubiquitous protists and important predators of bacteria. As such they have a strong impact on the structure of microbial communities. Several bacteria developed strategies to survive digestion by amoebae and are even able to multiply within amoebae. This includes bacterial symbionts such as Protochlamydia amoebophila and Simkania negevensis as well as opportunistic pathogens like Legionella pneumophila, the causative agent of an atypical pneumonia called Legionnaire’s disease.
Our goal is to shed light on the molecular and physiological interactions between amoebae, their symbionts and opportunistic human pathogens. This project contributes to a better understanding of the role of amoebae and other microorganisms in water-borne disease outbreaks.
The overall goal of our research is to shed light on molecular and physiological interactions during infection of symbiont-containing amoebae with L. pneumophila. Elucidating the mechanisms of a symbiont-mediated defense against a human pathogen will help to better understand the role of amoebae and other microbes in disease outbreaks in general and in particular the ecology of L. pneumophila. In addition we investigated the host-specific response of another environmental chlamydia, S. negevensis, that similar to L. pneumophila can live in amoebae as well as in humans to better understand the host-specific response of chlamydial symbionts and their potential to become opportunistic human pathogens themselves.
Co-infection experiments had shown that populations of the model amoeba Acanthamoeba castellanii Neff containing the symbiont P. amoebophila recovered sustainably from a L. pneumophila infection compared to control cultures without the symbiont. To test if the observed symbiont-mediated defense against L. pneumophila was a general phenomenon in amoebae-chlamydia symbioses, we infected freshly isolated amoebae from the environment with and without P. amoebophila with L. pneumophila. We monitored symbiont and pathogen distribution and abundance in the host cells qualitatively and quantitatively over time to document infection and inhibition processes. Our experiments showed that freshly isolated environmental amoebae with symbionts survive a legionellae infection while the same amoeba strains lacking symbionts eventually died. Continuing this path we conducted a transcriptome experiment at different time points during the co-infection experiments with L. pneumophila and the chlamydial symbiont in amoebae to reveal the mechanisms of interaction between the partners over the course of an infection. Analysing the data with respect to the response of L. pneumophila during infection of amoebae revealed on a global level that co-infection with the symbiont leads to a developmental arrest of L. pneumophila. This supports what we have seen on the phenotypic level, namely that the presence of symbiont P. amoebophila interrupts directly (or indirectly) L. pneumophila’s life cycle and highlights that symbiotic interactions between chlamydial symbionts and amoeba hosts impact the ecology and spread of the human pathogen L. pneumophila.
Using the established transcriptome analysis work flow we studied another environmental chlamydia, S. negevensis. We characterized the life cycle of S. negevensis in the amoeba A. castellanii Neff as well as in the human HeLa cell line. We quantified the cell numbers of S. negevensis at distinctive developmental stages during infection and observed differences in the life cycle of S. negevensis depending of the host. Our transcriptome experiments at distinctive developmental stages during S. negevensis infection in amoebae and the human HeLa cells confirmed these observations and revealed host-specific differences in gene-expression.

During my first year as a Marie Skłodowska-Curie fellowship recipient I received training on how to cultivate and infect amoebae with and without symbionts with legionellae in the lab from members of my host group in Vienna. In addition Lena Konig, a PhD candidate in my host group, introduced me to the computational analyses of transcriptome data.
Furthermore, the fellowship got me the opportunity to finish and publish two papers from previous projects. Currently a 3rd manuscript is under revision summarizing the results of the symbiont-mediated defense against L. pneumophila in amoebae. A 4th manuscript describing the host-specific response of S. negevensis is being prepared. Furthermore, the results of this project have been presented at the Fast Scientific Break for young researchers at the Pasteur Institute in Paris, France, in 2018 and at the Congrès SFM - Société Française de Microbiologie in Paris, France, in 2017.
I have also co-supervised a master student, Sara Escola, during her master thesis, and I participated at KinderUni 2017(1) and 2018(2), a summer school initiative of the University of Vienna to inform school kids about science, and the European Researchers’ night 2017(3) to explain and advertise science to members of the public.

Legionellae are opportunistic human pathogens infecting thousands of people annually in Europe according to the European Center for Disease Prevention and Control (ECDC(1)) and are thus an important public health issue. These bacteria are ubiquitous in many natural and man-made water systems, where they survive as free-living organisms in biofilms, or intracellularly within a variety of protozoa, such as amoebae. Indeed, amoebae are known to be a main route for legionellae spread and replication, and our research of pathogen ecology is therefore key to understand legionellae outbreaks and develop strategies to prevent such outbreaks.
Our research on the response of amoebae and their bacterial symbionts to legionellae infection provides a unique opportunity to investigate a novel aspect of legionellae spread with potential for its control in the environment and already led to a collaboration with Prof. Dr. Carmen Buchrieser’s research group at the Pasteur Institute in Paris, France, one of the world-leading labs on legionellae. This interaction is scientifically rewarding for the present project and additionally broadened my professional network to researchers working in the field of pathogenic bacteria and infectious diseases.
The research on S. negevensis contributed first insights into host-specific response of chlamydial symbionts and represents an important contribution to a better understanding of the potential of chlamydial symbionts to become opportunistic human pathogens themselves.

(1) ECDC SURVEILLANCE REPORT Legionnaires’ disease in Europe 2012
Infection cycle of L. pneumophila in amoebae