Periodic Reporting for period 3 - NCI-CAD (Neutrophil - Chlamydia interactions at the crossroad of adaptation and defence)
Okres sprawozdawczy: 2022-10-01 do 2024-03-31
Polymorphonuclear neutrophils (PMNs) are a major subset of innate immune cells that are part of the first line immune defence against bacterial infections. Chlamydia trachomatis, a major human pathogen, manipulate PMNs during the infection process and subvert them to become unresponsive to a broad range of stimuli, including Chlamydia themselves. Even more surprising, although PMNs are extremely short lived, Chlamydia not only survive PMN exposure but can also surprisingly exploit PMN itself as host cell for replication. It remains completely unclear how PMNs are converted to host cells for obligate intracellular bacteria.
Incidences of sexually transmitted diseases (STI) have increased during the past decades with a concomitant rapid spread of antibiotic resistant bacteria. Chlamydia trachomatis is the most frequent cause of bacterial STIs with more than 130 millions per year. These infections often remain asymptomatic and are consequently not diagnosed and treated, resulting in the subsequent development of severe chronic pathologies and an enormous economic burden for health systems. The reason for the asymptomatic nature of chlamydial infection is currently unknown but may well be connected to the subversion of PMNs.
We identified a chlamydial secreted protease (CPAF) to be the bacterial effector responsible for preventing the activation of the non-stimulated PMNs. In addition, the chlamydial secreted deubiquitinase Cdu1 is required for intracellular adaptation of Chlamydia, indicating that PMNs may posses antibacterial cell-autonomous defence strategies based on the host ubiquitin system. The objective of the current project is the detailed investigation of how Chlamydia subvert PMNs in the course of infection.
Incidences of sexually transmitted diseases (STI) have increased during the past decades with a concomitant rapid spread of antibiotic resistant bacteria. Chlamydia trachomatis is the most frequent cause of bacterial STIs with more than 130 millions per year. These infections often remain asymptomatic and are consequently not diagnosed and treated, resulting in the subsequent development of severe chronic pathologies and an enormous economic burden for health systems. The reason for the asymptomatic nature of chlamydial infection is currently unknown but may well be connected to the subversion of PMNs.
We identified a chlamydial secreted protease (CPAF) to be the bacterial effector responsible for preventing the activation of the non-stimulated PMNs. In addition, the chlamydial secreted deubiquitinase Cdu1 is required for intracellular adaptation of Chlamydia, indicating that PMNs may posses antibacterial cell-autonomous defence strategies based on the host ubiquitin system. The objective of the current project is the detailed investigation of how Chlamydia subvert PMNs in the course of infection.
We have purified the CPAF protease, the relevant effector of Chlamydia to identify the targets cleaved on the PMN surface. In addition, we have successfully established the infection of mouse ER-Hoxb8 neutrophils to use them as a test system for gene knock out in neutrophils.
We have intensively studied the effect of C. trachomatis infection on the PMN biology. Infection extents the survival of human PMNs in vitro by activating a network of signalling molecules. These included molecules of apoptosis control (Mcl-1) and metabolic reprogramming (c-Myc) and a signalling pathway thus far not connected to neutrophil survival. An intriguing result was the observation that the average lifespan of PMNs could be extended four- to fivefold by manipulating these pathways and viable Chlamydia could be recovered even after such long infection periods.
The metabolic reprogramming of PMNs by C. trachomatis infection has been studied monitoring the gene expression of the bacteria and the PMNs simultaneously by dual-RNA seq. Purifying high quality RNA suitable for sequencing analysis turned out to be challenging and required several steps of adjustment. Samples from different timepoints and technical repeats have meanwhile been successfully obtained and send for sequencing.
To monitor the interaction of C. trachomatis with PMNs in vivo, we have established a mouse infection model in cooperation with the Max-Planck Research group of Georg Gasteiger. Association of Chlamydia with immune cells of the mouse genital tract has been analysed by FACS analysis. Chlamydia and immune cells in genital tract sections were also analysed by advanced microscopy. We followed in this work package an unexpected observation of an interferon-gamma (IFN-gamma) inducing signalling pathways that is initiated by primary human epithelial cells (but not tumour cells) and by neutrophils. Dependent on this observation we will design experiments to understand the interplay of epithelial cells and PMNs to orchestrate IFN-gamma responses in vivio. The respective knockout mouse model has already been established.
Additionally, complex cell culture models have been established to monitor the transmission of infectious C. trachomatis from PMNs to epithelial cells. This model will help to test our hypothesis that PMNs function as vehicles to spread Chlamydia in the female reproductive tract.
We have intensively studied the effect of C. trachomatis infection on the PMN biology. Infection extents the survival of human PMNs in vitro by activating a network of signalling molecules. These included molecules of apoptosis control (Mcl-1) and metabolic reprogramming (c-Myc) and a signalling pathway thus far not connected to neutrophil survival. An intriguing result was the observation that the average lifespan of PMNs could be extended four- to fivefold by manipulating these pathways and viable Chlamydia could be recovered even after such long infection periods.
The metabolic reprogramming of PMNs by C. trachomatis infection has been studied monitoring the gene expression of the bacteria and the PMNs simultaneously by dual-RNA seq. Purifying high quality RNA suitable for sequencing analysis turned out to be challenging and required several steps of adjustment. Samples from different timepoints and technical repeats have meanwhile been successfully obtained and send for sequencing.
To monitor the interaction of C. trachomatis with PMNs in vivo, we have established a mouse infection model in cooperation with the Max-Planck Research group of Georg Gasteiger. Association of Chlamydia with immune cells of the mouse genital tract has been analysed by FACS analysis. Chlamydia and immune cells in genital tract sections were also analysed by advanced microscopy. We followed in this work package an unexpected observation of an interferon-gamma (IFN-gamma) inducing signalling pathways that is initiated by primary human epithelial cells (but not tumour cells) and by neutrophils. Dependent on this observation we will design experiments to understand the interplay of epithelial cells and PMNs to orchestrate IFN-gamma responses in vivio. The respective knockout mouse model has already been established.
Additionally, complex cell culture models have been established to monitor the transmission of infectious C. trachomatis from PMNs to epithelial cells. This model will help to test our hypothesis that PMNs function as vehicles to spread Chlamydia in the female reproductive tract.
A new signalling pathway relevant for the lifespan of PMNs has been identified. Manipulating this pathway extends the lifespan of PMNs four- to fivefold. Interestingly, these long-lived PMNs can harbour infective C. trachomatis. We expect to understand the extended survival of Chlamydia under these conditions in detail until the end of the project.
An unexpected signalling circuit initiated by Chlamydia-infected primary epithelial cells affecting IFN-gamma signalling has been identified. Evidence has been obtained that this signalling pathway also affects the interaction of C. trachomatis with PMNs in vivo. We will use knockout mice to investigate the impact of this signalling pathway for in vivo infection.
The first dual RNA-seq experiments of PMNs infected with C. trachomatis have been performed. These data together with the currently prepared scRNA-seq analysis will provide a comprehensive understanding of the metabolic reprogramming and the respective neutrophil subpopulation relevant for long-term survival in PMNs.
An unexpected signalling circuit initiated by Chlamydia-infected primary epithelial cells affecting IFN-gamma signalling has been identified. Evidence has been obtained that this signalling pathway also affects the interaction of C. trachomatis with PMNs in vivo. We will use knockout mice to investigate the impact of this signalling pathway for in vivo infection.
The first dual RNA-seq experiments of PMNs infected with C. trachomatis have been performed. These data together with the currently prepared scRNA-seq analysis will provide a comprehensive understanding of the metabolic reprogramming and the respective neutrophil subpopulation relevant for long-term survival in PMNs.