Host interactions of intracellular Staphylococcus aureus

Staphylococcus aureus causes a variety of diseases ranging from abscesses to endocarditis and sepsis. Treatment options are rapidly dwindling due to the emergence of methicillin-resistant S. aureus (MRSA). During infections, S. aureus is challenged by defensive strategies of the host. For example, the bacteria are taken up by phagocytic immune cells, which usually are able to remove a majority of the bacteria. However, some bacteria may persist in the host either by neutralizing host immune strategies or by hiding inside tissue cells. The latter are able to internalize the pathogen and thus constitute so-called non-professional phagocytes (NPPC). Cytotoxic S. aureus strains often escape from the resulting endocytic vesicles and replicate in the cytoplasm and subsequently cause host cell death. The involved bacterial effectors and host factors in this complex interplay and the underlying molecular mechanisms are largely unknown.

The research group uses fluorescent reporters in both, host cells and the pathogen, to monitor and analyze the infection dynamics. Further, the group employs transposon mutant screens coupled to next-generation sequencing techniques to identify bacterial virulence factors required for establishment of infection in specific intracellular and extracellular infection niches.

One major research focus of the group currently is the interaction of S. aureus with sphingolipids on the host cell surface, the resulting bacterial uptake, and the intracellular fate of the bacteria. S. aureus produces a variety of plasma membrane-active toxins, as wells as a bacterial sphingomyelinase and various other virulence factors, which modulate or permeabilize cellular membranes at the host cell surface or within endosomes. The group focuses on altered bacterail virulence upon changes in sphingolipid content of host membranes.
Together with Prof. Dr. Christian Stigloher (Imaging Core Facility) we want to understand S. aureus infections on host membranes and their dependencies on sphingolipids on an organismal level for which Prof. Stigloher has established the genetically tractable model organism C. elegans as an infection model for S. aureus.

Due to the fruitful cooperation with the organic Chemistry Department of Prof. Dr. Jürgen Seibel and the Bioimaging department of Prof. Dr. Markus Sauer, both members of the RTG2581 consortium, we now can use modified or functionalized sphingolipids to visualize S. aureus-infected cells and the respective host membranes with Expansion Microscopy (ExM) at high spatial resolution with standard confocal microscopy equipment (see also below).

The funding for this project is provided by the Deutsche Forschungsgemeinschaft (DFG) within the Research Training Group RTG2581 “SPHINGOINF”.
 

Another major focus of the laboratory is the dissemination of the pathogen in the host.
Together with Prof. Dr. Eva Media at the HZI Braunschweig we investigate so called Molecular Decision Points underlying the systemic spreading of Staphylococcus aureus through the infected host organism. This collaborative research is based on the observation that S. aureus is able to disseminate via the bloodstream of the host from an initial local site of infection to distant organs. Thereby the pathogen can cause progressive, chronic, or recurrent infections. When S. aureus is in the bloodstream it initially passes through the liver, which constitutes the first line of defence.Liver-resident macrophages, so-called Kupffer cells, thereby filter the bacteria out of the circulation.
When blood stream phagocytes and Kupffer cells fail to eliminate the pathogen completely, the bacteria can disseminate to secondary organs, such as kidneys and bones where cells of the host immune system wall off the bacteria by abscess formation.
However, also abscesses fail to contain S. aureus completely which may result in recurrence of infection. We here want to identify critical host factors, the so-called Molecular Decision Points which control the capacity of phagocytes to clear S. aureus from the bloodstream. Within our project C04 in the Collaborative Research Center DECIDE we have the ideal research conditions to investigate this important field of S. aureus infection biology.
 

Ref.:

• Blättner S, Das S, Paprotka K, Eilers U, Krischke M, Kretschmer D, Remmele CW, Dittrich M, Müller T, Schuelein-Voelk C, Hertlein T, Mueller MJ, Huettel B, Reinhardt R, Ohlsen K, Rudel T, Fraunholz MJ (2016) Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes. PLoS Pathogens 12:e1005857
• Grosz, M., Kolter, J., Paprotka, K., Winkler, A.C., Schafer, D., Chatterjee, S.S., Geiger, T., Wolz, C., Ohlsen, K., Otto, M., Rudel, T., Sinha, B. and Fraunholz, M. (2014). Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin alpha. Cell Microbiol 16, 451-465.

Host cell death caused by intracellular Staphylococcus aureus is influenced by calcium levels and the bacterial cysteine protease Staphopain A

Once in the host cytoplasm, S. aureus perturbs intracellular calcium levels, replicates (YouTube video by Dr. Kathrin Stelzner https://www.youtube.com/watch?v=OqCVZ_S10rw) and shortly thereafter infected host cells die. Together with the group of Thomas Rudel, we demonstrated that host cell death of cytoplasmic S. aureus requires the bacterial cysteine protease staphopain A (ScpA), however the targets of which in the host cytoplasm currently are unknown.

Refs.:

• Stelzner K, Winkler AC, Liang C, Boyny A, Ade CP, Dandekar T, Fraunholz MJ, Rudel T (2020) Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death. mBio 11(6):e02250-20
• Stelzner K, Boyny A, Hertlein T, Sroka A, Moldovan A, Paprotka K, Kessie D, Mehling H, Potempa J, Ohlsen K, Fraunholz MJ, Rudel T (2021) Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells. PLoS Pathogens 17(9):e1009874.

Host plasma membrane permeabilization by staphylococcal α-toxin results in activation of sphingomyelinase dependent membrane repair mechanisms

Extracellular S. aureus further uses one of its pore-forming toxins, α-toxin, to induce calcium influx into host cytoplasm. Permeabilized host cell membranes are repaired by a mechanism involving endocyctosis of damaged membranes, as well as recruitment of membrane repair enzymes such as acid sphingomyelinase (ASM), which is located in host lysosomes. Thus, the host cell uses a conserved repair mechanism already described for other toxins to reseal membranes damaged by staphylococcal α-toxin. We currently investigate the contribution of host sphingolipids to S. aureus cellular microbiology within GRK2581 SPHINGOINF.

Ref.:

• Krones D, Rühling M, Becker KA, Kunz TC, Sehl C, Paprotka K, Gulbins E, Fraunholz M (2021) Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line. Frontiers in Microbiology 12:694489

Expansion Microscopy of Staphylococcus aureus requires the use of a specific protease cocktail

Since many of the groups research questions require fluorescence microscopy, we investigated, if so-called Expansion microscopy (ExM) may be applicable to S. aureus-infected cells. ExM circumvents the diffraction limitation of classical microscopy techniques by swelling the sample. Therein, the rigidity of the staphylococcal cell wall was overcome by application of specific proteases and now enables us to image host-pathogen interactions with at least four-fold higher resolution with standard confocal fluorescence microscopes. This work was possible due to the collaborations within the GRK2581 SPINGOINF (see above).

Ref:

• Kunz TC, Rühling M, Moldovan A, Paprotka K, Kozjak-Pavlovic V, Rudel T, Fraunholz M (2021) The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy. Frontiers in Cellular and Infection Microbiology 11:644750

Associations

We are proud to be associated with the DFG priority program SPP2225 EXIT, which is a consortium of researchers that investigate exit strategies of intracellular pathogens. SPP2225 thereby explores convergently evolved exit pathways employed by bacterial, parasitic and fungal pathogens with relevance for human health.