gms | German Medical Science

The discovery of penicillin initiated the golden age of antibiotic discovery. Antibiotics are central to the treatment and prevention of bacterial infections and have underpinned advances in cancer chemotherapy, surgery, the survival of pre-term infants and organ transplantation. Penicillin belongs to the beta-lactam antibiotics, one of the most prescribed antibiotic class. Nevertheless, the effects, besides inhibiting the penicillin-binding proteins, remains to be elucidated. In this work, we used chlamydiae as a model organism to get insides into this process. Chlamydiae are obligate intracellular “minimal” bacteria and causal agents of human and animal diseases with major economic and public health concern. For a long time the host range of chlamydiae has been underestimated. Recently, we discovered Chlamydia-related bacteria in great apes from Central Africa identified our closest relatives as novel hosts paving the way to understand the evolutionary adaption of chlamydiae to humans [1].

Several fascinating aspects distinguish the chlamydiae from other eubacteria. The most striking one is their extraordinary biphasic developmental cycle. In general, during the life cycle, chlamydiae alternate between elementary body (EB) and reticulate body (RB). The extracellular EBs (0,1 µm in size) are responsible for the dissemination of the infection whereas the RBs are important for replication. After attachment to an eukaryotic cells EBs get internalized and remain in special vacuoles termed inclusions. Within the inclusion EBs differentiate into the intracellular RBs (1 µm in size). These undergo repeated cycles of binary fission before they differentiate back to EBs and are released to start a new infection cycle [2]. Under stressful conditions, chlamydiae can enter a non-infectious but viable state, known as persistence. In vitro persistence is characterized by aberrant bodies (enlarged, non-dividing reticulate bodies), which are not capable of differentiating into EBs and remain associated with their host over long periods of time. Several stressors, including interferon-γ-induced tryptophan deprivation, iron chelation, and exposure to antimicrobial agents can induce chlamydial persistence [3], [4]. After removal of the stressor chlamydia can turn back into their normal cell cycle. Because of their intracellular lifestyle they do not need a cell wall (peptidoglycan) to resist osmotic challenges and for more than 20 years the structure could not been detected. However, despite the apparently missing cell wall, these pathogens are unexpectedly sensitive to cell wall biosynthesis inhibitors which is known as “Chlamydial anomaly”. In 2014, Maurelli and coworkes revealed evidence for the presence of circularly shaped peptidoglycan-like structures in chlamydiae [5].

The goal of this work is to gain more insights into the “Chlamydial anomaly” on a cellular and molecular level. For this reason a fluorescence-microscopy based system has been established to analyze cellular effects of cell wall targeting antibiotics on the chlamydial life cycle in a cell culture model. Specially the treatment with different beta-lactams, beside penicillin, and beta-lactamase inhibitors was investigated. The tested compounds resulted in varying numbers and morphological types of persisting cells in these cell wall lacking bacteria. For a better understanding of this phenomena proteins of the peptidoglycan biosynthesis pathway were investigated on a molecular level. In particular cell wall hydrolases like AmiA, the homolog of a hydrolase important for cytokinesis in E. coli. AmiA from Chlamydia pneumoniae was functionally analyzed using complementation experiments in the heterologous host E. coli as well as biochemical assays with the recombinant enzyme. The chlamydial AmiA restores the function and separates daughter cells in an E. coli amidase mutant. Interestingly, purified AmiA accepted both polymeric peptidoglycan as well as monomeric lipid II (the cell wall precursor) as a substrate. Noteworthy, AmiA showed an unexpected novel dual activity acting as amidase and penicillin-sensitive carboxypeptidase [6]. Functional conservation of AmiA implicates a role of this enzyme in chlamydial cell division. Additionally, the processing of lipid II caused by AmiA might subvert Nod2 receptor-mediated host response to muropeptides, contributing to long-term residence of chlamydiae in the host. However, a better understanding of the chlamydial lipid II processing and cell division machineries is needed to gain further insight into host response modulation and impact of persistent chlamydiae in patients. Further insights into the molecular mechanism on the basic of these findings could help to develop new treatment strategies against active and persistent chlamydial infections.

Note: A. Klöckner and C. Otten contributed equally.