Biology subjects

Brötz-Oesterhelt, H.

Publications and source records attributed to Brötz-Oesterhelt, H..

2 recordsLinked to original sources

bioRxiv2023

GlnA3Mt is able to glutamylate spermine but it is not essential for the detoxification of spermine in Mycobacterium tuberculosis

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host immune response. Since polyamines, which are synthesized by infected macrophages are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with toxic spermine. The actinomycete Streptomyces coelicolor, closely related to M. tuberculosis makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using a glutamine synthetase-based in vitro enzymatic activity assay we determined that GlnA3Mt (Rv1878) is a gamma-glutamylspermine synthetase. In an in vitro phosphate release assay we showed that purified His-Strep-GlnA3Mt as well as native GlnA3Mt prefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Subsequent RNAsequencing of M. tuberculosis bacteria revealed that the gene cluster consisting of the efflux pump-encoding rv3065-rv3066-rv3067 genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations. IMPORTANCEAntibiotics for the treatment of Mycobacterium tuberculosis infections attack classical bacterial targets, such as the cell envelope or the ribosome. Upon M. tuberculosis infection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamyl-sperminesynthetase GlnA3 may be responsible for resistance against toxic spermine. Here we show that the mycobacterial glutamyl-sperminesynthetase indeed can inactivate spermine by glutamylation. However, GlnA3 is probably not the only resistance mechanism since a glnA3 mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3Mt as well as of the spermine transporter in the pathogenicity of M. tuberculosis is of special interest for their validation as new targets of novel anti-tubercular drugs.

microbiology↗

bioRxiv2022

In vivo and In vitro Characterization of the ClpC AAA+ ATPase of Chlamydia trachomatis

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and in vivo approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated {beta}-casein. In vivo experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, the in vivo data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of anti-chlamydial agents. SignificanceChlamydia trachomatis is an obligate intracellular pathogen and the worlds leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new anti-chlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for anti-chlamydial compounds.

microbiology↗