Biology subjects

Bachmaier, S.

Publications and source records attributed to Bachmaier, S..

3 recordsLinked to original sources

bioRxiv2021

A divergent protein kinase A in the human pathogen Leishmania is associated with developmental morphogenesis

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote development into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastea. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of PKAR3 or PKAC3 null mutants lose their elongated shape and are round but remain flagellated. Truncation of an N-terminal formin homology-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 kinase activity via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARI{beta} and PKARII{beta} binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 does not bind cAMP but nucleoside analogs with a very high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape via PKA activity.

cell biology↗

bioRxiv2020

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

3A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N-terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4 kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

biochemistry↗

bioRxiv2020

Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei

The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout ({Delta}pepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the {Delta}pepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the {Delta}pepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the {Delta}pepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.

microbiology↗