Biology subjects

Bachmaier, S.

Publications and source records attributed to Bachmaier, S..

4 recordsLinked to original sources

bioRxiv2023

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in T. brucei

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by targeted RNAi knockdown and highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology. ImportanceTrypanosomes are major pathogens of humans and livestock. In addition they have been invaluable as a model system to investigate new biological systems, and not just of protozoa. Equally, they are known to have a lot of unique biology and biochemistry. One example of this is signal transduction by cyclic nucleotides. Some elements, including phosphodiesterases and the catalytic domains of its dozens of adenylate cyclase isoforms, are highly conserved, while the absence of G-proteins, a cAMP-responsive protein kinase A and other known effector types suggests a unique cAMP-dependent pathway, which as yet is mostly uncharacterised. Here, we identify a set of ten Trypanosoma brucei proteins, all localised to its flagellum, that appear to be involved in the production of cAMP, or in mediating its cellular effects. These cAMP Response Proteins (CARPs) were mostly unique to trypanosomes, suggesting a completely novel pathway. Two of the CARPs were shown to bind cAMP and were found to possess structurally conserved cyclic nucleotide binding domains.

microbiology↗

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↗