Biology subjects

Eisenreich, W.

Publications and source records attributed to Eisenreich, W..

8 recordsLinked to original sources

bioRxiv2026

Trehalose metabolism and its impact on PrfA activity in Listeria monocytogenes

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

microbiology↗

bioRxiv2024

The AusAB non-ribosomal peptide synthase in Staphylococcus aureus preferentially incorporates exogenous phenylalanine and tyrosine into the aureusimine natural products

Non-ribosomal peptide synthases (NRPS) are modular multidomain enzymes, responsible for the biosynthesis of various secondary metabolites, in a mRNA-template independent manner. They are predominantly present in bacteria and fungi, where they synthesize a variety of products, including antibiotics, siderophores, toxins and signalling molecules. The human pathogen Staphylococcus aureus possesses one single NRPS, AusA, highly conserved in all sequenced S. aureus strains. AusA incorporates the aromatic amino acids (AAA) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAA) valine and leucine into three cyclic dipeptides collectively called aureusimines: phevalin, tyrvalin and leuvalin. By using targeted metabolomics, we found that AusA preferentially incorporates phenylalanine and tyrosine from an exogenous source into aureusimines, whereas the source of valine can be either endo- or exogenous. Upon cultivation in a chemically defined medium (CDM) lacking phenylalanine, the amino acid was not incorporated into phevalin, despite de novo phenylalanine biosynthesis. Tyrosine production remained unaffected. Conversely, upon cultivation in medium lacking tyrosine, tyrvalin production was not detected, despite tyrosine de novo biosynthesis. Phevalin production, however, remained unaltered. By contrast, omission of valine in the culture medium not only resulted in de novo valine biosynthesis but also was accompanied by phevalin production. To our knowledge, this is the first report of a selective incorporation of AAAs by a bacterial NRPS, which provides useful basis for linking bacterial cell metabolic status to the biosynthesis of secondary metabolites. IMPORTANCEPeptide and protein synthesis are fundamental processes in nature, which are largely mediated by the ribosomal machinery. An alternative pathway for peptide synthesis is non-ribosomal mRNA-template independent synthesis, performed by so-called non-ribosomal peptide synthases (NRPS). NRPSs are multi-enzyme complexes, which serve the simultaneous role of template and biosynthetic machinery. They are mostly found in bacteria and fungi and are responsible for the biosynthesis of many pharmacologically significant products, including antibiotics, anticancer compounds or immunosuppressants. The human pathogen S. aureus possesses one such NRPS, AusA, which synthesizes three cyclic dipeptides termed "aureusimines" using the aromatic amino acids phenylalanine and tyrosine, and the branched-chain amino acid valine. Although the biological role of aureusimines remains unknown, AusA appears to play a role in the interaction of S. aureus with the host. In addition, owing to its minimal canonical NRPS structure and autonomous function (i.e. most NRPS pathways require the assembly of several NRPS proteins), AusA represents an excellent model system for studying such molecular assembly lines. Our observation is, to our knowledge, the first report of a NRPS incorporating phenylalanine and tyrosine only from exogenous sources (e.g. environment, culture medium), but not from de novo "self-made" pool. This opens up new avenues in understanding and modulating the function of NRPSs (e.g. for biotechnological purposes).

microbiology↗

bioRxiv2023

Helicobacter pylori modulates heptose metabolite biosynthesis and heptose-dependent innate immune host cell activation by multiple mechanisms

Heptose metabolites including ADP-heptose are involved in bacterial lipopolysaccharide and cell envelope biosynthesis. Recently, heptoses were also identified to have potent pro-inflammatory activity on human cells as novel microbe-associated molecular patterns. The gastric pathogenic bacterium Helicobacter pylori produces heptose metabolites which it transports into human cells through its Cag type 4 secretion system. Using H. pylori as a model, we have addressed the question, how pro-inflammatory ADP-heptose biosynthesis can be regulated by the bacteria. We have characterized the inter-strain variability and regulation of heptose biosynthesis genes and the modulation of heptose metabolite production by H. pylori, which impact cell-autonomous pro-inflammatory human cell activation. HldE, a central enzyme of heptose metabolite biosynthesis, showed strong sequence variability between strains, and was also strain-variably expressed. Transcript amounts of genes in the hldE gene cluster displayed intra-strain and inter-strain differences, were modulated by host cell contact and the presence of the cag pathogenicity island, and were affected by carbon starvation regulator A (CsrA). We reconstituted four steps of the H. pylori LPS heptose biosynthetic pathway in vitro using recombinant purified GmhA, HldE and GmhB proteins. On the basis of one- and two-dimensional NMR spectroscopy and mass spectrometry, the structures of major reaction products were identified as {beta}-D-ADP-heptose and {beta}-heptose-1-monophosphate. A pro-inflammatory heptose-monophosphate variant was also identified for the first time as a novel cell-active product in H. pylori bacteria. Separate purified HldE subdomains and variant HldE allowed to uncover additional strain variation in generating heptose metabolites.

microbiology↗

bioRxiv2022

The large GTPase Sey1/atlastin mediates lipid droplet- and FadL-dependent intracellular fatty acid metabolism of Legionella pneumophila

The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). The endoplasmic reticulum (ER)-resident large fusion GTPase Sey1/atlastin promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we show that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or {otimes}sey1 revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or {otimes}sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we discovered that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or {otimes}sey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for {otimes}sey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i) L. pneumophila wild-type in D. discoideum Ax3 but not in {otimes}sey1 mutant amoeba and (ii) L. pneumophila wild-type but not {otimes}fadL mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum {otimes}sey1 and L. pneumophila {otimes}fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.

microbiology↗

bioRxiv2022

A central role of sibling sRNAs NgncR_162/163 in main metabolic pathways of Neisseria gonorrhoeae

Bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in very few cases has the role in metabolism been demonstrated. We performed here a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 and their impact on the metabolism of the major human pathogen Neisseria gonorrhoeae. These sRNA have previously been shown to control genes of the citric acid and methylcitrate cycle by post-transcriptional negative regulation. By transcriptome analysis we expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially the uptake of glycine, branched chain amino acids and phenylalanine. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and hence probably affect biosynthesis of nucleotides, vitamins and other amino acids via the supply of C1-units. Metabolic flux analysis demonstrated a bipartite metabolism with glucose degradation providing intermediates for anabolic pathways, while energy metabolism via the citric acid cycle is mainly driven by amino acids, which feed into the cycle. Thus, by combined RNA-seq and metabolomics we significantly extended the regulon of NgncR_162/163 and demonstrate their role in the regulation of central metabolic pathways of the gonococcus. ImportanceNeisseria gonorrhoeae is a major human pathogen which infects more than 100 million people every year. An alarming development is the emergence of gonococcal strains resistant against virtually all of the antibiotics used for their treatment. Despite the medical importance and the vanishing treatment options of gonococcal infections, the bacterial metabolism and its regulation is only ill defined until today. We investigate here the regulation of the gonococcal metabolism by two previously studied sRNAs, NgncR_162/163 using RNA-seq and metabolomics. The results provided in this study demonstrate the regulation of transport processes and metabolic pathways involved in the biosynthesis of nucleotides, vitamins and amino acids by NgncR_162/163. Combined transcriptome and metabolome analyses provide a thus far unreached depth in the regulation of metabolic pathways by the neisserial sibling sRNAs and may therefore also be suitable for functional analysis of a growing number of other bacterial metabolic sRNA regulators.

microbiology↗

bioRxiv2021

Exploring the interaction network of a synthetic gut bacterial community

A key challenge in microbiome research is to predict functionality from microbial community composition. As central microbiota functions are determined by bacterial community networks it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments, as well as in community batch culture. Metabolomics analysis of spent culture supernatant of individual strains in combination with genome-informed pathway reconstruction provided insights into the metabolic potential of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both, exploitative and interference competition in vitro. In particular, Enterococcus faecalis KB1 was identified as important driver of community composition by affecting the abundance of several other consortium members. Together, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network, which serves as knowledge base for future mechanistic studies.

microbiology↗

bioRxiv2019

A central role of glutamine in Chlamydia infection

Obligate intracellular bacteria like Chlamydia trachomatis undergo a complex developmental cycle between infectious non-replicative (EBs) and non-infectious replicative (RBs) forms. EBs shortly after entering a host cell transform to RBs, a crucial process in infection, initiating chlamydial replication. As Chlamydia fail to replicate outside the host cell it is currently unknown how the transition from EBs to RBs is initiated. Here we show in a cell-free approach in axenic media that uptake of glutamine by the bacteria is critical to initiate EB-RB transition. These bacteria utilize glutamine to synthesize cell wall peptidoglycan which has recently been detected in the septa of replicating intracellular Chlamydia. The increased requirement for glutamine in infected cells is achieved by reprogramming the glutamine metabolism in a c-Myc-dependent manner. Glutamine was effectively taken up by the glutamine transporter SLC1A5 and metabolized via glutaminase. Interference with this metabolic reprogramming limited growth of Chlamydia. Intriguingly, Chlamydia failed to produce progeny in SLC1A5 knockout mice. Thus, we report on the central role of glutamine for the development of an obligate intracellular pathogenic bacterium and the reprogramming of host glutamine metabolism, which may provide a basis for innovative anti-infective strategies.

microbiology↗

bioRxiv2017

Lipid transfer from plants to arbuscular mycorrhiza fungi

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1{omega}5 FAs. Using isotopolog profiling we demonstrate that 13C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts.

plant biology↗