7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Unexpected diversity of autotrophic carbon fixation pathways in Desulfovibrionales

Microbial metabolism & biochemistry

Autotrophic CO2 fixation is the most essential biosynthetic process and the basis of primary production. Today, seven pathways of inorganic carbon assimilation are known. This diversity of autotrophy represents the diversity of microorganisms and their ecological niches, whereas autotrophic pathways are often specific to particular taxonomic groups. The order Desulfovibrionales consists mainly of anaerobic sulfate-reducing bacteria, some of which are autotrophic. Our analysis of Pseudodesulfovibrio profundus (Desulfovibrionaceae) genome showed the presence of the genes for the key enzymes of the Calvin-Benson cycle. We were able to confirm that this bacterium is capable of growing autotrophically, while enzyme assays and proteomic analysis revealed that the bacterium does indeed use the Calvin-Benson cycle. A related Desulfovibrio desulfuricans (Desulfovibrionaceae) was shown to fix CO2 via the recently described reductive glycine pathway [1]. Here, we found that the acetyl phosphate formed in this pathway is further metabolized via phosphate acetyltransferase and not through acetate kinase/acetyl-CoA synthetase reactions, as it was initially proposed based on proteomics data [1].Furthermore, we found that halophilic Desulfovermiculus halophilus (Desulfohalobiaceae) uses the Wood-Ljundgdahl (WL) pathway for growth. We have further analyzed the efficiency of the autotrophic metabolism as a function of biomass production versus H2S production and found the highest efficiency in D. desulfuricans, while P. profundus was the least efficient species. The efficiency of carbon fixation in D. halophilus using the WL pathway was lower than that of D. desulfuricans, probably due to the energetic costs of adaptation to halophilic conditions. Our analysis revealed considerable diversity of autotrophic metabolism in Desulfovibrionales. The evolution of autotrophic pathways in this bacterial group will be discussed.[1] Sánchez-Andrea et al., Nat Comm 11:5090 (2020)

Oral Presentation

Carbon utilization of Salmonella Typhimurium in different mouse models and on the family level of Enterobacteriaceae

Salmonella Typhimurium (S. Tm) is a common foodborne pathogen and a well-established pathogenic Enterobacterium to study colonization and infection of mammalian hosts. S. Tm invades the gastrointestinal tract and causes inflammation, which drastically changes the gut luminal environment, providing S. Tm with inorganic electron acceptors to bloom and outcompete the resident microbiota (1). However, it is largely unknown which carbon sources S. Tm utilizes at the onset of invasion. For this reason, mutant pools representing the metabolic capacity of S. Tm specifically sugar degradation were constructed, with each mutant labeled with a wild-type isogenic standardized hybrid (WISH) tag (2). The WISH tag is unique for each mutant and can be quantified by qPCR or Illumina sequencing, allowing us to study the fitness of several mutants. To modulate niche competition, the mutant pool will be studied in different mouse models and challenged in the presence of different competitors. Host physiology, e.g., colonization resistance and microbiota composition are known to impact S. Tm colonization (3). In a complementary approach, we looked at the family level of Enterobacteriaceae and identified the core and the accessory genome. Our focus was specifically on carbon utilization, leading to the identification of a core metabolic network centered around simple sugars and mixed acid fermentation. This observation aligns seamlessly with our in vivo data concerning S. Tm. Our investigation promises to uncover crucial insights into the significant metabolic pathways governing the initial growth of S. Tm. Additionally, we aim to identify shared metabolic traits within the family of Enterobacteriaceae that are imperative for invading mammalian hosts. By combining both species and family-level approaches, we aspire to unravel key metabolic characteristics inherent to Enterobacteriaceae.(1) Rogers, A. W., Tsolis, R. M., &amp; Bäumler, A. J. (2021). Microbiol. Mol. Biol. Rev, 85(1), e00027-19.(2) Daniel, B. et al, (2024). Nat. Microbiol, accepted.(3) Herzog, M. K. M., ... &amp; Hardt, W. D. (2023). Gut microbes, 15(1), 2172667.

Unveiling diverse strategies: glucose-1,6-bisphosphate synthesis in bacteria

Glucose-1,6-bisphosphate (Glc-1,6-BP) is a key regulator of central carbon metabolism. While the origin of Glc-1,6-BP in vertebrates is well investigated, the existence of a bacterial Glc-1,6-BP synthase was confirmed only recently. Investigation of the photoautotrophic cyanobacterium Synechocystis revealed a cryptic secondary phosphoglucomutase (SynPGM2) acting as a Glc-1,6-BP synthase. Despite displaying limited phosphoglucomutase (PGM) activity, SynPGM2 efficiently converts fructose-1,6-bisphosphate and glucose-1-phosphate/glucose-6-phosphate to Glc-1,6-BP. Bacteroides salyersiae encodes a homolog of SynPGM2, catalyzing the same Glc-1,6-BP synthesis reaction. Since both enzymes belong to the same conserved domain (CD) αPHM subfamily, it was concluded that bacterial Glc-1,6-BP is specifically formed by members of the cd05800 subfamily [1]. Remarkably, the B. salyersiae enzyme is annotated as the primary PGM, suggesting that heterotrophic bacteria might not use a secondary specialized enzyme for Glc-1,6-BP synthesis.Here, we aim to ascertain if heterotrophic bacteria exclusively utilize their main PGM for Glc-1,6-BP synthesis and if Glc-1,6-BP synthesis is limited to PGM of the cd05800 subfamily. For this, heterologously expressed PGM enzymes of various heterotrophic bacteria and several CD subfamilies were screened for Glc-1,6-BP synthase activity via coupled enzyme assays and HPLC-MS analysis.Our results show that Glc-1,6-BP synthesis extends beyond the cd5800 subfamily and can also be found within the cd05801, cd05799 and cd03089 subfamilies. Additionally, only some heterotrophic bacteria like Yersinia enterocolitica and Enterococcus faecium employ their primary PGM for Glc-1,6-BP synthesis. Others, like Escherichia coli, possess a secondary Glc-1,6-BP-producing PGM. While Glc-1,6-BP was mainly formed via the SynPGM2 reaction mechanism, the Enterococcus faecium and Yersinia enterocolitica PGM could also perform the reaction of the mammalian Glc-1,6-BP synthase. In summary, we could show that bacteria employ diverse strategies to produce the same essential regulator.[1] Neumann, N., S. Friz, and K. Forchhammer,2022. mBio, 13 (4): p. e01469-01422.

MraY from Pseudomonas aeruginosa is inhibited by uridyl-peptide antibiotics

Introduction Antibiotic resistance is one of the most serious health threats and the therapeutic options to treat infections caused by multidrug-resistant strains are seriously compromised. Especially for infections caused by Pseudomonas aeruginosa isolates which have emerged resistance towards all carbapenems, aminoglycosides, and fluoroquinolones, novel antibiotics with new targets or unprecedented mechanisms of action are urgently needed. Integral component of the drug development process is the analysis of the mechanism of action of an antibiotic, as well as identification of the molecular target. Without this detailed knowledge, rational drug design is strongly hampered. Uridyl-peptide antibiotics (UPA) are a promising group of nucleoside natural products, which show potent activity against P. aeruginosa. An important molecular target of this compound class is the phospho-N-acetylmuramoyl-pentapeptide-transferase MraY, which catalyses the transfer of phospho-MurNAc pentapeptide to the membrane-standing lipid carrier undecaprenyl phosphate resulting in the formation of Lipid I.Methods &amp; Results Using a fluorometric assay with the heterologously expressed MraY from P. aeruginosa, eight UPA's were characterized, and all showed IC50 values in the nanomolar range. Further, a second periplasmatic binding site was identified and confirmed by using site-directed mutagenesis resulting in significant elevation of the IC50 compared to the wildtype MraY, making UPA's the first antibiotics with a dual mode of action against the MraY of P. aeruginosa. To corroborate the data, a bioinformatical approach was used, by calculating the ΔG of the wildtype MraY and the mutants with AutoDock Vina. The theoretical binding showed very similar results to the biological data. Additionally, we could identify LPS synthesis of Pseudomonas as a new target of the UPA's, by establishing an in vitro assay with wbpL of P. aeuruginosa.Conclusion We could further characterize UPA's by the identification of a second periplasmatic binding site and a new mode of action by inhibiting LPS biosynthesis, making UPA's a promising new antibotic class.

Professor

Staphylococcus aureus and skin infection: A proteomic journey through metabolic pathways

ObjectivesSkin infections by methicillin-resistant Staphylococcus aureus (MRSA) present significant challenges due to their virulence and antibiotic resistance. This study aims to characterize the proteome variation of S. aureus during skin infection to identify new therapeutic targets.MethodsUsing in vivo (mouse model) and 3D in vitro skin models, we studied the proteome of S. aureus strains ST398 and JE2 (USA300) during skin infection. Mice were infected with 107 CFU mid-exponential phase cultures of MRSA onto a skin lesion by scrapping the dorsal back part, and skin samples were collected at 2- and 4- days post-infection for bacterial count and protein extraction. The 3D in vitro model, simulating human skin, was infected with 108 CFU of the same MRSA strains. After 2 days, we collected and extracted proteins from the tissues for analysis via shotgun proteomics, employing a Q-Exactive mass spectrometer. The data from this analysis were processed using SEQUEST-HT in Proteome Discoverer software. We applied a TMT labeling approach for the in vivo model and a label-free (LFQ) approach for the in vitro model.Results and conclusionKEGG mapping tool identified pathways involved in infection in both models, including glycolysis and gluconeogenesis, pentose phosphate pathway, and pyruvate metabolism, while glycine serine and threonine metabolism pathway was seen solely in the in vivo model. In addition, we attempted to understand strain-specific adaptations to the infection, highlighting an increase in the arginine biosynthesis pathway observed in JE2 in both tissue models, along with differences in S. aureus infection protein expression between the two strains, such as the upregulation of femX and femB genes in JE2 which are involved in peptidoglycan biosynthesis.This research enhances our understanding of protein expression levels during skin infection. By broadening our comprehension of Staphylococcus aureus biology, our work paves the way for developing targeted therapeutic interventions, thereby offering a meaningful contribution to the fight against antimicrobial resistance

PD Dr.

Underground metabolism in the gut: Degradation of Nε-carboxymethyllysine (CML) in Escherichia coli

Amino acids undergo numerous of enzymatic or non-enzymatic post-translational modifications. Nε-Carboxymethyllysine (CML) results from the condensation of a reducing sugar with the free amino group of lysine, in the so called Maillard reaction or glycation (Maillard et al., 1912). This non-enzymatic event is responsible for the flavour and colour of thermally processed foods. Up to 11.3 mg of CML per kg of protein are ingested daily by humans, but the limited absorption in the gastrointestinal tract suggests a major role in its degradation by colonic bacteria (Delgado-Andrade et al., 2012, Lassak et al., 2023). When exposing E. coli to CML the predominant degradation product is carboxymethylcadaverine (Hellwig et al., 2019). However, no enzyme in the CML metabolism is known to date. We have now unveiled an unexpected high promiscuity of the ornithine decarboxylase SpeC toward many lysine derivatives including CML. The enzyme is the first in an underground metabolism, enabling E. coli to utilize CML as sole nitrogen source. We further discovered that proton consumption in the CML decarboxylation reaction helps to counteract the mild acid pH in the colon. Investigating the molecular &quot;players&quot; of CML metabolism in E. coli will help understand how glycated amino acids influence the ability of gut bacteria to thrive and compete, and gain insight about the effect those compounds and their degradation products have on our health.

Oral Session
Microbial Metabolism & Biochemistry 2

Appointment

Chair

Topic

Programme

Oral SessionMicrobial Metabolism & Biochemistry 2

Appointment

Chair

Topic

Programme

Oral Session
Microbial Metabolism & Biochemistry 2