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Salinomycin (SAL), originally identified for its potent antibacterial properties, has recently garnered attention for its remarkable activity against a variety of cancer types. Beyond its direct cytotoxic effects on cancer cells, SAL can also enhance the efficacy of anti-CD20 immunotherapy in B-cell malignancies, both in vitro and in vivo. Despite these promising findings, the precise molecular mechanisms underlying SAL’s anticancer action remain poorly understood. Here, we demonstrate that even at low concentrations (0.25–0.5 mM), SAL disrupts mitochondrial membrane potential and induces oxidative stress in Burkitt lymphoma. Further investigations uncovered that SAL shifts cellular metabolism from mitochondrial respiration to aerobic glycolysis. Additionally, metabolomic profiling identified SAL-induced arginine depletion as a key metabolic alteration. These findings provide new insights into SAL’s multifaceted mechanisms of action and support its potential as an adjunctive therapy in cancer treatment.

Metabolomics is a comprehensive analysis of metabolites within a biological system. It offers insights into understanding of cellular processes at a molecular level. Bacteria, with their relatively simple structure and rather fast growth rates are good candidates for metabolomics studies. Other advantages of using metabolomics in research involving bacteria are: the plasticity of bacterial metabolism as well as an easy control over experimental conditions. This allows rapid collection of data and facilitates the interpretation of the results. Bacteria cultures can be maintained under highly controlled in vitro conditions. This is crucial for metabolomics, where even minor variations can lead to significant differences in metabolic profiles. However, challenges such as complex data analysis, dynamic metabolic changes, resolving multiple pathways, standardization issues, need for proper controls, and resource requirements can be experienced.

The aim of this thesis is to study bacterial interaction with each other and with chemical compounds from the perspective of their metabolism and to improve the approaches to metabolite analysis in bacteria. Specifically, this thesis includes studies of bacterial metabolism under various experimental conditions, including different growth phases, co-culture of species, and drug exposure. 

Understanding how bacteria respond to drugs at the metabolic level can aid in the development of new therapeutic methods. Paper I and Paper III studied the lipidome of Streptococcus pneumoniae and the metabolome of Mycobacterium tuberculosis (Mtb) to identify metabolic pathways that are altered in response to drug exposure and, hence, may be responsible for drug resistance. 

Bacteria often exist in complex microbial communities. Interspecies interactions may reflect in their metabolism over different growth phases. Metabolomics can extract these interactions by identifying metabolites that differ between single species and consortia. Paper II investigates the influence of co-culture and growth phase on metabolite patterns in Actinobacteria, showing adaptive strategies for growth, stress survival, and environmental interaction, providing insights into bacterial physiology.  

Finally, to improve annotation of bacterial metabolites, a study design was established for a comprehensive database over bacterial metabolites. The design covers ten species of bacteria. The key factors such as growth phase, nutrient availability, approaches to cultivation and sampling, biomass harvesting, and high-throughput LC-MS/MS metabolomics were considered and samples were prepared with multiple replicates and in a carefully controlled manner, aiming to catalogue and characterize diverse bacterial metabolites (Paper V). Paper IV proves advantages of Orthogonal Projections to Latent Structures Effect Projections (OPLS-EP), a paired multivariate analysis that has been used here for the first time to improve model performance of bacterial datasets. Programming was used to develop new code instruments and methodology for metabolomics data treatment and analysis. 

In conclusion, this thesis provides insights into metabolism of bacterial systems under various conditions, such as growth stages, co-cultures, and drug exposure as well as evaluation of different tools for metabolomics analyses to study bacterial physiology.

Metabolomik är den omfattande analysen av metaboliter inom ett biologiskt system. Det ger insikt i cellulära processer på molekylär nivå. Bakterier, med sin relativt enkla struktur och snabba tillväxttakt är bra kandidater för metabolomik. Fördelarna i dessa system är bakteriernas snabba tillväxt och att de experimentella förhållandena är enkla att kontrollera i denna typ av försök. Detta möjliggör snabb insamling av data och enklare tolkning av resultaten. Bakteriekulturer kan bibehållas under mycket kontrollerade in vitro-förhållanden. Detta är avgörande för metabolomik, där även mindre variationer i experimentella förhållanden kan leda till betydande skillnader i metaboliska profiler. Men utmaningar finns, t ex när det gäller den komplexa dataanalysen, dynamiska förändringar i metabolismen, standardiseringsproblem, brus i mätdata, och vilka resurser som finns att tillgå när det gäller instrumentering.

Syftet med denna studie var att använda och utvärdera olika metabolomik- verktyg (GC-MS och LC-MS, olika databearbetningsmetoder mm) och använda dem för att titta på bakteriell metabolism under olika förhållanden, såsom tillväxtfas, experimentella förhållanden, samlevnad, och läkemedelsexponering. 

Att förstå hur bakteriers metabolism påverkas av läkemedel kan bidra till utvecklingen av nya behandlingsmetoder. Artikel I och III studerade metabolitsammansättningen hos Streptococcus pneumoniae och Mycobacterium tuberculosis (Mtb) och identifierade metaboliska vägar som förändras som svar på läkemedelsexponering och som även kan vara ansvariga för resistens. 

Bakterier finns ofta i komplexa mikrobiella samhällen. Interaktioner mellan arter påverkar, i dessa fall, deras metaboliska aktivitet under olika tillväxtfaser. Metabolomik kan kartlägga dessa interaktioner genom att identifiera metaboliter som påverkas under samlevnad. Artikel II undersöker inflytandet av samlevnad och tillväxtfas på metabolitmönster i Actinobacteria. Dessa studier visar adaptiva strategier för tillväxt, stressöverlevnad och interaktion med omgivande miljö, vilket ger nya insikter inom bakteriell fysiologi.  

För att förbättra framtida metabolomikanalyser upprättades en studiedesign för att utveckla en omfattande databas för bakteriella metaboliter. Designen är utformad för att optimera bakteriell odling, provtagning, mängd biomassa som erhålls, och efterföljande LC-MS/MS analyser. Databasen syftar till att katalogisera och karakterisera olika bakteriella metaboliter (Artikel V). Artikel IV visar fördelarna med ”Orthogonal Projections to Latent Structures Effect Projection” (OPLS-EP), en ny typ av parad multivariatanalys som förbättrar modellprestanda för bakteriella metabolitdata. Nya verktyg har även utformats i form av datorkod och databehandlingsmetoder. 

Sammanfattningsvis ger denna avhandling både insikter i 1) metabolismen hos bakteriella system under olika förhållanden, såsom tillväxtförhållanden, samlevnad, och läkemedelsexponering samt, 2) tillämpning och utvärdering av olika analysverktyg för metabolomikstudier av mikrobiella system.

Introduction: Tuberculosis (TB) treatment typically involves a tailored combination of four antibiotics based on the drug resistance profile of the infecting strain. The increasing drug resistance of Mycobacterium tuberculosis (Mtb) requires the development of novel antibiotics to ensure effective treatment regimens. Gallium (Ga) is being explored as a repurposed drug against TB due to its ability to inhibit Mtb growth and disrupt iron metabolism. Given the potential interactions between Ga and established antibiotics, we investigated how a combination of Ga with levofloxacin (Lfx) or linezolid (Lzd) affects the growth and metabolome of a multidrug-resistant (MDR) Mtb clinical strain.

Methods: Mtb was cultured using a BACTEC 960 system with concentrations of Ga ranging from 125 to 1,000 μM and with 250 to 500 μM of Ga combined with 0.125 mg/L of Lfx or Lzd. For metabolome analysis, the antibacterials were used at concentrations that inhibited the growth of bacteria without causing cell death. Metabolites were extracted from Mtb cells and analyzed using chromatography-mass spectrometry.

Results: The MDR Mtb strain exhibited a dose-dependent response to Ga. Notably, the enhancement in growth inhibition was statistically significant for the Ga/Lfx combination compared to Ga alone, while no such significance was observed for Ga/Lzd. Moreover, exposure to Ga/Lfx or Ga/Lzd resulted in distinct metabolite profiles. Ga treatment increased the level of aconitate, fumarate, and glucose in the cells, suggesting the inhibition of iron-dependent aconitase and fumarate hydratase, as well as disruption of the pentose phosphate pathway. The levels of glucose, succinic acid, citric acid, and hexadecanoic acid followed a similar pattern in cells exposed to Ga and Ga/Lfx at 500 μM Ga but exhibited different trends at 250 μM Ga.

Discussion: In the presence of Lfx, the Mtb metabolome changes induced by Ga are more pronounced compared to those observed with Lzd. Lfx affects nucleic acids and transcription, which may enhance Ga-dependent growth inhibition by preventing the metabolic redirection that bacteria typically use to bypass iron-dependent enzymes.

Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus. 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT. These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae.