Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H2S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.

Broad-spectrum anti-cancer activity of fused human arginase variants.

The rapid increase in cancer cases worldwide necessitates the development of novel therapeutic approaches. Therapies targeting cancer's altered metabolism, especially those that deplete critical amino acids, have emerged as promising ones, some of which are already being used in clinical practice and many others are under development. This study reports the anti-cancer activity of two novel fused human arginase I (FHA) variants, FHA-3 and FHA-12, assessed using the NCI-60 human tumor cell line panel. Both variants have demonstrated a range of potencies in a single-dose assay (10 µM), but FHA-3 was found to be more potent with significant growth inhibition in most tested cell lines. To calculate 50% growth inhibition (GI50), FHA-3 was further evaluated in a five-dose assay, where notable anti-cancer activity was observed across the nine cancer types of the NCI-60 panel. Our results demonstrated the broad-spectrum anti-cancer activity of novel FHA variants, with FHA-3 being the most potent. Further studies elucidating its efficacy in animal models will help explore its therapeutic potential.

Synthesis of aspartic acid and tyrosine by the fire blight pathogen Erwinia amylovora is not required for proliferation on apple flower stigmas or virulence in fruitlets.

AIMS: The Gram-negative bacterium Erwinia amylovora (Ea) is the causal agent of fire blight, a devastating disease of apples and pears. In the fire blight disease cycle, Ea grows in different plant tissues, each presenting a distinct nutrient environment. Here, we investigate the ability of aspartate and tyrosine double auxotroph Ea lines to proliferate on apple flower stigma surfaces representing the epiphytic growth stage of Ea and in developing fruitlets representing one endophytic growth stage of Ea. METHODS AND RESULTS: Heterologous complementation studies in an Escherichia coli aspartate and tyrosine auxotroph verify that Ea aspartate aminotransferase (AspC) and tyrosine aminotransferase (TyrB) act as aspartate and tyrosine amino transferases. Growth analysis reveals that Ea aspC tyrB mutants multiply to near-wild-type levels on apple flower stigmas and immature fruitlets. CONCLUSIONS: Ea AspC and TyrB are reciprocally complementing for aspartate and tyrosine synthesis in Ec and in Ea. Ea aspC  and  tyrB mutants obtain sufficient aspartate and tyrosine to support multiplication on stigma surfaces and virulence in immature fruitlets.

Heme auxotrophy in abundant aquatic microbial lineages.

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms. Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment. These heme auxotroph groups are largely limited to pathogens, symbionts, or microorganisms living in nutrient-replete conditions, whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms. Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme, based on the experimental or genomic evidence. We found that two recently cultivated acI isolates require exogenous heme for their growth. One of the cultured acI isolates also exhibited auxotrophy for riboflavin. According to whole-genome analyses, all (n = 20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and Marine groups IIb and III of Euryarchaeota Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity.

Development and characterization of fused human arginase I for cancer therapy.

Recombinant human arginase I (rhArg I) have emerged as a potential candidate for the treatment of varied pathophysiological conditions ranging from arginine-auxotrophic cancer, inflammatory conditions and microbial infection. However, rhArg I have a low circulatory half-life, leading to poor pharmacokinetic and pharmacodynamic properties, which necessitating the rapid development of modifications to circumvent these limitations. To address this, polyethylene glycol (PEG)ylated-rhArg I variants are being developed by pharmaceutical companies. However, because of the limitations associated with the clinical use of PEGylated proteins, there is a dire need in the art to develop rhArg I variant(s) which is safe (devoid of limitations of PEGylated counterpart) and possess increased circulatory half-life. In this study, we described the generation and characterization of a fused human arginase I variant (FHA-3) having improved circulatory half-life. FHA-3 protein was engineered by fusing rhArg I with a half-life extension partner (domain of human serum albumin) via a peptide linker and was produced using P. pastoris expression system. This purified biopharmaceutical (FHA-3) exhibits (i) increased arginine-hydrolyzing activity in buffer, (ii) cofactor - independency, (iii) increased circulatory half-life (t1/2) and (iv) potent anti-cancer activity against human cancer cell lines under in vitro and in vivo conditions.

The addition of arginine deiminase potentiates Mithramycin A-induced cell death in patient-derived glioblastoma cells via ATF4 and cytochrome C.

BACKGROUND: Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy. METHODS: In this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth. RESULTS: MitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen. CONCLUSIONS: Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials.

Metabolic and genetic basis for auxotrophies in Gram-negative species.

Auxotrophies constrain the interactions of bacteria with their environment, but are often difficult to identify. Here, we develop an algorithm (AuxoFind) using genome-scale metabolic reconstruction to predict auxotrophies and apply it to a series of available genome sequences of over 1,300 Gram-negative strains. We identify 54 auxotrophs, along with the corresponding metabolic and genetic basis, using a pangenome approach, and highlight auxotrophies conferring a fitness advantage in vivo. We show that the metabolic basis of auxotrophy is species-dependent and varies with 1) pathway structure, 2) enzyme promiscuity, and 3) network redundancy. Various levels of complexity constitute the genetic basis, including 1) deleterious single-nucleotide polymorphisms (SNPs), in-frame indels, and deletions; 2) single/multigene deletion; and 3) movement of mobile genetic elements (including prophages) combined with genomic rearrangements. Fourteen out of 19 predictions agree with experimental evidence, with the remaining cases highlighting shortcomings of sequencing, assembly, annotation, and reconstruction that prevent predictions of auxotrophies. We thus develop a framework to identify the metabolic and genetic basis for auxotrophies in Gram-negatives.

Myristate can be used as a carbon and energy source for the asymbiotic growth of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi, forming symbiotic associations with land plants, are obligate symbionts that cannot complete their natural life cycle without a host. The fatty acid auxotrophy of AM fungi is supported by recent studies showing that lipids synthesized by the host plants are transferred to the fungi, and that the latter lack genes encoding cytosolic fatty acid synthases. Therefore, to establish an asymbiotic cultivation system for AM fungi, we tried to identify the fatty acids that could promote biomass production. To determine whether AM fungi can grow on medium supplied with fatty acids or lipids under asymbiotic conditions, we tested eight saturated or unsaturated fatty acids (C12 to C18) and two ß-monoacylglycerols. Only myristate (C14:0) led to an increase in the biomass of Rhizophagus irregularis, inducing extensive hyphal growth and formation of infection-competent secondary spores. However, such spores were smaller than those generated symbiotically. Furthermore, we demonstrated that R. irregularis can take up fatty acids in its branched hyphae and use myristate as a carbon and energy source. Myristate also promoted the growth of Rhizophagus clarus and Gigaspora margarita Finally, mixtures of myristate and palmitate accelerated fungal growth and induced a substantial change in fatty acid composition of triacylglycerol compared with single myristate application, although palmitate was not used as a carbon source for cell wall biosynthesis in this culture system. Our findings demonstrate that myristate boosts the asymbiotic growth of AM fungi and can also serve as a carbon and energy source.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

NamZ1 and NamZ2 from the Oral Pathogen Tannerella forsythia Are Peptidoglycan Processing Exo-ß-N-Acetylmuramidases with Distinct Substrate Specificities.

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-ß-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-N-acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic ß-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic ß-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-ß-N-acetylmuramidases. IMPORTANCE Two exo-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

Cooperation increases robustness to ecological disturbance in microbial cross-feeding networks.

Microorganisms mainly exist within complex networks of ecological interactions. Given that the growth and survival of community members frequently depend on an obligate exchange of essential metabolites, it is generally unclear how such communities can persist despite the destabilising force of ecological disturbance. Here we address this issue using a population dynamics model. In contrast to previous work that suggests the potential for obligate interaction networks to emerge is limited, we find the opposite pattern: ecological disturbance favours both specific network topologies and cooperative cross-feeding among community members. These results establish environmental perturbations as a key driver shaping the architecture of microbial interaction networks.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation.

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Loss of Dihydroxyacid Dehydratase Induces Auxotrophy in Bacillus anthracis.

Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that ΔilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to nonsupplemented wild type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and ΔilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis, and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts. IMPORTANCE Anthrax disease, caused by B. anthracis, can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.

Metabolic implication of tigecycline as an efficacious second-line treatment for sorafenib-resistant hepatocellular carcinoma.

Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.

Resource allocation explains lactic acid production in mixed-culture anaerobic fermentations.

Lactate production in anaerobic carbohydrate fermentations with mixed cultures of microorganisms is generally observed only in very specific conditions: the reactor should be run discontinuously and peptides and B vitamins must be present in the culture medium as lactic acid bacteria (LAB) are typically auxotrophic for amino acids. State-of-the-art anaerobic fermentation models assume that microorganisms optimise the adenosine triphosphate (ATP) yield on substrate and therefore they do not predict the less ATP efficient lactate production, which limits their application for designing lactate production in mixed-culture fermentations. In this study, a metabolic model taking into account cellular resource allocation and limitation is proposed to predict and analyse under which conditions lactate production from glucose can be beneficial for microorganisms. The model uses a flux balances analysis approach incorporating additional constraints from the resource allocation theory and simulates glucose fermentation in a continuous reactor. This approach predicts lactate production is predicted at high dilution rates, provided that amino acids are in the culture medium. In minimal medium and lower dilution rates, mostly butyrate and no lactate is predicted. Auxotrophy for amino acids of LAB is identified to provide a competitive advantage in rich media because less resources need to be allocated for anabolic machinery and higher specific growth rates can be achieved. The Matlab™ codes required for performing the simulations presented in this study are available at https://doi.org/10.5281/zenodo.4031144.

Arginine-deprivation-induced oxidative damage sterilizes Mycobacterium tuberculosis.

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.