Deletion of Rv2571c confers resistance to arylamide compounds in Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, is an urgent global health problem requiring new drugs, new drug targets and an increased understanding of antibiotic resistance. We have determined the mode of resistance to a series of arylamide compounds in M. tuberculosis We isolated M. tuberculosis resistant mutants to two arylamide compounds which are inhibitory to growth under host-relevant conditions (butyrate as a sole carbon source). Thirteen mutants were characterized, and all had mutations in Rv2571c; mutations included a premature stop codon and frameshifts as well as non-synonymous polymorphisms. We isolated a further ten strains with mutations in Rv2571c with resistance. Complementation with a wild-type copy of Rv2571c restored arylamide sensitivity. Over-expression of Rv2571c was toxic in both wild-type and mutant backgrounds. We constructed M. tuberculosis strains with an unmarked deletion of the entire Rv2571c gene by homologous recombination and confirmed that these were resistant to the arylamide series. Rv2571c is a member of the aromatic amino acid transport family and has a fusaric acid resistance domain which is associated with compound transport. Since loss or inactivation of Rv2571c leads to resistance, we propose that Rv2571c is involved in the import of arylamide compounds.

Gut Epithelial Metabolism as a Key Driver of Intestinal Dysbiosis Associated with Noncommunicable Diseases.

In high-income countries, the leading causes of death are noncommunicable diseases (NCDs), such as obesity, cancer, and cardiovascular disease. An important feature of most NCDs is inflammation-induced gut dysbiosis characterized by a shift in the microbial community structure from obligate to facultative anaerobes such as Proteobacteria This microbial imbalance can contribute to disease pathogenesis by either a depletion in or the production of microbiota-derived metabolites. However, little is known about the mechanism by which inflammation-mediated changes in host physiology disrupt the microbial ecosystem in our large intestine leading to disease. Recent work by our group suggests that during gut homeostasis, epithelial hypoxia derived from peroxisome proliferator-activated receptor γ (PPAR-γ)-dependent ß-oxidation of microbiota-derived short-chain fatty acids limits oxygen availability in the colon, thereby maintaining a balanced microbial community. During inflammation, disruption in gut anaerobiosis drives expansion of facultative anaerobic Enterobacteriaceae, regardless of their pathogenic potential. Therefore, our research group is currently exploring the concept that dysbiosis-associated expansion of Enterobacteriaceae can be viewed as a microbial signature of epithelial dysfunction and may play a greater role in different models of NCDs, including diet-induced obesity, atherosclerosis, and inflammation-associated colorectal cancer.

In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis.

Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis.

Inflammation boosts the availability of electron acceptors in the intestinal lumen, creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection increases intestinal levels of the amino acid aspartate. S. Tm used aspartate-ammonia lyase (aspA)-dependent fumarate respiration for growth in the murine gut only during inflammation. AspA-dependent growth advantage was abolished in the gut of germ-free mice and restored in gnotobiotic mice colonized with members of the classes Bacteroidia and Clostridia. Reactive oxygen species (ROS) produced during the host response caused lysis of commensal microbes, resulting in the release of microbiota-derived aspartate that was used by S. Tm, in concert with nitrate-dependent anaerobic respiration, to outcompete commensal Enterobacteriaceae. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent S. Tm expansion during colitis.

An early-life microbiota metabolite protects against obesity by regulating intestinal lipid metabolism.

The mechanisms by which the early-life microbiota protects against environmental factors that promote childhood obesity remain largely unknown. Using a mouse model in which young mice are simultaneously exposed to antibiotics and a high-fat (HF) diet, we show that Lactobacillus species, predominant members of the small intestine (SI) microbiota, regulate intestinal epithelial cells (IECs) to limit diet-induced obesity during early life. A Lactobacillus-derived metabolite, phenyllactic acid (PLA), protects against metabolic dysfunction caused by early-life exposure to antibiotics and a HF diet by increasing the abundance of peroxisome proliferator-activated receptor γ (PPAR-γ) in SI IECs. Therefore, PLA is a microbiota-derived metabolite that activates protective pathways in the small intestinal epithelium to regulate intestinal lipid metabolism and prevent antibiotic-associated obesity during early life.

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance.

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

5-Aminosalicylic Acid Ameliorates Colitis and Checks Dysbiotic Escherichia coli Expansion by Activating PPAR-γ Signaling in the Intestinal Epithelium.

5-Aminosalicylic acid (5-ASA), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, is a widely used first-line medication for the treatment of ulcerative colitis, but its anti-inflammatory mechanism is not fully resolved. Here, we show that 5-ASA ameliorates colitis in dextran sulfate sodium (DSS)-treated mice by activating PPAR-γ signaling in the intestinal epithelium. DSS-induced colitis was associated with a loss of epithelial hypoxia and a respiration-dependent luminal expansion of Escherichia coli, which could be ameliorated by treatment with 5-ASA. However, 5-ASA was no longer able to reduce inflammation, restore epithelial hypoxia, or blunt an expansion of E. coli in DSS-treated mice that lacked Pparg expression specifically in the intestinal epithelium. These data suggest that the anti-inflammatory activity of 5-ASA requires activation of epithelial PPAR-γ signaling, thus pointing to the intestinal epithelium as a potential target for therapeutic intervention in ulcerative colitis.IMPORTANCE An expansion of Enterobacterales in the fecal microbiota is a microbial signature of dysbiosis that is linked to many noncommunicable diseases, including ulcerative colitis. Here, we used Escherichia coli, a representative of the Enterobacterales, to show that its dysbiotic expansion during colitis can be remediated by modulating host epithelial metabolism. Dextran sulfate sodium (DSS)-induced colitis reduced mitochondrial activity in the colonic epithelium, thereby increasing the amount of oxygen available to fuel an E. coli expansion through aerobic respiration. Activation of epithelial peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling with 5-aminosalicylic acid (5-ASA) was sufficient to restore mitochondrial activity and blunt a dysbiotic E. coli expansion. These data identify the host's epithelial metabolism as a potential treatment target to remediate microbial signatures of dysbiosis, such as a dysbiotic E. coli expansion in the fecal microbiota.

High-fat diet-induced colonocyte dysfunction escalates microbiota-derived trimethylamine N-oxide.

A Western-style, high-fat diet promotes cardiovascular disease, in part because it is rich in choline, which is converted to trimethylamine (TMA) by the gut microbiota. However, whether diet-induced changes in intestinal physiology can alter the metabolic capacity of the microbiota remains unknown. Using a mouse model of diet-induced obesity, we show that chronic exposure to a high-fat diet escalates Escherichia coli choline catabolism by altering intestinal epithelial physiology. A high-fat diet impaired the bioenergetics of mitochondria in the colonic epithelium to increase the luminal bioavailability of oxygen and nitrate, thereby intensifying respiration-dependent choline catabolism of E. coli In turn, E. coli choline catabolism increased levels of circulating trimethlamine N-oxide, which is a potentially harmful metabolite generated by gut microbiota.

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors.

The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.IMPORTANCEMycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

Ribosomal protein L7/L12 is required for GTPase translation factors EF-G, RF3, and IF2 to bind in their GTP state to 70S ribosomes.

Ribosomal protein L7/L12 is associated with translation initiation, elongation, and termination by the 70S ribosome. The guanosine 5' triphosphate hydrolase (GTPase) activity of elongation factor G (EF-G) requires the presence of L7/L12, which is critical for ribosomal translocation. Here, we have developed new methods for the complete depletion of L7/L12 from Escherichia coli 70S ribosomes to analyze the effect of L7/L12 on the activities of the GTPase factors EF-G, RF3, IF2, and LepA. Upon removal of L7/L12 from ribosomes, the GTPase activities of EF-G, RF3, and IF2 decreased to basal levels, while the activity of LepA decreased marginally. Upon reconstitution of ribosomes with recombinant L12, the GTPase activities of all GTPases returned to full activity. Moreover, ribosome binding assays indicated that EF-G, RF3, and IF2 require L7/L12 for stable binding in the GTP state, and LepA retained > 50% binding. Lastly, an EF-G∆G' truncation mutant possessed ribosome-dependent GTPase activity, which was insensitive to L7/L12. Our results indicate that L7/L12 is required for stable binding of ribosome-dependent GTPases that harbor direct interactions to the L7/L12 C-terminal domains, either through a G' domain (EF-G, RF3) or a unique N-terminal domain (IF2). Furthermore, we hypothesize this interaction is concomitant with counterclockwise ribosomal intersubunit rotation, which is required for translocation, initiation, and post-termination.