Glutamine is required for M1-like polarization in response to Mycobacterium tuberculosis infection

In response to Mycobacterium tuberculosis infection, macrophages mount early proinflammatory and antimicrobial responses similar to those observed in M1 macrophages classically activated by LPS and IFN-γ. A metabolic reprogramming to HIF-1-mediated uptake of glucose and its metabolism by glycolysis is required for M1-like polarization, but little is known about other metabolic programs driving M1-like polarization during M. tuberculosis infection. Identification and quantification of labeling patterns of U 13 C glutamine and U 13 C glucose-derived metabolites demonstrated that glutamine, rather than glucose, is catabolized in both the oxidative and reductive TCA cycle of M1-like macrophages, thereby generating signaling molecules that include succinate, biosynthetic precursors such as aspartate, and the antimicrobial metabolite itaconate. This conclusion is corroborated by diminished M1 polarization via chemical inhibition of glutaminase (GLS), the key enzyme of the glutaminolysis pathway, and by genetic deletion of GLS in infected macrophages. Furthermore, characterization of the labeling distribution pattern of U 15 N glutamine in M1-like macrophages indicates that glutamine serves as a nitrogen source for the synthesis of intermediates of purine and pyrimidine metabolism plus amino acids including aspartate. Thus, the catabolism of glutamine, as an integral component of metabolic reprogramming in activating macrophages, fulfills the cellular demand for bioenergetic and biosynthetic precursors of M1-like macrophages. Knowledge of these new immunometabolic features of M1-like macrophages is expected to advance the development of host-directed therapies that will enhance bacterial clearance and prevent immunopathology during tuberculosis.

Itaconate: an antimicrobial metabolite of macrophages

Itaconate is a conjugated 1,4-dicarboxylate produced by macrophages. This small molecule has recently received increasing attention due to its role in modulating the immune response of macrophages upon exposure to pathogens. Itaconate has also been proposed to play an antimicrobial function; however, this has not been explored as intensively. Consistent with the latter, itaconate is known to show antibacterial activity in vitro and was reported to inhibit isocitrate lyase, an enzyme required for survival of bacterial pathogens in mammalian systems. Recent studies have revealed bacterial growth inhibition under biologically relevant conditions. In addition, an antimicrobial role for itaconate is substantiated by the high concentration of itaconate found in bacteria-containing vacuoles, and by the production of itaconate-degrading enzymes in pathogens such as Salmonella enterica ser. Typhimurium, Pseudomonas aeruginosa, and Yersinia pestis. This review describes the current state of literature in understanding the role of itaconate as an antimicrobial agent in host-pathogen interactions.

ISOLATION OF POTENTIAL ANTIMICROBIAL METABOLITE FROM ENDOPHYTIC BACILLUS AMYLOLIQUEFACIENS DL06 OF CARNIVOROUS PLANT DROSERA BURMANNII VAHL.

Objectives: Exploitation of bacterial endophytes for production of antimicrobial substances has led to the discovery of novel natural metabolites of diverse chemical nature. The present study focuses attention toward optimization of cultural conditions for production of antimicrobial compound(s) by an endophytic bacterium DL06 followed by its extraction and partial purification. Methods: The leaf endophytic bacterium Bacillus amyloliquefaciens DL06 (GenBank Accession no. MK696415, Microbial Culture Collection Accession no. 4186) isolated from carnivorous plant Drosera burmannii has been identified as a potent producer of antimicrobial metabolite following agar cup assay against several test bacterial and fungal strains. Cultural conditions for production of antimicrobials were optimized by “one variable at a time” method. The active fraction was isolated and purified partially using solvent extraction, thin-layer chromatography, and high performance liquid chromatography (HPLC) analysis. Results: B. amyloliquefaciens DL06 produced maximum antimicrobial compound in tryptic soy broth and Davis–Mingioli’s medium when grown under shake culture. Production of the antimicrobial metabolite has been optimized for the inoculum density, aeration, temperature, pH as well as carbon, and nitrogen sources. The antimicrobial metabolite was extracted from the cell-free culture filtrate in butanol and partially purified by silica gel column chromatography and HPLC. Conclusions: The antimicrobial metabolite, tentatively identified as quercetin showed broad spectrum bioactivity affecting several fungi and a number of Gram-positive and Gram-negative bacteria.

Antimicrobial metabolite profiling of Nigrospora sphaerica from Adiantum philippense L.

Background Endophyte bestows beneficial aspects to its inhabiting host, along with a contribution to diverse structural attributes with biological potential. In this regard, antimicrobial profiling of fungal endophytes from medicinal plant Adiantum philippense revealed bioactive Nigrospora sphaerica from the leaf segment. Chemical and biological profiling through TLC–bioautography and hyphenated spectroscopic techniques confirmed the presence of phomalactone as an antimicrobial metabolite. Results The chemical investigation of the broth extract by bioassay-guided fractionation confirmed phomalactone as a bioactive antimicrobial secondary metabolite. The antimicrobial activity of phomalactone was found to be highest against Escherichia coli by disc diffusion assay. The MIC was found to be significant against both Escherichia coli and Xanthomonas campestris in the case of bacteria and dermatophyte Candida albicans at 150 μg/ml, respectively. Conclusions Overall, the results highlighted the antimicrobial potential of phomalactone from the endophyte Nigrospora sphaerica exhibiting a broad spectrum of antimicrobial activity against human and phytopathogenic bacteria and fungi. This work is the first report regarding the antibacterial activity of phomalactone.

Application of Optimized and Validated Agar Overlay TLC–Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest

The agar overlay TLC–bioautography is one of the crucial methods for simultaneous in situ detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC–bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial capability of ethyl acetate extract and the purified compound coriloxin was determined by disc diffusion, minimal inhibitory concentration and agar overlay TLC–bioautography assay. The visible LOD of coriloxin antimicrobial activity was found at 10 μg for Escherichia coli and 20 μg for both Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as the relative standard deviation is less than 6.56%, which proved that this method was precise. The accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with RSD values 0.94–2.30%, respectively. The overall findings of this investigation suggest that agar overlay TLC–bioautography assay is a suitable and acceptable method for the in situ determination of antimicrobial pharmaceuticals.

Characterization of Pediococcus acidilactici PFC69 and Lactococcus lactis PFC77 Bacteriocins and Their Antimicrobial Activities in Tarhana Fermentation

Tarhana is a traditional cereal product fermented by lactic acid bacteria (LAB) and yeast strains that has gained special interest recently as an infant nutrition. Tarhana contains wheat flour, yogurt, and various vegetables that might create a microbiological toxicological risk, especially for Bacillus cereus and Staphylococcus aureus. In this study, characterization of the metabolites responsible for antibacterial activity of Pediococcus acidilactici PFC69 and Lactococcus lactis PFC77 strains obtained from tarhana was performed, and antibacterial effects were detected against B. cereus ATCC 11778 and S. aureus ATCC 29213 during the fermentation. A total of 12,800 AU/mL antibacterial activity was observed for the supernatants of the PFC69 and PFC77 strains that were found to be stable at high temperature and in low pH conditions and sensitive to proteases, suggesting the antimicrobial metabolite is a bacteriocin. These bacteriocins were further purified and their molecular sizes were determined as 4.5 and 3.5 kDa, respectively. Importantly, inoculation of PFC69 and PFC77 to tarhana dough significantly decreased B. cereus ATCC 11778 and S. aureus ATCC 29213 amounts from the fifth day of fermentation compared to the control dough samples. P. acidilactici PFC69 and L. lactis PFC77 strains were concluded as bioprotective cultures for tarhana and these strains were offered for other cereal-based fermentations.

Small molecule resensitizes Salmonella to itaconate and decreases bacterial proliferation in macrophages

Antimicrobial resistance is a global health crisis offering little reprieve. The situation urgently calls for new drug targets and therapies for infections. We have previously suggested a different approach to treat infections, termed bacterio-modulation, in which a compound modulates the bacterial response to the host immune defense. Herein we show that monocytes infected with Salmonella enterica spp. Typhimurium can be cured using non-antimicrobial compounds that resensitize the bacterium to itaconate, a macrophage-derived antimicrobial metabolite. We propose that this represents a novel strategy to treat infections.