Most mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis.

Steryl esters (SEs) are important storage compounds in many eukaryotes and are often prominent components of intracellular lipid droplets. Here, we demonstrate that selected Actino- and Proteobacteria growing on sterols are also able to synthesize SEs and to sequester them in cytoplasmic lipid droplets. We found cholesteryl ester (CE) formation in members of the actinobacterial genera Rhodococcus, Mycobacterium, and Amycolatopsis, as well as several members of the proteobacterial Cellvibrionales order. CEs maximally accumulated under nitrogen-limiting conditions, suggesting that steryl ester formation plays a crucial role for storing excess energy and carbon under adverse conditions. Rhodococcus jostii RHA1 was able to synthesize phytosteryl and cholesteryl esters, the latter reaching up to 7% of its cellular dry weight and 69% of its lipid droplets. Purified lipid droplets from RHA1 contained CEs, free cholesterol, and triacylglycerols. In addition, we found formation of CEs in Mycobacterium tuberculosis when it was grown with cholesterol plus an additional fatty acid substrate. This study provides a basis for the application of bacterial whole-cell systems in the biotechnological production of SEs for use in functional foods and cosmetics. IMPORTANCE Oleaginous bacteria exhibit great potential for the production of high-value neutral lipids, such as triacylglycerols and wax esters. This study describes the formation of steryl esters (SEs) as neutral lipid storage compounds in sterol-degrading oleaginous bacteria, providing a basis for biotechnological production of SEs using bacterial systems with potential applications in the functional food, nutraceutical, and cosmetic industries. We found cholesteryl ester (CE) formation in several sterol-degrading Actino- and Proteobacteria under nitrogen-limiting conditions, suggesting an important role of this process in storing energy and carbon under adverse conditions. In addition, Mycobacterium tuberculosis grown on cholesterol accumulated CEs in the presence of an additional fatty acid substrate.

Staphyloferrin B (SB) is an iron-chelating siderophore produced by in invasive infections. Proteins for SB biosynthesis and export are encoded by the gene cluster, in which SbnI, a member of the ParB/Srx superfamily, acts as a heme-dependent transcriptional regulator of the locus. However, no structural or functional information about SbnI is available. Here, a crystal structure of SbnI revealed striking structural similarity to an ADP-dependent free serine kinase, SerK, from the archaea We found that features of the active sites are conserved, and biochemical assays and P NMR and HPLC analyses indicated that SbnI is also a free serine kinase but uses ATP rather than ADP as phosphate donor to generate the SB precursor-phospho-l-serine (OPS). SbnI consists of two domains, and elevated -factors in domain II were consistent with the open-close reaction mechanism previously reported for SerK. Mutagenesis of Glu and Asp in SbnI disclosed that they are required for kinase activity. The only known OPS source in bacteria is through the phosphoserine aminotransferase activity of SerC within the serine biosynthesis pathway, and we demonstrate that an mutant is a serine auxotroph, consistent with a function in l-serine biosynthesis. However, the mutant strain could produce SB when provided l-serine, suggesting that SbnI produces OPS for SB biosynthesis These findings indicate that besides transcriptionally regulating the locus, SbnI also has an enzymatic role in the SB biosynthetic pathway.

The standard treatment for chronic hepatitis C (pegylated interferon and ribavirin) causes challenging physical and psychological side effects. The current pilot study evaluated the efficacy of a brief, telephone-based, cognitive-behavioral self-management intervention designed to address mood and quality of life within a sample of veterans on antiviral treatment for hepatitis C. Results from this pilot study support the feasibility of this telehealth intervention, showing that veterans were highly satisfied with the content of the intervention and compliant with the telephone calls. Findings further indicate that symptoms of depression and anxiety and mental health quality of life either remained stable or improved in those participants who received the brief telephone intervention, while those receiving usual care showed significant declines in mood and mental health quality of life. The findings from this study provide evidence that a brief, clinician-administered phone intervention may help individuals on antiviral therapy for hepatitis C to cope more effectively with the negative treatment side effects.

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis , shortens treatment in rodents relative to standard of care drugs.