Summary During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

Mycobacterium tuberculosis (Mtb) senses and adapts to acidic environments during the course of infection. Acidic pH-dependent adaptations include the induction of metabolic genes associated with anaplerosis and growth arrest on specific carbon sources. Here we report that deletion of isocitrate lyase or phosphoenolpyruvate carboxykinase results in reduced growth at acidic pH and altered metabolite profiles, supporting that remodeling of anaplerotic metabolism is required for pH-dependent adaptation. Mtb cultured at pH 5.7 in minimal medium containing glycerol as a single carbon source exhibits an acid growth arrest phenotype, where the bacterium is non-replicating but viable and metabolically active. The bacterium assimilates and metabolizes glycerol and maintains ATP pools during acid growth arrest and becomes tolerant to detergent stress and the antibiotics isoniazid and rifampin. A forward genetic screen identified mutants that do not arrest their growth at acidic pH, including four enhanced acid growth (eag) mutants with three distinct mutations in the proline-proline-glutamate (PPE) gene MT3221 (also named ppe51). Overexpression of the MT3221(S211R) variant protein in wild type Mtb results in enhanced acid growth and reduced drug tolerance. These findings support that acid growth arrest is a genetically controlled, adaptive process and not simply a physiological limitation associated with acidic pH.

Low-density lipoprotein receptor–related proteins 5 and 6 (Lrp5 and Lrp6) are co-receptors of Wnt ligands and play important roles in Wnt/β-catenin signal transduction. Mice homozygous for a germline deletion of Lrp6 die at birth with several associated defects, while Lrp5-deficient mice are viable. Here we conditionally deleted Lrp5 and/or Lrp6 in the mouse gut (gut−/−) by crossing mice carrying floxed alleles of Lrp5 and Lrp6 to a strain expressing Cre recombinase from the villin promoter (villin-Cre). The changes in morphology, differentiation and Wnt signal transduction were validated using immunohistochemistry and other staining. Consistent with observations in mice carrying a homozygous germline deletion in Lrp5, intestinal development in Lrp5gut−/− mice was normal. In addition, mice homozygous for villin-Cre–induced deletion of Lrp6 (Lrp6gut−/−) were viable with apparently normal intestinal differentiation and function. However, mice homozygous for villin-Cre inactivated alleles of both genes (Lrp5gut−/−;Lrp6gut−/−) died within one day of birth. Analysis of embryonic Lrp5gut−/−;Lrp6gut−/− intestinal epithelium showed a progressive loss of cells, an absence of proliferation, and a premature differentiation of crypt stem/precursor cells; no notable change in differentiation was observed in the embryos lacking either gene alone. Further immunohistochemical studies showed that expression of the Wnt/β-catenin target, cyclin D1, was specifically reduced in the intestinal epithelium of Lrp5gut−/−;Lrp6gut−/− embryos. Our data demonstrate that Lrp5 and Lrp6 play redundant roles in intestinal epithelium development, and that Lrp5/6 might regulate intestinal stem/precursor cell maintenance by regulating Wnt/β-catenin signaling.

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