Lanbo Shi

Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence

The lung is the primary target of infection with Mycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication of M. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes of M. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-γ and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for α-crystallin (acr), rv2626c, and rv2623 and decreased for superoxide dismutase C (sodC), sodA, and fibronectin-binding protein B (fbpB). This pattern of M. tuberculosis transcription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001) Annu. Rev. Microbiol. 55, 139–163] associated with adaptation of tubercle bacilli to hypoxia in vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-γ gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages; sodC, sodA, and fbpB transcription showed no decrease; and acr, rv2626c, and rv2623 transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature of M. tuberculosis as it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.

Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration

Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus of Mycobacterium tuberculosis was conducted in vivo in the infected mouse lung, and in vitro in bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and the aa3-type cytochrome c oxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochrome bd oxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation of M. tuberculosis to host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with a cydC mutant (defective in the cytochrome bd oxidase-associated transporter) at the transition to chronic infection provided initial evidence that the bd oxidase pathway is required for M. tuberculosis adaptation to host immunity. In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochrome bd oxidase expression increased, but cytochrome c oxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism during M. tuberculosis growth arrest in vitro and in vivo will guide manipulation of in vitro conditions to model bacterial adaptation to host immunity.

The temporal response of the Mycobacterium tuberculosis gene regulatory network during growth arrest

The virulence of Mycobacterium tuberculosis depends on the ability of the bacilli to switch between replicative (growth) and non‐replicative (dormancy) states in response to host immunity. However, the gene regulatory events associated with transition to dormancy are largely unknown. To address this question, we have assembled the largest M. tuberculosis transcriptional‐regulatory network to date, and characterized the temporal response of this network during adaptation to stationary phase and hypoxia, using published microarray data. Distinct sets of transcriptional subnetworks (origons) were responsive at various stages of adaptation, showing a gradual progression of network response under both conditions. Most of the responsive origons were in common between the two conditions and may help define a general transcriptional signature of M. tuberculosis growth arrest. These results open the door for a systems‐level understanding of transition to non‐replicative persistence, a phenotypic state that prevents sterilization of infection by the host immune response and promotes the establishment of latent M. tuberculosis infection, a condition found in two billion people worldwide.

Antibody Profiles Characteristic of Mycobacterium tuberculosis Infection State

ABSTRACT The relationship between specific antibody profiles and tuberculosis (TB) state was investigated by measuring serum antibody levels to six Mycobacterium tuberculosis antigens in human subjects grouped into four diagnostic categories: active disease, inactive (past) tuberculosis, latent infection without radiographic chest abnormalities, and infection free. Statistical data analyses showed that the latter two groups were serologically indistinguishable and that active tuberculosis and inactive tuberculosis were characterized by different antibody profiles. Antibodies to the 38-kDa antigen, alanine dehydrogenase, and Rv2626c were associated with active TB, while antibodies to the 16-kDa antigen, ferredoxin A, and ESAT-6 were associated with inactive TB. Thus, the targets of the immune response vary with tuberculosis state. The correlation between bacterial antigen production and infection stage was investigated in mice infected with M. tuberculosis by bacterial transcription profiling. It was found that levels of transcripts encoding the six M. tuberculosis antigens varied during infection. Together, the data indicate that antigen composition of tubercle bacilli varies with stage of infection and that immunoprofiling can distinguish between tuberculosis states.

Carbon flux rerouting during Mycobacterium tuberculosis growth arrest

A hallmark of the Mycobacterium tuberculosis life cycle is the pathogens ability to switch between replicative and non‐replicative states in response to host immunity. Transcriptional profiling by qPCR of ∼ 50 M. tuberculosis genes involved in central and lipid metabolism revealed a re‐routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity‐induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms.

Effect of Growth State on Transcription Levels of Genes Encoding Major Secreted Antigens of Mycobacterium tuberculosis in the Mouse Lung

ABSTRACT Arrest of the multiplication of Mycobacterium tuberculosis caused by expression of adaptive immunity in mouse lung was accompanied by a 10- to 20-fold decrease in levels of mRNAs encoding the secreted Ag85 complex and 38-kDa lipoprotein. esat-6 mRNA levels were high throughout infection. The data imply that multiplying and nonreplicating tubercle bacilli have different antigen compositions.

Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs.

To elucidate the little-known bioenergetic pathways of host immune cells in tuberculosis, a granulomatous disease caused by the intracellular pathogen Mycobacterium tuberculosis, we characterized infected murine lung tissue by transcriptomic profiling and confocal imaging. Transcriptomic analysis revealed changes of host energy metabolism during the course of infection that are characterized by upregulation of key glycolytic enzymes and transporters for glucose uptake, and downregulation of enzymes participating in the tricarboxylic acid cycle and oxidative phosphorylation. Consistent with elevated glycolysis, we also observed upregulation of a transporter for lactate secretion and a V type H+ -ATPase involved in cytosolic pH homeostasis. Transcription profiling results were corroborated by immunofluorescence microscopy showing increased expression of key glycolytic enzymes in macrophages and T cells in granulomatous lesions. Moreover, we found increased mRNA and protein levels in macrophages and T cells of hypoxia inducible factor 1 alpha (HIF-1α), the regulatory subunit of HIF-1, a master transcriptional regulator. Thus, our findings suggest that immune cells predominantly utilize aerobic glycolysis in response to M. tuberculosis infection. This bioenergetic shift is similar to the Warburg effect, the metabolic signature of cancer cells. Finding immunometabolic changes during M. tuberculosis infection opens the way to new strategies for immunotherapy against tuberculosis.

Regulation of Central Metabolism Genes of Mycobacterium tuberculosis by Parallel Feed-Forward Loops Controlled by Sigma Factor E (σE)

Cells respond to external stimuli through networks of regulatory interactions. The human pathogen Mycobacterium tuberculosis responds to stress encountered during infection by arresting multiplication and implementing critical metabolic changes that lead to or sustain the nonreplicative state. Much of this differentiation program is recapitulated when M. tuberculosis cultures are subjected to gradual oxygen depletion in vitro. Here we report that hypoxic induction of critical central metabolism genes in the glyoxylate shunt (icl1) and in the methylcitrate cycle (gltA1) involves both global and local regulators. The global regulators are accessory sigma factors σ(B) for icl1 and σ(E) for gltA1. The local regulators are the products of two paralogous genes mapping at positions adjacent to the corresponding effector gene or operon. We call these genes lrpI and lrpG (for local regulatory protein of icl1 and gltA1). We also found that (i) each sigma factor controls the corresponding local regulator, (ii) both global and local regulators are required for effector gene induction, and (iii) the occurrence of sigma factor control of effector gene induction is independent of its control over the corresponding local regulator. Together, these data indicate that induction of icl1 and gltA1 utilizes parallel feed-forward loops with an AND input function. Both feed-forward loops are affected by σ(E), since this sigma factor is part of the gltA1 loop and controls sigB in the icl1 loop. Feed-forward loops may critically contribute to the cellular developmental program associated with M. tuberculosis dormancy.

In vitro model of mycobacterial growth arrest using nitric oxide with limited air.

ABSTRACT An in vitro model of mycobacterial growth arrest was developed using Mycobacterium bovis BCG. When an exponentially growing culture was transferred to an evacuated tube, growth continued; treatment with a source of nitric oxide (diethylenetriamine-nitric oxide adduct [DETA-NO] at 50 μM) halted growth immediately, and aeration restored growth. When the period of growth arrest exceeded 4 h, a time lag occurred before aeration could restore growth. The lag time was maximal (24 h) after 16 h of growth arrest. These time lags indicated that one transition period was required for cells to achieve full arrest of growth and another for them to recover fully from growth arrest. DETA-NO-induced growth arrest failed to protect from the lethal effects of anaerobic shock, which caused rapid lysis of both growing and growth-arrested cells. While growth arrest had little effect on the lethal action of rifampin, it eliminated isoniazid lethality. Growth arrest reduced but did not eliminate fluoroquinolone lethality. Two fluoroquinolones, moxifloxacin and gatifloxacin, were equally lethal to exponentially growing cells, but moxifloxacin was more active during growth arrest. This difference is attributed to the fluoroquinolone C-7 ring structure, the only difference between the compounds. Collectively these data characterize a new system for halting mycobacterial growth that may be useful for evaluating new antituberculosis agents.

Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence.

Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation.