Sabine Ehrt

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages Insights into the Phagosomal Environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogens cell envelope.

The spectrum of latent tuberculosis: rethinking the biology and intervention strategies

Immunological tests provide evidence of latent tuberculosis in one third of the global population, which corresponds to more than two billion individuals. Latent tuberculosis is defined by the absence of clinical symptoms but carries a risk of subsequent progression to clinical disease, particularly in the context of co-infection with HIV. In this Review we discuss the biology of latent tuberculosis as part of a broad range of responses that occur following infection with Mycobacterium tuberculosis, which result in the formation of physiologically distinct granulomatous lesions that provide microenvironments with differential ability to support or suppress the persistence of viable bacteria. We then show how this model can be used to develop a rational programme to discover effective drugs for the eradication of M. tuberculosis infection.

Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.

Mycobacterial survival strategies in the phagosome: defence against host stresses

Infections with Mycobacterium tuberculosis remain a major cause of disease and death in humans. Among the factors that contribute to M. tuberculosiss success as a pathogen is its ability to withstand potentially bactericidal host defences and to resist elimination by an activated immune system. This resistance to killing by the host is in part due to the low permeability of the mycobacterial cell envelope for many toxic molecules. In addition, it depends upon the detoxification of reactive oxygen and reactive nitrogen molecules produced by the host, the repair of the damage these molecules cause and maintenance of a neutral intrabacterial pH within acidic environments. The latter three mechanisms are the focus of this review.

Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection

Metabolic adaptation to the host niche is a defining feature of the pathogenicity of Mycobacterium tuberculosis (Mtb). In vitro, Mtb is able to grow on a variety of carbon sources, but mounting evidence has implicated fatty acids as the major source of carbon and energy for Mtb during infection. When bacterial metabolism is primarily fueled by fatty acids, biosynthesis of sugars from intermediates of the tricarboxylic acid cycle is essential for growth. The role of gluconeogenesis in the pathogenesis of Mtb however remains unaddressed. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the first committed step of gluconeogenesis. We applied genetic analyses and 13C carbon tracing to confirm that PEPCK is essential for growth of Mtb on fatty acids and catalyzes carbon flow from tricarboxylic acid cycle–derived metabolites to gluconeogenic intermediates. We further show that PEPCK is required for growth of Mtb in isolated bone marrow–derived murine macrophages and in mice. Importantly, Mtb lacking PEPCK not only failed to replicate in mouse lungs but also failed to survive, and PEPCK depletion during the chronic phase of infection resulted in mycobacterial clearance. Mtb thus relies on gluconeogenesis throughout the infection. PEPCK depletion also attenuated Mtb in IFNγ-deficient mice, suggesting that this enzyme represents an attractive target for chemotherapy.

Metabolomics of Mycobacterium tuberculosis Reveals Compartmentalized Co-Catabolism of Carbon Substrates

Metabolic adaptation to the host environment is a defining feature of the pathogenicity of Mycobacterium tuberculosis (Mtb), but we lack biochemical knowledge of its metabolic networks. Many bacteria use catabolite repression as a regulatory mechanism to maximize growth by consuming individual carbon substrates in a preferred sequence and growing with diauxic kinetics. Surprisingly, untargeted metabolite profiling of Mtb growing on ¹³C-labeled carbon substrates revealed that Mtb could catabolize multiple carbon sources simultaneously to achieve enhanced monophasic growth. Moreover, when co-catabolizing multiple carbon sources, Mtb differentially catabolized each carbon source through the glycolytic, pentose phosphate, and/or tricarboxylic acid pathways to distinct metabolic fates. This unusual topologic organization of bacterial intermediary metabolism has not been previously observed and may subserve the pathogenicity of Mtb.

A membrane protein preserves intrabacterial pH in intraphagosomal Mycobacterium tuberculosis

Acidification of the phagosome is considered to be a major mechanism used by macrophages against bacteria, including Mycobacterium tuberculosis (Mtb). Mtb blocks phagosome acidification, but interferon-γ (IFN-γ) restores acidification and confers antimycobacterial activity. Nonetheless, it remains unclear whether acid kills Mtb, whether the intrabacterial pH of any pathogen falls when it is in the phagosome and whether acid resistance is required for mycobacterial virulence. In vitro at pH 4.5, Mtb survived in a simple buffer and maintained intrabacterial pH. Therefore, Mtb resists phagolysosomal concentrations of acid. Mtb also maintained its intrabacterial pH and survived when phagocytosed by IFN-γ–activated macrophages. We used transposon mutagenesis to identify genes responsible for Mtbs acid resistance. A strain disrupted in Rv3671c, a previously uncharacterized gene encoding a membrane-associated protein, was sensitive to acid and failed to maintain intrabacterial pH in acid in vitro and in activated macrophages. Growth of the mutant was also severely attenuated in mice. Thus, Mtb is able to resist acid, owing in large part to Rv3671c, and this resistance is essential for virulence. Disruption of Mtbs acid resistance and intrabacterial pH maintenance systems is an attractive target for chemotherapy.

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the worlds population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-γ–deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtbs genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.

Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon

An estimated one-third of the worlds population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Here, we demonstrate that, unlike wild-type M. tuberculosis, a strain of M. tuberculosis disrupted in the mce1 operon was unable to enter a stable persistent state of infection in mouse lungs. Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain. Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration. Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor α, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4. The mce1 mutant strain complemented with the mce1 genes stimulated tumor necrosis factor α and NO production by murine macrophages at levels stimulated by the wild-type strain. These observations indicate that the mce1 operon mutant is unable to stimulate T helper 1-type immunity in mice. The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism. The mce1 operon of M. tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host.

Protection from Alzheimer's-like disease in the mouse by genetic ablation of inducible nitric oxide synthase

Brains from subjects who have Alzheimers disease (AD) express inducible nitric oxide synthase (iNOS). We tested the hypothesis that iNOS contributes to AD pathogenesis. Immunoreactive iNOS was detected in brains of mice with AD-like disease resulting from transgenic expression of mutant human β-amyloid precursor protein (hAPP) and presenilin-1 (hPS1). We bred hAPP-, hPS1-double transgenic mice to be iNOS+/+ or iNOS−/−, and compared them with a congenic WT strain. Deficiency of iNOS substantially protected the AD-like mice from premature mortality, cerebral plaque formation, increased β-amyloid levels, protein tyrosine nitration, astrocytosis, and microgliosis. Thus, iNOS seems to be a major instigator of β-amyloid deposition and disease progression. Inhibition of iNOS may be a therapeutic option in AD.