Philip D. Butcher

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.

Benzothiazinones Kill Mycobacterium tuberculosis by Blocking Arabinan Synthesis

Ammunition for the TB Wars Tuberculosis is a major human disease of global importance resulting from infection with the air-borne pathogen Mycobacterium tuberculosis, which is becoming increasingly resistant to all available drugs. An antituberculosis benzothiazinone compound kills mycobacterium in infected cells and in mice. Makarov et al. (p. 801) have identified a sulfur atom and nitro residues important for benzothiazinones activity and used genetic methods and biochemical analysis to identify its target in blocking arabinogalactan biosynthesis during cell-wall synthesis. The compound affects the same pathway as ethambutol, and thus a benzothiazinone drug has the potential to become an important part of treatment of drug-resistant disease and, possibly, replace the less effective ethambutol in the primary treatment of tuberculosis. An isomerase required for cell-wall synthesis is a target for an alternative drug lead for tuberculosis treatment. New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-β-d-ribose 2′-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays

Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.

Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.

Background Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. Methods and Findings Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. Conclusion As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

Dissection of ESAT-6 System 1 of Mycobacterium tuberculosis and Impact on Immunogenicity and Virulence

ABSTRACT The dedicated secretion system ESX-1 of Mycobacterium tuberculosis encoded by the extended RD1 region (extRD1) assures export of the ESAT-6 protein and its partner, the 10-kDa culture filtrate protein CFP-10, and is missing from the vaccine strains M. bovis BCG and M. microti. Here, we systematically investigated the involvement of each individual ESX-1 gene in the secretion of both antigens, specific immunogenicity, and virulence. ESX-1-complemented BCG and M. microti strains were more efficiently engulfed by bone-marrow-derived macrophages than controls, and this may account for the enhanced in vivo growth of ESX-1-carrying strains. Inactivation of gene pe35 (Rv3872) impaired expression of CFP-10 and ESAT-6, suggesting a role in regulation. Genes Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877 encoding an ATP-dependent chaperone and translocon were essential for secretion of ESAT-6 and CFP-10 in contrast to ppe68 Rv3873 and Rv3876, whose inactivation did not impair secretion of ESAT-6. A strict correlation was found between ESAT-6 export and the generation of ESAT-6 specific T-cell responses in mice. Furthermore, ESAT-6 secretion and specific immunogenicity were almost always correlated with enhanced virulence in the SCID mouse model. Only loss of Rv3865 and part of Rv3866 did not affect ESAT-6 secretion or immunogenicity but led to attenuation. This suggests that Rv3865/66 represent a new virulence factor that is independent from ESAT-6 secretion. The present study has allowed us to identify new aspects of the extRD1 region of M. tuberculosis and to explore its role in the pathogenesis of tuberculosis.

High-Dose Rifapentine with Moxifloxacin for Pulmonary Tuberculosis

BACKGROUND Tuberculosis regimens that are shorter and simpler than the current 6-month daily regimen are needed. METHODS We randomly assigned patients with newly diagnosed, smear-positive, drug-sensitive tuberculosis to one of three regimens: a control regimen that included 2 months of ethambutol, isoniazid, rifampicin, and pyrazinamide administered daily followed by 4 months of daily isoniazid and rifampicin; a 4-month regimen in which the isoniazid in the control regimen was replaced by moxifloxacin administered daily for 2 months followed by moxifloxacin and 900 mg of rifapentine administered twice weekly for 2 months; or a 6-month regimen in which isoniazid was replaced by daily moxifloxacin for 2 months followed by one weekly dose of both moxifloxacin and 1200 mg of rifapentine for 4 months. Sputum specimens were examined on microscopy and after culture at regular intervals. The primary end point was a composite treatment failure and relapse, with noninferiority based on a margin of 6 percentage points and 90% confidence intervals. RESULTS We enrolled a total of 827 patients from South Africa, Zimbabwe, Botswana, and Zambia; 28% of patients were coinfected with the human immunodefiency virus. In the per-protocol analysis, the proportion of patients with an unfavorable response was 4.9% in the control group, 3.2% in the 6-month group (adjusted difference from control, -1.8 percentage points; 90% confidence interval [CI], -6.1 to 2.4), and 18.2% in the 4-month group (adjusted difference from control, 13.6 percentage points; 90% CI, 8.1 to 19.1). In the modified intention-to-treat analysis these proportions were 14.4% in the control group, 13.7% in the 6-month group (adjusted difference from control, 0.4 percentage points; 90% CI, -4.7 to 5.6), and 26.9% in the 4-month group (adjusted difference from control, 13.1 percentage points; 90% CI, 6.8 to 19.4). CONCLUSIONS The 6-month regimen that included weekly administration of high-dose rifapentine and moxifloxacin was as effective as the control regimen. The 4-month regimen was not noninferior to the control regimen. (Funded by the European and Developing Countries Clinical Trials Partnership and the Wellcome Trust; RIFAQUIN Current Controlled Trials number, ISRCTN44153044.).

Differential expression of mycobacterial proteins following phagocytosis by macrophages

Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.

Probing Host Pathogen Cross-Talk by Transcriptional Profiling of Both Mycobacterium tuberculosis and Infected Human Dendritic Cells and Macrophages.

Background Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. Methodology/Principal Findings In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. Conclusions/Significance This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

Mycobacterial P1-Type ATPases Mediate Resistance to Zinc Poisoning in Human Macrophages

Summary Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P1-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.