Mtb Pathogenesis Research Articles

New tricks for old dogs: countering antibiotic resistance in tuberculosis with host-directed therapeutics.

Despite the availability of Mycobacterium tuberculosis (Mtb) drugs for over 50 years, tuberculosis (TB) remains at pandemic levels. New drugs are urgently needed for resistant strains, shortening duration of treatment, and targeting different stages of the disease, especially for treatment during human immunodeficiency virus co-infection. One solution to the conundrum that antibiotics kill the bacillus yet select for resistance is to target the host rather than the pathogen. Here, we discuss recent progress in so-called 'host-directed therapeutics' (HDTs), focusing on two general mechanistic strategies: (i) HDTs that disrupt Mtb pathogenesis in macrophages and (ii) immunomodulatory HDTs that facilitate protective immune responses that kill Mtb or reduce deleterious responses that exacerbate disease. HDTs hold significant promise as adjunctive therapies in that they are less likely to engender resistance, will likely have efficacy against antibiotic-resistant strains, and may have activity against non-replicating Mtb. However, TB is a complex and variegated disease, and human populations exhibit significant diversity in their immune responses to it, which presents a complicated landscape for HDTs to navigate. Nevertheless, we suggest that a detailed mechanistic understanding of drug action, together with careful selection of disease stage targets and dosing strategies may overcome such limitations and allow the development of HDTs as effective adjunctive treatment options for TB.

The Mycobacterium tuberculosis Clp Gene Regulator Is Required for in Vitro Reactivation from Hypoxia-induced Dormancy

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease worldwide and is the causative agent of tuberculosis (Chao, M. C., and Rubin, E. J. (2010) Annu. Rev. Microbiol. 64, 293-311). Throughout infection, Mtb encounters a variety of host pressures. Thus, responding to these host stresses via the induction of multiple regulatory networks is needed for survival within the host. The Clp protease gene regulator, Rv2745c (clgR), is induced in response to environmental stress conditions, implicating its potential role in Mtb pathogenesis. Transcriptional activation of genes downstream of Rv2745c occurs in a condition-dependent manner. Our isogenic Mtb:ΔRv2745c mutant expresses a significantly different phenotype upon reaeration conditions. Transcriptional analysis revealed differential gene expression profiles relative to wild-type Mtb. Rv2745c is strongly induced in response to hypoxic and reaeration conditions, implicating a role of Rv2745c in vivo during both establishment of infection and reactivation. We found dysregulation of downstream genes within both the σ(H)/σ(E) regulon as well as the dosR regulon in the isogenic mutant, Mtb:ΔRv2745c. Upon hypoxic and reaeration conditions, Clp protease induction occurred within wild-type Mtb, indicating that activation of clgR, which subsequently leads to Clp protease induction, is crucial for degradation of misfolded proteins and ultimately survival of Mtb upon specific stress conditions. Our data indicate the diverse response of Rv2745c, σ(H) and σ(E) in response to a variety of stress conditions. Activation of Rv2745c in response to various stress conditions leads to differential activation of downstream genes, indicating the diverse role of Rv2745c and its importance for Mtb survival in vivo.

Proteins purified from Mycobacterium tuberculosis MDR and Susceptible clinical isolates: Identification by proteomics approach

Aims and objectivesTuberculosis (TB) remains a major global health problem. The incidence of multidrug-resistant tuberculosis (MDR-TB) has increased in recent years. To combat the disease, novel intervention strategies effective against drug-resistant and susceptible subpopulations of Mycobacterium tuberculosis are urgently required as adducts in present treatment regimen. This study has employed a proteomic approach, which is a direct method, to identify proteins from resistant M. tuberculosis isolates compared with sensitive isolates. MethodsThe clinical isolates M.tuberculosis strains were provided by the TB bank of Pasteur Institute of Iran. The bacilli were cultured on 7H9 broth with ADC enrichment at 37°C for 3–4weeks (late log phase). Cells were washed with PBS, suspended in sonication buffer (10mM DTT, 1μg/ml DNAse, 100mM Glycerol, 10mM Triton X100, 20mM EDTA, 50mM Tris-Hcl, 1mM PMSF, 0.02% sodium azide, pH 7.4) and then were subjected to sonication for 1h at 50Hz, in ice, and subsequently centrifuged at 5000rpm for 45min at −4°C. Proteins were precipitated by adding refrigerated ethanol or saturated ammonium sulfate to the supernatant. Pellet resuspended in PBS and then dialyzed for 24h against PBS pH 7.4. IEF was carried out using the Ettan IPGphor 3 isoelecteric focusing system. Immobilized pH gradient IPG strips of pH 4–7 and length 11cm were rehydrated overnight at 20°C with 500μg protein which was mixed with rehydration buffer. Proteins were separated in second dimension on 12% SDS-PAGE in a vertical electrophoretic dual gel. 2D gels were analyzed using Image Master, Melanie 7.0 software. Mass spectrometry was performed using Autoflex II TOF/TOF. Protein sequences were retrieved from Tuberculist server hosted by Pasteur Institute, Paris. ResultsMass spectrometry and bioinformatic characterization of both drug-resistant and sensitive isolates of M. tuberculosis showed that the majority of commonly expressed/upregulated proteins belonged to the cellular metabolism and respiration category (Rv0866, Rv3057c, Rv3248c, Rv1133c, Rv0462, and Rv1876). One hypothetical protein (Rv2744c) and two membrane and cell wall fraction proteins (Rv0379, Rv1886c) were identified. Protein spot 1 was 60kDa chaperonin-2/GroEl-2 (Rv0440). This essential gene prevents misfolding and promotes the refolding and proper assembly of unfolded/misfolded polypeptides generated under stress conditions. Further, another spot Rv2013c (HSP16.3/HSPX) has been shown to be induced under oxygen-deficient conditions. Its role in maintenance of long-term viability during latent, asymptomatic infections and in replication during initial infection has also been proposed. ConclusionSuch information could be helpful for the development of newer therapeutic agents or of diagnostic markers for better treatment or diagnosis of TB. This study extends the list of the potential determinants of differences in virulence between the two isolates (MDR and susceptible TB) and adds to the current understanding of MTB pathogenesis.

Agents of change - concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling.

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two-component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O-phosphorylation) in prokaryotes such as Mtb. However, as novel functions and components of bacterial O-phosphorylation are identified, distinct differences between pro- and eukaryotic phosphosignalling systems become apparent. The emerging picture of O-phosphorylation in Mtb is complicated, goes beyond the eukaryotic paradigms, and shows the limitations of viewing bacterial phosphosignalling within the confines of the 'eukaryotic-like' model. Here, we summarize recent findings about Ser/Thr and the recently discovered Tyr phosphorylation pathways in Mtb, highlight the similarities and differences between eukaryotic and prokaryotic O-phosphorylation, and pose additional questions about signalling components, pathway organization, and ultimately, the cellular roles of O-phosphorylation in Mtb physiology and pathogenesis.

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources.

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.

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σH/σE regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.

A novel mechanism of host cell death by ESAT-6 like protein of Mycobacterium tuberculosis

Background: Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), manipulates the host immune responses for its survival. Several secretary proteins exported through ESX secretion system modulate the antibacterial effector functions of macrophages. Here we attempted to functionally elucidate the role of one of the previously uncharacterized ESAT-6 family protein in Mtb pathogenesis. Methods & Materials: Mtb gene is cloned and expressed in Mycobacterium smegmatis. Invasion assay is performed in HeLa cells where as survival assays were carried out in both mouse and human macrophages. Survival mechanisms were studied by quantifying free radicals formation and host survival rate. Induction of autophagic pathway is studied by the expression of LC3. Nuclear and chromosomal aberrations were studied using micronucleus assay. The level of cytokine expressions were measured using qPCR. Results: Recombinant Mycobacterium smegmatis strain expressing ESAT-6 family protein showed more invasion in human epithelial cells (HeLa) and survival in mouse macrophages (RAW264.7) and human monocyte derived macrophages (THP-1). The survival mechanistic studies revealed, infection induced host cell death and also up regulation of the level of nitric oxide, inducible nitric oxide synthase, reactive oxygen species, superoxide dismutase and catalase activities. Moreover, Mycobacterium smegmatis expressing ESAT-6 infection reduced T-cell survival, caused loss of mitochondrial membrane integrity, activated autophagy and also induced the expression of NF-κB and late endosomal markers such as Rab10, LAMP-2, Cathepsin-B and Cathepsin-Z in macrophages. An up regulation of both pro-inflammatory (TNF-α, IL-12) and anti-inflammatory (IL-10, TGF-β) cytokines was also observed after infection in macrophages. Genome stability studies showed that recombinant Mycobacterium smegmatis strain expressing ESAT-6 family protein infection led to several chromosomal abnormalities such as micronuclei formation in cytokinesis blocked cells, chromosomal malsegregation and nuclear fragmentation. Conclusion: We envision that this study offers a novel mechanism of host cell death by Mtb infection. Taken together, this study has identified a novel ESAT-6 like family protein that acts as virulence as well as an immunomodulatory factor to evade killing by host, and thus it may provide a novel drug target.

Towards the functional characterization and inhibition of hydrolase enzymes in mycobacteria (972.5)

The emergence of multiple and extremely drug‐resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) in humans, threatens the usefulness of our current therapeutic arsenal. As a result, there is an increasing need to identify novel mycobacterial targets that may lead to shorter, more effective treatment regimens. One promising group of potential targets is the serine hydrolases, which have diverse activities, but use a conserved nucleophilic serine residue for catalysis. In Mtb, many SHs have essential functions for Mtb survival, which makes the SH family a promising candidate for drug screening in mycobacteria. Using activity‐based probe profiling, the SH proteomes were visualized in three mycobacterial species that serve as models for virulent Mtb. Distinct populations of proteins were detect in the cell envelope versus the cytosol and between species. Importantly, mass spectrometry analysis of the SH proteome from attenuated Mtb identified known SHs, bioinformatically predicted SHs, and unannotated proteins as SHs. Furthermore, pre‐treatment of attenuated Mtb lysates with small‐molecule inhibitors resulted in concentration‐dependent inhibition of select enzymes. In conclusion, these data suggests that interspecies differences in the mycobacterial SH proteome can aid in understanding Mtb pathogenesis and that using activity‐based probe profiling will help identify selective inhibitors that have potential antimicrobial activity or can be used for characterizing newly annotated SHs. Therefore in the future, we will pursue small‐molecule screens to identify inhibitors that will aid in the functional characterization of these newly annotated SHs. Grant Funding Source: Supported by NIGMS T32‐GM008444, NIGMS T32‐GM092714, and the American Lung Association

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.

Card Regulation of Mycobacterial Transcription Initiation

A staggering 30% of the world's population is infected with latent tuberculosis (WHO 2010). As such, Mycobacterium tuberculosis (Mtb) represents a major global burden to public health, and understanding its pathology is crucial to improving therapies. CarD is a transcription factor essential for Mtb survival and pathogenesis. CarD is known to bind both RNA polymerase (RNAP) as well as DNA. Weakening CarD's interactions with RNAP improves efficacy of Rifampicin, which demonstrates the immediate clinical potential of CarD as a therapeutic target. The mechanism of CarD's interaction with mycobacterial RNAP and DNA during transcription is unknown, although ChIP-seq data shows CarD is most often located on promoter DNA throughout the genome suggesting that CarD is involved in initiation of transcription. CarD is also found at all rRNA promoters, so it is possible that CarD's essentiality arises from its role in regulation of rRNA levels during growth and latency. Therefore, to understand this essential component of Mtb pathogenesis, we aim to elucidate the mechanism of CarD-mediated regulation of transcription initiation at mycobacterial rRNA promoters.

Pupylation as a signal for proteasomal degradation in bacteria

Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that certain bacteria use a tagging pathway functionally related to ubiquitination in order to target proteins for proteasomal degradation. However, functional analogies do not translate into structural or mechanistic relatedness. Bacterial Pup, unlike eukaryotic Ub, does not adopt a β-grasp fold, but is intrinsically disordered. Furthermore, isopeptide bond formation in the pupylation process is carried out by enzymes evolutionary descendent from glutamine synthetases. While in eukaryotes, the proteasome is the main energy-dependent protein degradation machine, bacterial proteasomes exist in addition to other architecturally related degradation complexes, and their specific role along with the role of pupylation is still poorly understood. In Mycobacterium tuberculosis (Mtb), the Pup–proteasome system contributes to pathogenicity by supporting the bacterium's persistence within host macrophages. Here, we describe the mechanism and structural framework of pupylation and the targeting of pupylated proteins to the proteasome complex. Particular attention is given to the comparison of the bacterial Pup–proteasome system and the eukaryotic ubiquitin–proteasome system. Furthermore, the involvement of pupylation and proteasomal degradation in Mtb pathogenesis is discussed together with efforts to establish the Pup–proteasome system as a drug target. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Mycobacterium tuberculosis proteasomes, pupylation and pathogenesis

Prokaryotic ubiquitin‐like protein, Pup, specifically conjugates to proteasome substrates in the human pathogen Mycobacterium tuberculosis (Mtb). Like ubiquitylation, pupylation occurs on lysines, but proceeds by a different chemistry. I will discuss recent developments in the characterization of the Pup pathway. The Pupproteasome system is essential for the pathogenesis of Mtb, thus a better understanding of this pathway will hopefully allow us to develop new drugs to treat one of the world's most devastating diseases.

Glucose Phosphorylation Is Required for Mycobacterium tuberculosis Persistence in Mice

Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. 13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host–pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.

Effect of carbon monoxide on Mycobacterium tuberculosis pathogenesis

The intracellular pathogen Mycobacterium tuberculosis (Mtb) is exposed to multiple host antimicrobial pathways, including toxic gases such as superoxide, nitric oxide and carbon monoxide (CO). To survive, mycobacteria evolved mechanisms to resist the toxic environment, and in this review we focus on a relatively new field, namely, the role of macrophage heme oxygenase and its enzymatic product CO in Mtb pathogenesis. In particular, we focus on (i) the induction of heme oxygenase during Mtb infection and its relevance to Mtb pathogenesis, (ii) the ability of mycobacteria to catabolize CO, (iii) the transcriptional reprogramming of Mtb by exposure to CO, (iv) the general antimicrobial properties of CO and (v) new genetic evidence characterizing the ability of Mtb to resist CO toxicity. Developing a complete molecular and genetic understanding of the pathogenesis of Mtb is essential to its eventual eradication.

Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis

BackgroundThe sequence of the pathogen Mycobacterium tuberculosis (Mtb) strain H37Rv has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other Mtb strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of Mtb pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of Mtb and M. bovis, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes, and Bifidobacterium longum.ResultsOur results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four.ConclusionsOur analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of Mtb in a comparative context, and are available online and through TBDB.org.

Interleukin-17A differentially modulates BCG induction of cytokine production in human blood macrophages

The pathogenesis of Mtb depends in part on cytokine cross-regulation between macrophages and T cells in host immunity. Th17 cells produce IL-17A to induce granuloma formation and to restrict mycobacterial dissemination. IL-17A also mediates cytokine responses induced by proinflammatory cytokines such as TNF-α. Our previous results showed that BCG induces IL-6, IL-10, and TNF-α via activity of protein kinases, including dsRNA-activated serine/threonine protein kinase and glycogen synthase kinase-3 in primary human monocytes. Therefore, we investigated whether IL-17A, upon its induction by BCG, plays an additional role to aid the production of downstream proinflammatory cytokines in macrophages. Here, we showed that IL-17A enhanced IL-6 mRNA and protein levels inducible by BCG in a time- and dose-dependent manner, whereas it had no effect on IL-10 and TNF-α production. We also demonstrated that IL-17A activated the phosphorylation of ERK1/2 triggered by BCG. With the use of a specific chemical inhibitor of a MAPK/ERK-activating kinase (MEK1/2), we confirmed the correlation between the enhanced ERK1/2 activation and augmented IL-6 production. Additionally, we revealed that IL-17A acts in concert with BCG-induced TNF-α to enhance the level of IL-6 synthesis. Taken together, our results suggest a significant role of IL-17A to serve as a modulator of cytokine expression in innate immune response during mycobacterial infection.

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation.

Targeting the role of N-terminal methionine processing enzymes in Mycobacterium tuberculosis

The discovery of anti-tuberculosis agents that target new pathways is crucial for effective short-term TB therapy that will limit the development of resistance. The clinical significance of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, latent TB and Human Immunodeficiency Virus co-infection in tuberculosis patients have made the development of new antimycobacterials more imperative. A better understanding of the major pathways that are involved in the pathogenesis, survival, and dormancy of Mtb will aid in the identification of new drug targets. Here, we review the N-terminal methionine excision (NME) pathway as a potential drug target during host infection with M. tuberculosis. The removal of the N-terminal methionine is a requirement for some proteins prior to post-translational modifications and processing. Therefore, an understanding of the physiological relevance of the two families of enzymes at the center of NME - peptide deformylases and methionine aminopeptidases - has the prospect of adding novel targets and antimycobacterials to the pipeline.

MARCO, TLR2, and CD14 Are Required for Macrophage Cytokine Responses to Mycobacterial Trehalose Dimycolate and Mycobacterium tuberculosis

Virtually all of the elements of Mycobacterium tuberculosis (Mtb) pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6′-dimycolate (TDM/cord factor). TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88)-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs), implying involvement of toll-like receptors (TLRs). However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO), a class A scavenger receptor, is utilized preferentially to “tether” TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-κB)-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA), which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-κB signaling to occur. Consistent with these observations, macrophages from MARCO−/− or MARCO−/−SRA−/− mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2) and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow–derived) may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO−/− mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding receptors for TDM, explain the differential response to TDM of various macrophage populations, which differ in their expression of the scavenger receptors, and identify MARCO as a novel component required for TLR signaling.