Aisha Farhana

Redox homeostasis in mycobacteria: the key to tuberculosis control?

Mycobacterium tuberculosis (Mtb) is a metabolically flexible pathogen that has the extraordinary ability to sense and adapt to the continuously changing host environment experienced during decades of persistent infection. Mtb is continually exposed to endogenous reactive oxygen species (ROS) as part of normal aerobic respiration, as well as exogenous ROS and reactive nitrogen species (RNS) generated by the host immune system in response to infection. The magnitude of tuberculosis (TB) disease is further amplified by exposure to xenobiotics from the environment such as cigarette smoke and air pollution, causing disruption of the intracellular prooxidant–antioxidant balance. Both oxidative and reductive stresses induce redox cascades that alter Mtb signal transduction, DNA and RNA synthesis, protein synthesis and antimycobacterial drug resistance. As reviewed in this article, Mtb has evolved specific mechanisms to protect itself against endogenously produced oxidants, as well as defend against host and environmental oxidants and reductants found specifically within the microenvironments of the lung. Maintaining an appropriate redox balance is critical to the clinical outcome because several antimycobacterial prodrugs are only effective upon bioreductive activation. Proper homeostasis of oxido-reductive systems is essential for Mtb survival, persistence and subsequent reactivation. The progress and remaining deficiencies in understanding Mtb redox homeostasis are also discussed.

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Background Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive. Methodology/Principal Findings Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA. Conclusion Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell.

Iron acquisition, assimilation and regulation in mycobacteria

Iron is as crucial to the pathogen as it is to the host. The tuberculosis causing bacillus, Mycobacterium tuberculosis (M.tb), is an exceptionally efficient pathogen that has evolved proficient mechanisms to sequester iron from the host despite its thick mycolate-rich outer covering and a highly impermeable membrane of phagolysosome within which it persists inside an infected host macrophage. Further, both overindulgence and moderation of iron inside a host are a threat to mycobacterial persistence. While for removing iron from the host reservoirs, mycobacteria synthesize molecules that have several times higher affinity for iron than their host counterparts, they also synthesize molecules for efficient storage of excess iron. This is supported by tightly regulated iron dependent global gene expressions. In this review we discuss the various molecules and pathways evolved by mycobacteria for an efficient iron metabolism. We also discuss the less investigated players, like iron responsive proteins and iron responsive elements in mycobacteria, and highlight the lacunae in our current understanding of iron acquisition and utilization in mycobacteria with an ultimate aim to make iron metabolism as a possible anti-mycobacterial target.

Iron sulfur cluster proteins and microbial regulation: implications for understanding tuberculosis

All pathogenic and nonpathogenic microbes are continuously exposed to environmental or endogenous reactive oxygen and nitrogen species, which can critically effect survival and disease. Iron-sulfur [Fe-S] cluster containing prosthetic groups provide the microbial cell with a unique capacity to sense and transcriptionally respond to diatomic gases (e.g. NO and O2) and redox-cycling agents. Recent advances in our understanding of the mechanisms for how the FNR and SoxR [Fe-S] cluster proteins respond to NO and O2 have provided new insights into the biochemical mechanism of action of the Mycobacterium tuberculosis (Mtb) family of WhiB [Fe-S] cluster proteins. These insights have provided the basis for establishing a unifying paradigm for the Mtb WhiB family of proteins. Mtb is the etiological agent for tuberculosis (TB), a disease that affects nearly one-third of the worlds population.

Mycobacterium tuberculosis WhiB3: a novel iron-sulfur cluster protein that regulates redox homeostasis and virulence.

SIGNIFICANCE Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can persist in a latent state for decades without causing overt disease. Since latent Mtb is refractory to current antimycobacterial drugs, the discovery and characterization of the biological mechanisms controlling the entry, maintenance, and emergence from latent infection is critical to the development of novel clinical therapies. RECENT ADVANCES Recently, Mtb WhiB3, a member of the family of intracellular iron-sulfur (Fe-S) cluster proteins has emerged as a redox sensor and effector molecule controlling several aspects of Mtb virulence. WhiB3 was shown to contain a 4Fe-4S cluster that specifically reacts with important host gases (O(2) and NO), and exogenous and endogenous metabolic signals to maintain redox balance. Notably, the concept of reductive stress emerged from studies on WhiB3. CRITICAL ISSUES The detailed mechanism of how WhiB3 functions as an intracellular redox sensor is unknown. Sustaining Mtb redox balance is particularly important since the bacilli encounter a large number of redox stressors during infection, and because several antimycobacterial prodrugs are effective only upon bioreductive activation in the mycobacterial cytoplasm. FUTURE DIRECTIONS How Mtb WhiB3 monitors its internal and external surroundings and modulates endogenous oxido-reductive pathways which in turn alter Mtb signal transduction, nucleic acid and protein synthesis, and enzymatic activation, is mostly unexplored. Modern expression, metabolomic and proteomic technologies should provide fresh insights into these yet unanswered questions.

Environmental Heme-Based Sensor Proteins: Implications for Understanding Bacterial Pathogenesis

SIGNIFICANCE Heme is an important prosthetic group required in a wide array of functions, including respiration, photosynthesis, metabolism, O(2) transport, xenobiotic detoxification, and peroxide production and destruction, and is an essential cofactor in proteins such as catalases, peroxidases, and members of the cytochrome P450 superfamily. Importantly, bacterial heme-based sensor proteins exploit the redox chemistry of heme to sense environmental gases and the intracellular redox state of the cell. RECENT ADVANCES The bacterial proteins FixL (Rhizobium ssp.), CooA (Rhodospirillum rubrum), EcDos (Escherichia. coli), RcoM (Burkholderia xenovorans), and particularly Mycobacterium tuberculosis (Mtb) DosS and DosT have emerged as model paradigms of environmental heme-based sensors capable of detecting multiple gases including NO, CO, and O(2). CRITICAL ISSUES How the diatomic gases NO, CO, or O(2) bind to heme iron to generate Fe-NO, Fe-CO, and Fe-O(2) bonds, respectively, and how the oxidation of heme iron by O(2) serves as a sensing mechanism that controls the activity of key proteins is complex and largely unclear. This is particularly important as many bacterial pathogens, including Mtb, encounters three overlapping host gases (NO, CO, and O(2)) during human infection. FUTURE DIRECTIONS Heme is an important prosthetic group that monitors the microbes internal and external surroundings to alter signal transduction or enzymatic activation. Modern expression, metabolomic and biochemical technologies combined with in vivo pathogenesis studies should provide fresh insights into the mechanism of action of heme-based redox sensors.

In-Vitro Helix Opening of M. tuberculosis oriC by DnaA Occurs at Precise Location and Is Inhibited by IciA Like Protein

Background Mycobacterium tuberculosis (M.tb), the pathogen that causes tuberculosis, is capable of staying asymptomatically in a latent form, persisting for years in very low replicating state, before getting reactivated to cause active infection. It is therefore important to study M.tb chromosome replication, specifically its initiation and regulation. While the region between dnaA and dnaN gene is capable of autonomous replication, little is known about the interaction between DnaA initiator protein, oriC origin of replication sequences and their negative effectors of replication. Methodology/Principal Findings By KMnO4 mapping assays the sequences involved in open complex formation within oriC, mediated by M.tb DnaA protein, were mapped to position −500 to −518 with respect to the dnaN gene. Contrary to E. coli, the M.tb DnaA in the presence of non-hydrolysable analogue of ATP (ATPγS) was unable to participate in helix opening thereby pointing to the importance of ATP hydrolysis. Interestingly, ATPase activity in the presence of supercoiled template was higher than that observed for DnaA box alone. M.tb rRv1985c, a homologue of E.coli IciA (Inhibitor of chromosomal initiation) protein, could inhibit DnaA-mediated in-vitro helix opening by specifically binding to A+T rich region of oriC, provided the open complex formation had not initiated. rIciA could also inhibit in-vitro replication of plasmid carrying the M.tb origin of replication. Conclusions/Significance These results have a bearing on the functional role of the important regulator of M.tb chromosomal replication belonging to the LysR family of bacterial regulatory proteins in the context of latency.

Exposure to cigarette smoke impacts myeloid-derived regulatory cell function and exacerbates airway hyper-responsiveness

Cigarette smoking enhances oxidative stress and airway inflammation in asthma, the mechanisms of which are largely unknown. Myeloid-derived regulatory cells (MDRC) are free radical producing immature myeloid cells with immunoregulatory properties that have recently been demonstrated as critical regulators of allergic airway inflammation. NO (nitric oxide)-producing immunosuppressive MDRC suppress T-cell proliferation and airway-hyper responsiveness (AHR), while the O2•− (superoxide)-producing MDRC are proinflammatory. We hypothesized that cigarette smoke (CS) exposure may impact MDRC function and contribute to exacerbations in asthma. Exposure of bone marrow (BM)-derived NO-producing MDRC to CS reduced the production of NO and its metabolites and inhibited their potential to suppress T-cell proliferation. Production of immunoregulatory cytokine IL-10 was significantly inhibited, while proinflammatory cytokines IL-6, IL-1β, TNF-α and IL-33 were enhanced in CS-exposed BM-MDRC. Additionally, CS exposure increased NF-κB activation and induced BM-MDRC-mediated production of O2•−, via NF-κB-dependent pathway. Intratracheal transfer of smoke-exposed MDRC-producing proinflammatory cytokines increased NF-κB activation, reactive oxygen species and mucin production in vivo and exacerbated AHR in C57BL/6 mice, mice deficient in Type I IFNR and MyD88, both with reduced numbers of endogenous MDRC. Thus CS exposure modulates MDRC function and contributes to asthma exacerbation and identifies MDRC as potential targets for asthma therapy.

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

ABSTRACT Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells. To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.