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Nitric Oxide 59 (2016) 28e41

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Nitric Oxide

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Review

The emerging role of gasotransmitters in the pathogenesis oftuberculosis

Krishna C. Chinta a, Vikram Saini a, e, Joel N. Glasgow a, James H. Mazorodze b,Md. Aejazur Rahman b, Darshan Reddy c, Jack R. Lancaster Jr. d, Adrie J.C. Steyn a, b, e, *

a Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USAb KwaZulu-Natal Research Institute for TB and HIV (KRITH), Durban, South Africac Department of Cardiothoracic Surgery, Nelson R Mandela School of Medicine, University of KwaZulu Natal, Durban, South Africad Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USAe UAB Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, USA

a r t i c l e i n f o

Article history:Received 16 June 2016Accepted 30 June 2016Available online 4 July 2016

Keywords:Mycobacterium tuberculosisGasotransmitterNitric oxideCarbon monoxideHydrogen sulfideHeme oxygenase-1Reactive oxygen speciesReactive nitrogen speciesHypoxia

* Corresponding author. Department of MicrobioloBirmingham, Birmingham, AL, USA.

E-mail addresses: adrie.steyn@k-rith.org, asteyn@u

http://dx.doi.org/10.1016/j.niox.2016.06.0091089-8603/© 2016 The Authors. Published by Elsevier

a b s t r a c t

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen and the second largest contrib-utor to global mortality caused by an infectious agent after HIV. In infected host cells, Mtb is faced with aharsh intracellular environment including hypoxia and the release of nitric oxide (NO) and carbonmonoxide (CO) by immune cells. Hypoxia, NO and CO induce a state of in vitro dormancy where Mtbsenses these gases via the DosS and DosT heme sensor kinase proteins, which in turn induce a set of ~47genes, known as the Mtb Dos dormancy regulon. On the contrary, both iNOS and HO-1, which produceNO and CO, respectively, have been shown to be important against mycobacterial disease progression. Inthis review, we discuss the impact of O2, NO and CO on Mtb physiology and in host responses to Mtbinfection as well as the potential role of another major endogenous gas, hydrogen sulfide (H2S), in Mtbpathogenesis.© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291.1. Global TB burden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291.2. Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291.3. Overview of Mtb physiology and disease dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2. Role of oxygen in Mtb pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.2. O2 gradients during Mtb infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.3. Reactive oxygen species (ROS) and Mtb physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3. Nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.2. Role of NO in host response to Mtb infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.3. Role of NO in Mtb physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4. Carbon monoxide (CO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.2. Role of CO in bacterial pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.3. Role of CO in Mtb physiology and pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

gy, University of Alabama at

ab.edu (A.J.C. Steyn).

Inc. This is an open access article u

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

K.C. Chinta et al. / Nitric Oxide 59 (2016) 28e41 29

5. Hydrogen sulfide (H2S) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345.2. A role for H2S in TB? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

6. Mtb gas sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366.1. The Mtb Dos dormancy regulon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366.2. Mtb iron-sulfur (Fe-S) cluster proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376.3. Other gas-sensing systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

7. Future challenges and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

1. Introduction

1.1. Global TB burden

The World Health Organization (WHO) has estimated that one-third of the world’s population is infected with Mycobacteriumtuberculosis (Mtb), the bacterium that causes tuberculosis disease(TB). In 2014, ~1.5 million people died from TB, making Mtb thesecond deadliest pathogen of the 21st century after human im-munodeficiency virus (HIV) [1]. HIV/AIDS exacerbates the global TBburden, as is evidenced by that fact that of the 9.6 million peoplenewly infected in 2014, ~1.1 million (12%) were HIV positive [1].Bacille Calmette-Guerin (BCG), the only vaccine against TB, iseffective only in children and does not provide consistent protec-tion in vaccinated adults. TB can be treated with existing drugs;however, the lengthy treatment regimen of 6e9months often leadsto patient noncompliance, which can result in the development ofdrug-resistant TB. Globally, an estimated 5% of active TB cases aremulti-drug resistant TB (MDR-TB), defined as resistance to isoniazidand rifampicin, the two most potent first-line anti-TB drugs. In2013, an estimated 480,000 people worldwide developed MDR-TB,of which an estimated 190,000 died [1,2]. Together with HIVco-infection, the emergence of MDR-TB represents a growingchallenge to global public health that threatens to undermine thegains achieved through the last 80 years of medical advances.Clearly, it is of great importance to better understand the molecularmechanisms ofMtb pathogenesis in order to developmore effectivedrugs and therapies against this global threat.

1.2. Scope

In this review, we aim to provide a new perspective on the roleof gasotransmitters in TB, specifically nitric oxide (NO), carbonmonoxide (CO) and the newly emerging gas, hydrogen sulfide(H2S). In addition, wewill discuss the role of molecular oxygen (O2),including hypoxia, as the concentration of O2 plays a major role inTB disease progression. An emphasis is placed on the in vivo sourcesof these gases, and their roles in Mtb physiology and TB disease.This is followed by discussion of the mechanisms whereby Mtbsenses and responds to these gases, including the Dos dormancythree-component system, the more recently described SenX-RegXtwo-component system, and iron-sulfur (Fe-S) cluster proteins.This review will not attempt in-depth descriptions of the chemicalproperties of these gases, but certain properties will be discussedwhere appropriate. For in-depth reviews on the chemical proper-ties of these gases, we refer the reader to several excellent reviewarticles [3e7]. This review will focus on the role of these host-derived intracellular gases in regulating Mtb disease progressionwith special focus on their specific roles in Mtb dormancy and inregulating host immune responses against TB.

1.3. Overview of Mtb physiology and disease dynamics

Mtb is a rod-shaped, acid-fast, obligate aerobewith awaxy lipid-rich cell wall and extremely slow growth rate (doubling time of~18 h in culture) [8]. Analysis of the Mtb genome has identifieddedicated machinery for lipid biosynthesis, numerous virulencefactors and sensor proteins that allow Mtb to respond to, and sur-vive in, the hostile environment within host cells [9]. Mtb physi-ology is unique as studies have shown thatMtb can survive for up to12 years in sealed tubes and remain fully virulent [10]. Duringinfection Mtb is exposed to a wide range of host substratesincluding organic acids, virtually all amino acids, nucleic acid pre-cursors, nucleotides, lipids and numerous carbohydrates [11]. Thus,it is clear that Mtb must adjust its metabolism in response to theavailability of environmental nutrients during the different stagesof infection.

Mtb infection typically begins with the inhalation of Mtb-con-taining droplets aerosolized by an infected person [12]. The humanlung has a total alveolar surface area of ~70m2, and an adult inhalesand exhales ~11,500 L of air each day [13]. Since the only route ofMtb transmission is by inhalation, the lungs are the primary site ofinfection. The first step of infection is the uptake of bacilli byalveolar macrophages (AM) by phagocytosis and the formation ofphagosomes. Within the AM, fusion of phagosomes with acidiclysosomes is critical for bactericidal activity [12]. However, Mtbuniquely blocks phagolysosome fusion and acidification, therebyeliciting the rapid recruitment of neutrophils, inflammatorymonocytes, interstitial macrophages and T-cells in the lungs fol-lowed by formation of cellular aggregates called granulomas.Containment ofMtbwithin granulomas results in an asymptomaticlatent TB infection (LTBI), which is clinically defined by a reactive TBskin test without clinical symptoms of disease or radiologicalfindings [14]. Mtb granulomas have been shown to be hypoxic inguinea pig and non-human primate (NHP) models and in a specificmouse model of TB [15,16] (Fig. 1).

The occurrence of LTBI highlights a unique aspect of Mtb phys-iology that has been extensively studied, but is not fully under-stood; that is, the ability ofMtb to persist for years or decades in thehost in a genetically controlled state of low metabolic activityreferred to as dormancy [17]. As an obligate aerobe, Mtb requiresoxygen for growth, and therefore, it is rational to conclude that thelow oxygen environment of the TB granuloma contributes to thismetabolic shutdown of the bacillus. Studies in the 1950s showedthat open (oxygen rich) lesions from resected lung tissue fromhuman TB patients yielded actively growing Mtb that were pre-dominantly drug resistant. In contrast, Mtb cells isolated fromclosed (oxygen poor) lesions showed delayed growth, but weredrug sensitive despite being refractory to antimycobacterial drugtherapy [18,19]. This pointed to O2 as an important modulator ofMtb physiology and therapeutic intervention. Not surprisingly, a

Fig. 1. Schematic overview of Mtb infection. Mtb infection is initiated when an individual is exposed to aerosolized bacteria. Of those exposed, ~70% remain uninfected, whereas~30% develop primary infection. The host immune response to the primary infection is initiated by innate immune responses of macrophages and neutrophils via phagocytosis,upregulation of iNOS and HO-1 and production of ROS/RNS. This is followed by antigen presentation by dendritic cells, cell-mediated immunity and further infiltration of immunecells. These responses initially restrict bacterial replication and dissemination and lead to the formation of granulomas. In most cases, Mtb resides within the granuloma in a non-replicating dormant state leading to LTBI. Approximately 90% of individuals with LTBI remain asymptomatic while ~5% develop active TB disease, characterized by massive he-moptysis, dysregulated immune responses and extensive tissue damage.

K.C. Chinta et al. / Nitric Oxide 59 (2016) 28e4130

key objective in the TB field is to understand the mechanismswhereby Mtb persists in tissues for decades without replicating, tothen abruptly resume growth and cause disease. Acquiring suchknowledge will make a significant contribution towards newtherapeutic interventions.

2. Role of oxygen in Mtb pathogenicity

2.1. Overview

Prior to the advent of TB drug therapy, patients were treated insanatoria, where rest and fresh air were considered conducive torecovery [20]. The human host reaction to the bacillus was studied,andwhile many patients succumbed to the consumptive process, inother patients the bacilli were isolated and walled off by the body’sdefense mechanisms, leading to LTBI. One of the theories postu-lated was that the lower oxygen levels in these “walled off” areasinhibited the growth of the obligate aerobic mycobacterium, andthe concept of “resting the lung”while the infection took its coursebecame popularized. Various techniques to artificially collapse alung to render a relatively hypoxemic environment were thenproposed, and “collapse therapy” marked the birth of thoracicsurgery for the treatment of TB [21]. Methods of inducing “collapsetherapy” included instilling air, liquid or spherical objects into thechest cavity to prevent the lung from completely expanding. Bydepriving the organism of oxygen, it was thought that the diseaseprocess would be curtailed. The advent of chemotherapy for TBmarked the end of an era of collapse therapy for TB, and theattention of thoracic surgeons was then directed toward lung

resection for TB (Fig. 2).

2.2. O2 gradients during Mtb infection

It was believed that patients with pulmonary TB improved ifthey lived at altitudes above 2000 m due to the lower oxygentension [22]. Using radioisotope techniques, West and Dollery [22]measured regional perfusion and ventilation and calculated oxygentension in the upright human lung. They observed that the partialpressure of alveolar oxygen (PaO2) at sea level is approximately132mmHg at the level of the first anterior interspace, decreasing to89 mm Hg at the level of the fifth anterior [23]. These observationssupported the popular belief that oxygen tension is a majordeterminant of the apical localization of pulmonary TB. However,an alternate explanation is that the effect of gravity on the erecthuman lung leads to a reduced pulmonary artery blood flow in theapical and subapical regions leading to higher O2 tension [24,25].This reduced blood flow would impede tissue clearance and favoraccumulation of Mtb, making the apical lobes ideal for Mtb growthand proliferation [26]. Although it can be argued that conventionalin vitro tissue culture experiments expose Mtb to hyperoxic con-ditions, recent in vitro studies have shown that the intracellularenvironment ofMtb-infected primary humanmacrophages are alsohypoxic [27].

In the human TB lung, there is a gradient of oxygen tensionsfrom the upper apices to the lower lobes. The flow rate of blood inthe upper apex of the lung is reduced due to depressed pressurewithin the alveoli relative to pulmonary artery pressure in theupright position. Therefore, the apices have elevated oxygen

Fig. 2. Typical chest radiograph, HRCT and granuloma of a TB patient. (A) Plain chest radiograph of a 46 year old woman with active pulmonary tuberculosis, demonstrating athick walled cavity in the right lung with associated parenchymal opacification. (B, C) High resolution computed tomography (HRCT) images illustrate the location of the cavity inthe apical segment of the right lower lobe with the associated tree-in-bud appearance in the basal segments that are characteristic of active tuberculosis. (D, E, F) Right lowerlobectomy specimen with macroscopic features of active pulmonary tuberculous nodules with central caseous necrosis that is evident after sectioning. (G) Changes in partial oxygenpressure (pO2) upon Mtb infection. (H) Representative tuberculous granuloma with central necrotic core surrounded by immune cells which create a gradient of O2, NO, CO andpossibly H2S.

K.C. Chinta et al. / Nitric Oxide 59 (2016) 28e41 31

tensions despite reduced ventilation due to restricted blood flow[28]. Several studies have shown that Mtb-infected lungs havevarious oxygen tensions, all of which are below the normal rangeseen in naïve subjects. This hypoxic environment within the Mtb-infected lung has important implications for drug distribution andbioavailability. The interplay between the host andMtb requires in-depth analysis of various oxygen levels within the different com-partments of human tuberculous lung, which could provide newinsights into the underlying mechanisms of current anti-TB drugsactivity. Despite recent progress, the physiology of Mtb-infectedlung tissue is not yet fully resolved in terms of drug penetration andlevels at the site of infection [29]. Animal models have been usefulfor shedding light on the dynamics of granuloma formation and TBdisease progression. Granulomatous tissues in mouse models of TBhave features that resemble those in humans despite some subtledifferences [30]. Routine access to human tuberculous lung speci-mens for research purposes is unfortunately restricted by thepaucity of these biopsies, and represents a major hindrance to TBresearch. This is important as there is no single animal model thataccurately represents the full spectrum of human pulmonary TB.Another limitation of human TB lung studies is that the granuloma

is dynamic in nature; hence real-time experimentation is notpossible in the human host. Mice, guinea pigs, rabbits, and non-human primates have the ability to form well defined granulomasupon Mtb infection. Overall, it is clear that differential O2 concen-trations play critical roles in the Mtb pathogenesis.

2.3. Reactive oxygen species (ROS) and Mtb physiology

As a diffusible diatomic gas, O2 plays important roles in hostresponse to infection. As mentioned earlier, Mtb is exposed to lowO2 levels or hypoxic conditions within the lungs. Further, ROS andreactive nitrogen species (RNS) generated by host immune cellswithin the lung create a harsh intracellular environment againstMtb. The oxidative burst created by NADPH oxidase (NOX) of hostphagocytic cells via catalysis of a single electron reduction of O2generates O2�

─ and further reduction of onemore electron produceshydrogen peroxide (H2O2) by the activity of superoxide dismutase(SOD). Further, H2O2, in the presence of ferrous ions (Fe2þ) un-dergoes the Fenton reaction to produce hydroxyl radical (OH�).Overall, hypoxia and these free radicals constitute a strong hostinnate defense that can damage bacterial cell wall components,

K.C. Chinta et al. / Nitric Oxide 59 (2016) 28e4132

DNA, and proteins that protects the host against a wide range ofbacterial infections including Mtb [31e34]. Mice deficient in thecytosolic p47(phox) gene, which is required for O2�

─ production, aresusceptible to Mtb infection [35]. However, Mtb has evolved com-plex mechanisms that provide protection from the damaging ef-fects of ROS and hypoxia. Components of the Mtb cell wall,including lipoarabinomannan (LAM), mycolic acids and phenolicglycolic lipids (PGL-1), serve as effective ROS scavengers [36].Mycothiol and recently discovered ergothioneine act as redoxbuffers and protect Mtb from endogenous ROS [37,38]. Mtb alsopossesses several ROS-scavenging gene products, which contributeto ROS detoxification and enhance Mtb survival. Mtb katG (catalaseperoxidase) decomposes H2O2 into water and oxygen therebyprotecting cells from the damaging effects of H2O2 [39]. Mtb Mg/Feand Cu/Zn superoxide dismutases (sodA and sodC, respectively) alsoprotect the cells from oxidative burst and Mtb strains lacking thesegenes are susceptible to ROS-mediated killing in both macrophagesin vitro and in the murine model of TB [40e42]. Other genesincluding alkyl peroxide reductase genes (ahpC/D/E) and lipoamidedehydrogenase (lpd) also contribute to Mtb survival against host-generated ROS [43]. In response to hypoxic conditions generatedin the lung, the Mtb three-component heme sensor/regulator sys-tem (DosS/R/T) senses and binds to host O2 thereby causing tran-scriptional adaptation that results in upregulation of Mtb Dosdormancy regulon genes, which enables bacteria to persist in anon-replicative, low metabolic state [44] (see Section 6; Mtb gassensors).

3. Nitric oxide

3.1. Overview

Since its initial characterization in the 1980s as a potent vaso-dilator followed by its recognition as ‘molecule of the year’ in 1992[45], the gasotransmitter NO has been found to regulate a widearray of physiological functions such as neuromodulation, cellularsignaling, maintenance of redox homeostasis and as an effectormolecule during immune responses [46e49]. NO is producedendogenously by the nitric oxide synthase (NOS) enzyme family viaNADPH- and O2-dependent oxidation of L-arginine to L-citrullineand NO [50]. Three distinct mammalian NOS isoforms have beenidentified based on the location of their expression: NOS enzymesexpressed by endothelial cells (eNOS, also referred to as NOS3) andneurons (nNOS, also referred to as NOS1) are constitutivelyexpressed and act distinctively in response to intracellular calciumlevels in a calmodulin-dependent manner [51]. NO production forimmunological functions is mediated by a calcium-independentinducible form, iNOS [51,52]. iNOS is expressed predominantly incells of myeloid lineage including macrophages and neutrophilsand is generally induced by redox stress, exposure to microbialpathogens or bacterial endotoxins and pro-inflammatory cytokinessuch as IL-1, IFN-g and TNFa (52). NO is amajor RNS and reacts withatmospheric O2, superoxide anions (O2�

─), heme/non-heme ironand thiol groups (-SH) of proteins to regulate a wide array ofcellular signaling responses. One such response includes formationof peroxynitrite (ONOO─) upon reaction with O2�

─. NO and otherRNS including ONOO─ can modify bacterial DNA, proteins, andlipids in host cells as well as in invading pathogens. Further, NO alsointeracts with iron-sulfur clusters or heme groups of proteins. Ni-trite and nitrate, which were thought to be end products of NOmetabolism, can be recycled to produce NO. Bacteria in the gut havealso been shown to form nitric oxide under varying physiologicalconcentrations of O2 and L-arginine via acidification or reduction ofnitrite [53e55].

3.2. Role of NO in host response to Mtb infection

Cells of the innate immune systems including macrophages andneutrophils are crucial for host defense against many bacterial in-fections including Mtb. One of their major anti-bacterial strategiesincludes upregulation of iNOS expression and production of NO[56e58]. Genetic knock-out studies with macrophages isolatedfrom NADPH oxidase- or iNOS-deficient mice and in macrophagesisolated from TB patients provided early evidence that ROSincluding O2�

─, and RNS including NO are important for host de-fense against Mtb [35,59,60]. This was further substantiated byMacMicking et al., who demonstrated that iNOS-deficient mice arehighly susceptible toMtb infection and succumb to infectionwithin~45 days post infection [61]. Blocking iNOS functionwith inhibitorssuch as N6-(1-iminoethyl)-L-Lysine, aminoguanidine and NG-monomethyl-L-arginine resulted in rapid bacterial growth,increased disease pathology, high mortality and disease reac-tivation in murine TB models [62e64]. Similarly, inhibition of theiNOS-modulating cytokine TNFa also leads to Mtb reactivation in amurinemodel of latent TB [65]. Amore recent study has shown thatNO regulates TB immunopathology and tissue damage by regu-lating the levels of pro-inflammatory cytokine interleukin1b (IL1b)in both IFNg-dependent and independent manners [66]. Comparedto murine models, the protective role of NO in Mtb-infectedhumans is poorly understood. Nonetheless, immunohistochemicalanalysis of human TB lung tissue and AM revealed the presence ofiNOS, NO, nitrotyrosine and increased exhaled NO in TB patients,suggesting a role for NO in human TB [67e69]. Further, AM fromLTBI patients may kill Mtb in an iNOS-dependent manner [70,71].Similarly, Rich et al. reported that inhibition of intracellular Mtbgrowth correlated with elevated levels of NO and iNOS in AMfollowing Mtb infection [72]. Kuo et al. showed that increased NOgeneration by AM of TB patients plays an autoregulatory role inamplifying the synthesis of proinflammatory cytokines [73]. In thehuman promyelocytic cell line HL-60, vitamin D3 treatmentresulted in significant increase in NO production and enhancedinhibition of Mtb growth [74]. NO is also thought to be importantfor the formation of granulomas thereby protecting the host fromMtb dissemination [75]. Interestingly, when peripheral bloodmonocytes obtained from patients with active TB were infectedwith Mtb in vitro, increases in both NO and TNF-a levels wereobserved. However, the same cells obtained from patients withMDR-TB produced little NO and TNF-a following Mtb infection,suggesting a role for NO in TB-associated immunosuppression andTB drug resistance [76]. Overall these studies clearly suggest thatNO plays significant role in host response against Mtb.

3.3. Role of NO in Mtb physiology

As discussed above, iNOS/NO is an important component of thehost defense strategy against Mtb infection; however, Mtb hasdeveloped a number of complex mechanisms to resist, subvert andcounter the damaging effects of intracellular NO and other RNS.Darwin et al. have shown that the Mtb proteasome is crucial forbacterial resistance against host-generated oxidative and nitro-sative stress, likely through the degradation of stress-damagedproteins [77]. Other studies showed that the noxR1 and noxR3genes of Mtb confer protection against NO, RNS and ROS [78,79]Further, Mtb ahpc was protective during RNS-induced necrosis,apoptosis and also protected the bacterium from DNA damage bydetoxifying ONOO� [80]. Interestingly, transcriptomic analysis ofiNOS-deficient mice revealed that Mtb faces significant oxidativeand nitrosative stress within the phagosomal environment. How-ever, under these conditions Mtb undergoes drastic transcriptionaladaptation which enables the switch from aerobic to anaerobic

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respiration followed by dormancy [81]. Further, during hypoxia,nitrate is reduced byMtb to nitritewhich also assists in switching toanaerobic respiration and inducing dormancy [82]. Similarly, ni-trate respiration was also important for Mtb survival under acidicand nitrosative stress environments [83]. Voskuil et al. showed thatnon-toxic concentrations of NO inhibitMtb respiration and growth,and result in the induction of Mtb dormancy regulon genes [84].Similar to O2, NO is also sensed by the Mtb three-component sys-tem DosS/R/T, which causes Mtb to enter dormancy in vitro[44,85,86]. Interestingly, Cunningham-Bussel et al. have shown thatMtb also produces significant levels of nitrite during infection ofhuman macrophages, which assists its survival under hypoxicconditions by modulating bacterial respiration, ATP consumptionand transcriptional modulation of stress associated genes [87].Overall, these studies show that while NO is important for hostdefense, Mtb has evolved potent NO-mitigating machinery (Fig. 3).

4. Carbon monoxide (CO)

4.1. Overview

While initially regarded as a toxic environmental gas, CO is nowknown to be an enzymatic by-product of the heme oxygenase (HO)system and a key intracellular messenger that regulates numeroushost physiological responses [5]. HO enzymes convert free heme,sourced primarily from hemoglobin released from erythrocytes,into CO, free iron (Fe2þ) and biliverdin in equimolar ratios [88]. Twomajor HO enzymes have been characterized in mammals: theinducible form, HO-1, and a constitutive isoform, HO-2.While HO-2is constitutively expressed almost exclusively in the brain, HO-1 isubiquitously expressed at very low levels in almost all cell types,but its expression is highly induced by cellular redox stress, hyp-oxia, bacterial lipopolysaccharides andmany pathogenic challengesincluding mycobacterial species [5,89]. The HO system produces~86% of endogenous CO; the remainder is contributed by lipidperoxidation, bacteria, photo-oxidation, and xenobiotic

Fig. 3. Schematic illustration of the role of NO in Mtb disease. Mtb infection upregulates tNO. NO-mediated Mtb killing and modulation of host immune and inflammatory responsesO2

�─ to produce ONOO─ via NADPH oxidase. ONOO─ is pro-inflammatory, pro-oxidant and a pand increased disease pathology. NO also plays important roles in Mtb physiology where itresponse regulator DosR thereby inducing theMtb dormancy regulon. Also, NO can be converMtb stress-associated genes.

metabolism [5]. Verma et al. provided the first evidence that CO hasa physiological role and, like NO, acts as a signaling molecule toregulate cGMP production [90]. Additional studies showed CO to bea key regulator of vascular and cardiac functions [91], inflammation[92,93] and apoptosis [94,95]. Later studies revealed cytoprotectiveroles for CO, which eventually lead to evaluation of CO as a thera-peutic intervention in many pathophysiological conditions [96,97].

4.2. Role of CO in bacterial pathogenesis

While the functions of CO in host responses to bacterial in-fections have been studied, demonstration of a direct role for CO inbacterial physiology has so far been limited to its bactericidal ac-tivity in E. coli, Pseudomonas aeruginosa, and Staphylococcus aureus,which results in death of the bacillus [98,99]. Roles for CO in bac-terial pathogenesis have been shown in models of bacterial disease,including sepsis and malaria [100]. Administration of a carbonmonoxide releasing molecule (CO-RM) resulted in enhanced bac-terial clearance and improved outcomes in a HO-1-deficient mousemodel of severe sepsis [101]. CO has also been shown to enhancethe rate of macrophage phagocytosis of E. coli through upregulationof TLR-4 expression via p38 MAPK signaling [102]. A similar studyreported that macrophage-specific deletion of HO-1 increasesmacrophage death upon infection with E. coli and E. faecalis andthat this effect was reversed by administration of exogenous CO[103]. In malarial disease caused by the parasite Plasmodium,Pamplona et al., have shown that pharmacological induction of HO-1 or CO prevents experimental cerebral malaria (ECM) in mice byinhibiting the release of free heme. This prevents disruption of theblood brain barrier, and regulates CD8 T-cell responses [104].Similarly, Jeney et al. demonstrated that the HO/CO system, alongwith NO, provides disease tolerance during cerebral malaria viasuppression of T-cell responses [105]. Taken together, these studiesshow that host-derived CO plays an important role in host defenseand bacterial physiology.

he expression of iNOS, which catalyzes the conversion of L-arginine into L-citrulline andare crucial for host protection against Mtb infection. On the other hand, NO reacts withotent suppressor of T-cell responses and its overproduction leads to host tissue damageis sensed by the Mtb heme sensor kinase proteins DosS and DosT, which activate theted to NO2

─ or NO3─, which arrestMtb growth and lead to transcriptional adaptation of

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4.3. Role of CO in Mtb physiology and pathogenicity

One anti-bacterial strategy of host phagocytes is the upregula-tion of HO-1 and the production of CO via HO-1 enzymatic activity.However, unlike other bacterial pathogens, Mtb tolerates physio-logical levels of CO due to its expression of a CO-resistance gene, cor[106]. The first major advancement in our understanding of a rolefor CO in Mtb physiology came from studies showing that like O2and NO, CO can independently bind to Mtb heme sensor kinaseproteins DosS and DosT as confirmed by the formation of heme-carbonyl species upon exposure to CO [44]. Further, exposure ofMtb cells to CO-RM-derived CO or CO gas results in increasedexpression of dosR, hspX and fdxA genes of the Mtb dormancyregulon [44]. In vitro infection studies using macrophages isolatedfromwild-type and HO-1�/� mice showed that physiological levelsof host-generated CO induce a transcriptional profile of Mtbdormancy-inducing genes comparable to that of NO. This tran-scriptional regulation is specific to CO and not the other products ofHO-1 enzymatic activity [107,108]. These studies also demonstratedthat while induction of the dormancy regulon genes (discussedlater) by NO requires the activity of both heme sensor kinases DosSand DosT, the binding of CO to DosS alone is sufficient to induce theMtb dormancy regulon. These data demonstrate a direct role for COin initiation ofMtb dormancy, and that HO-1-derived CO and iNOS-derived NO may act in synchrony or independently to determinethe outcome of infection.

We and others have shown showed that HO-1 levels aresignificantly increased in macrophages in vitro and in the lungs ofmice followingMtb infection [107,108]. In the infected lung, HO-1 isupregulated in immune cells that surround granulomas, suggestingthat increased production of CO may be a host strategy to inducedormancy and restrict bacterial dissemination [108]. While thesestudies highlight the role of HO-1-derived CO inMtb dormancy, therole of CO in active TB disease is still unclear. Nonetheless, duringnon-tuberculous infections (NTM) caused byMycobacterium avium(M. avium), HO-1 confers host protection by suppressing theexpression of monocyte chemoattractant protein-1 (MCP-1) andchemokine receptor 2 (CCR2) resulting in the formation of compactgranulomas, thereby reducing bacterial dissemination. HO-1 alsocounteracts the cytotoxic effects of heme and inhibits apoptosis ofinfected macrophages [109,110]. While based on a HO-1-deficientmouse model of M. avium infection, these studies did not demon-strate a direct role for CO, a technically challenging task. However,since CO is known to have anti-apoptotic, anti-inflammatory andcytoprotective functions in macrophages when challenged withbacterial lipopolysaccharide (LPS), it is reasonable to postulate thatthe cytoprotective activity of HO-1 during M. avium infection isattributable to CO [111]. Further, these studies point to HO-1 and COas potential targets for therapeutic interventions against M. aviuminfection.

HO-1 and HO-1-derived CO play an important role in host anti-inflammatory and anti-oxidative responses during various patho-logical conditions [92,97]. Notably, active TB is characterized bydysregulated inflammatory responses such as heightened immunecell infiltration, increased levels of pro-inflammatory cytokines andhemorrhagic inflammation resulting in hemoptysis and extensivelung tissue damage [112e115]. Andrade et al. showed that HO-1levels are significantly elevated in the serum of patients withactive pulmonary TB compared to patients with LTBI, and suggestedthat elevated HO-1 levels could be a potential TB biomarker [116]. Alater study by the same group showed that increases in HO-1 levelsin TB patients corresponded with decreased expression of matrixmetalloproteinase-1 (MMP-1), which drives immunopathologyduring TB [117,118]. This inversely proportional expression of HO-1and MMP-1 was specific to TB and was not observed in other lung

diseases. The authors also demonstrated that CO specifically re-duces the expression of MMP-1 via suppression of c-Jun/AP-1activation and suggests a potential therapeutic role for CO in hostprotection against TB disease progression.

The dysregulated immune responses and increased influx ofimmune cells to the infection site during active TB results in sub-stantial oxidative and nitrosative stresses that likely result inextensive tissue damage. In contrast, dormant Mtb within granu-loma remain non-pathogenic and drug insensitive, leading to LTBI.These two disease phenotypes in TB are strikingly similar to ma-laria, wherein a clinically latent pre-erythrocytic stage is requiredto establish infection, and a subsequent erythrocytic stage isresponsible for active malarial disease [119]. Since application ofHO-1 and CO has been shown to protect mice frommalarial diseasepathology, a similar therapeutic strategy forMtb infectionmay havemerit.

Overall, while the role of CO in TB is not fully understood, it isclear that it plays a key role in both controlling mycobacterialdissemination and inducing dormancy in vitro. Further, as HO-1/COare important for host defense against oxidative and inflammatorydamage, their therapeutic application at the site of infection mayplay a significant role in minimizing exacerbated inflammation andoxidative damage during TB (Fig. 4).

5. Hydrogen sulfide (H2S)

5.1. Overview

H2S has recently emerged as the fourth physiologically impor-tant gas that mediates multiple processes in mammalian systemsincluding hypertension, atherosclerosis, heart failure, diabetes,inflammation, neurodegenerative diseases and asthma [120e126].H2S can be produced in the human body by resident microbes [127]or by dedicated enzymatic machinery consisting of three inde-pendent enzymes: cystathionine b-synthase (CBS), cystathionine g-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST)along with cysteine aminotransferase (CAT) [128] (Fig. 5). Theoverall chemical properties of sulfide (defined here as H2S þ HS�)relevant to biological systems are briefly outlined below; furtherdetails can be found in recent reviews on the biochemical proper-ties of sulfide [6,129e131]. H2S is a weak acid, with a pKa of ca. 7 forthe dissociation H2S 4 Hþ þ HS�, the second pKa is high enoughthat the sulfide anion (S2�) almost certainly has no biologicalrelevance. This means approximately 28% of sulfide is present asthe protonated nonelectrolyte species (H2S, hydrogen sulfide) at pH7.4. As an uncharged small lipophilic molecule, the fact that pro-tonation/deprotonation is extremely rapid means that membranesare quite permeable and so sulfide produced in one compartmentwill rapidly distribute within the biological milieu. The volatility ofH2S also demands relatively rapid exit to a headspace, requiringcareful attention to experimental configuration. The chemicalreactivity of sulfide is dominated by the fact that, like thiol, it is astrong nucleophile, much more so in the hydrosulfide anion form(HS�) than H2S. This means that HS� is the species that exerts themajority of (but not all) biochemical reactivity. The nucleophilicityof sulfide is reflected in its electronegativity, second only to oxygen(except for fluorine). Unlike oxygen, sulfur is able to utilize its 3dorbitals for an expanded valence shell thereby allowing multiplebonds and formal oxidation states ranging from �2 (e.g. H2S) to þ6(e.g. SO4

2�).Being electrophiles, transition metal ions react with sulfide by

both complete (oxidation-reduction) and partial (bonding) electrontransfer. The product of oxidation-reduction is the reduced metaland elemental sulfur (So). Elemental sulfur exists as a polymer, themost common configuration being S8. In the case of bonding, the

Fig. 5. Schematic illustration of endogenous H2S production and functions. H2S is produced endogenously by cystathionine-b-synthase (CBS), cystathionine-g-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). CBS and CSE use L-cysteine as the primary substrate with pyridoxal-50-phosphate as a cofactor. H2S formation through 3-MST isdownstream of cysteine aminotransferase (CAT), which catalyzes the reaction of L-cysteine with a-ketoglutarate leading to the formation of 3-mercaptopyruvate, the substrate usedby 3-MST to form H2S and pyruvate. H2S is important for numerous physiological responses including vasodilation, neuromodulation, and inflammation and can also act as an anti-oxidant.

Fig. 4. Schematic illustration of endogenous CO production and role in Mtb disease. CO is produced by HO-1 via heme catabolism. Other products of this enzymatic reactioninclude free iron (Fe2þ) and biliverdin, which is further converted into bilirubin. Iron further increases the expression of ferritin, an iron storage protein. CO, bilirubin and ferritin arepotent antioxidant molecules. Further, CO and bilirubin also play important roles in regulating inflammation, immune cell proliferation and host cell apoptosis. Mtb infectioninduces HO-1 expression thereby increasing endogenous CO, which could protect against heme-mediated tissue damage via its anti-oxidative, anti-proliferative and anti-apoptoticproperties. CO binds to Mtb heme sensor kinase proteins DosS and DosT, which like NO, induce the Mtb dormancy regulon.

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metal-sulfide bond is a special type of bond, called a coordinatebond. The toxicity of sulfide in humans is dominated by its re-actions with metals, specifically, with mitochondrial cytochromeoxidase at the site of O2 binding (the binuclear CuB/heme a3) andwith hemoglobin which, through the intermediacy of a highvalency heme-oxygen complex, produces sulfhemoglobin, whichcontains a covalent heme modification. Sulfide anion is also a keycomponent of a large class of iron proteins containing iron-sulfurcenters, which serves both oxidation/reduction and signalingroles. Undoubtedly, the best studied biological system of metal-sulfide interaction is in a symbiotic mollusk/bacterial symbiosis,where cytoplasmic hemoglobin in the host delivers both O2 and

sulfide to the bacterium for metabolism [132]. The properties of themollusk hemoglobin are elegantly tuned to carry these two mole-cules, involving configuration of the heme pocket and proximal anddistal protein residues.

Partially reduced oxygen species (O2�─, H2O2, and HO�) are also

electrophiles and react with sulfide. The rates of reaction, however,are highly variable, and in vivo these reactions are not likely to beimportant under most conditions due to the low concentrations ofthese species and sulfide. Finally, sulfide also reacts with otherelectrophilic species, of particular interest being the newlydiscovered species 8-nitro-cyclic GMP [133]. There is evidence thatthis reaction plays a key role in the signaling properties of this

K.C. Chinta et al. / Nitric Oxide 59 (2016) 28e4136

molecule. Sulfide also reacts with oxidized sulfur-containing spe-cies, for example, disulfide RSSR’. The product is thiol and persul-fide: HS� þ RSSR’ / RSS� þ R’S e (contrary to some statements inthe literature, sulfide does not react with thiol; in general, nucle-ophiles do not react with other nucleophiles). Similar further re-actions also produce polysulfur species R(S)nH, in great abundance.This is true for both small thiols (e.g. glutathione) and also proteincysteine thiol. Indeed, it may well be that these species are moreimportant functionally than sulfide itself. In spite of the lack ofrelevant data, there are observations about the role of H2S inmammals and other prokaryotes [127,134,135].

5.2. A role for H2S in TB?

It is not known whether H2S impacts the outcome of Mtbinfection and TB disease in humans. Regardless, the physicochem-ical properties of H2S, as well as its established role in modulatingimmunity point to a potentially important role for this gas in TB.Both CBS and CSE have been found critical for proper alveolariza-tion and functioning of the lung [136]. Since the human lung is theprimary organ of Mtb infection in which the bacillus can reside fordecades, it is reasonable to posit thatMtbmay havemechanisms forsensing and responding to H2S. Also, since CBS and CSE playessential roles in human health [136e138], it is plausible that H2Svia its immunomodulatory function is necessary for protectionagainst Mtb infection. Studies on the role of H2S on Mtb patho-genesis are eagerly awaited.

However, there are observations on the role of H2S in mammalsand other prokaryotes that can be extrapolated to comment on thepossible role that H2Smay play in TB disease. For example, exposingmice to low amounts of H2S induces a reversible hibernation-likestate characterized by lowering of body temperature, and reducedenergy expenditure in the form of reduced ATP consumption andlow metabolic rate (as evaluated by substantially decreased CO2production and O2 consumption) [139]. Induction of this hypo-metabolic state by H2S, referred to as suspended animation, is anoxygen-dependent phenomenon [140]. It is believed that the in-hibition of O2 utilization and induction of suspended animation inlow O2 conditions could have a host-protective role [140]. There areparallels that can be drawn from this H2S-induced suspended an-imation state and the characteristic features associated withdormancy inMtb. For example, during dormancyMtb is exposed tosevere environmental stresses (e.g., low pH, O2 and carbon sourcelimitation) that induce a metabolic state characterized bydecreased respiration, low ATP levels, reduced growth, low O2consumption and reduced metabolic rates.

H2S primarily affects mitochondrial respiration by targetingcomplex IV of the respiratory chain, i.e., cytochrome c oxidase [141].Based on historical studies using manometry [142] and in a morerecent drug study [143], Mtb is known to exhibit immense respi-ratory flexibility, which is a key requirement for maintaining anintracellular pathogenic lifestyle. It possesses two proton-pumpingcomplexes: NADH dehydrogenase I, which is dispensable forgrowth, and an aa3-type cytochrome c oxidase, which provides themeans of O2 reduction [144]. More importantly, Mtb also expressesa high-affinity alternate terminal oxidase, cytochrome bd oxidase,that can partially compensate for the loss in activity of the bc1-aa3complex [144]. Therefore, it will be of significant interest todetermine whether H2S affects Mtb respiration and consequentlyin vivo pathogenesis.

Lastly, there is a growing appreciation for the complex in-teractions between different intracellular gases such as NO, CO andH2S as they can all interact with the host (and potentially Mtb)electron transport chain [145e150]. Furthermore, NO and CO canalso regulate H2S levels by targeting the CBS heme prosthetic group

[151e153].

6. Mtb gas sensors

6.1. The Mtb Dos dormancy regulon

The success ofMtb as a pathogen is based in part on its ability toenter into dormancy, a genetically and physiologically controllednon-replicative state characterized by metabolic quiescence. In thisreversible state, Mtb can survive the harsh intracellular environ-ment inside the host [154]. Of note, dormancy in Mtb is a physio-logical state of persistence and should not be confused with TBlatency or LTBI, which are clinical descriptors indicating theabsence of TB disease symptoms in infected individuals. The tran-sition into dormancy occurs via marked changes in gene expres-sion, including up-regulation of the widely studied 47-gene Dosregulon in response to CO, NO or hypoxia [155,156]. In addition tothe genes of the Dos regulon, other gene sets, such as the enduringhypoxia response (EHR) genes, may play a role in dormancy;however molecular mechanisms of their regulation are largelyunknown [157,158]. Transcriptional analysis under hypoxic condi-tions in vitro revealed that even though ribosomal and metabolismgenes are downregulated under low-O2 conditions, the Dos regulongenes are significantly upregulated [159]. Similar to the response tohypoxia or low O2 levels, other host-generated stresses such as COand NO also upregulate these dormancy regulon genes. Whilemany genes of this regulon are still being characterized, they arepredicted to be involved in modulating Mtb physiology. Theseinclude genes with chaperone functions (acr or Rv2031c), ferre-doxin modulation (fdxA or Rv2007c), nitrate/nitrite transporter(nark2 or Rv 1737), ribonuclease reductase (nrdZ or Rv0570) andtriglyceride synthase (tgs1 or Rv3130) [19,160].

The genes of the dormancy regulon are upregulated when Mtbsenses host-generated hypoxia, NO and CO via the heme-containing kinases DosS and DosT, which along with a singleresponse regulator, DosR, comprise the DosR/S/T three-componentsystem [44,107,161]. As discussed earlier, our studies and othershave shown that the redox or ligation status of the heme iron inDosS and DosT modulates binding to O2, NO and CO either togetheror independently. This shows that these host-generated intracel-lular gases are central to Mtb disease progression [19,44]. This alsoemphasizes the fact that the success of Mtb as a pathogen can belargely attributed to its unique ability to sense and respond to NO,CO or lowO2. Several lines of evidence point to these diatomic gasesas modulators of dormancy. Firstly, Mtb is an obligate aerobe thatrequires oxygen as a terminal electron acceptor. However, the un-canny ability of Mtb to survive for more than a decade in test tubeswithout oxygen (or at least extremely low concentrations of oxy-gen) points to a novel aspect of Mtb adaptation to low oxygen en-vironments. Secondly, it has been conclusively demonstrated thatiNOS, and therefore NO, is crucial for protection of mice againstMtb[61]. Further studies in patients with active pulmonary TB confirmthe importance of NO in Mtb pathogenesis [35e76,162]. Unfortu-nately, despite several elegant in vitro studies [107,108,163] a clearlyestablished role for either HO-1 or CO in Mtb pathogenesis has yetto be demonstrated and represents an unexplored area ofinvestigation.

It should be noted that the lack of oxygen and the presence ofsufficient levels of NO and/or CO inhibit Mtb respiration and ulti-mately retard growth. An important difference between NO and COis that CO can only react with ferrous (Fe2þ) ironwhereas NO reactswith Fe2þ and Fe3þ. This raises the obvious question as to howconcentration gradients of oxygen, NO and CO, as well as thedifferent affinities of DosS and DosT for these gases contribute toMtb disease.

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Most studies showing the effect of gases on Mtb have beenperformed in vitro or in mouse models of TB and their relevance tohuman TB is yet to be established. However, recent studies in ourlaboratory using tissue samples from freshly resected humantuberculous lungs have clearly shown that significant levels of ROS/RNS are indeed present in human TB lungs. Interestingly ROS/RNSlevels were directly proportional to the extent of tissue damage,suggesting a gradual disease progression.

6.2. Mtb iron-sulfur (Fe-S) cluster proteins

Proteins that contain iron-sulfur cluster [Fe-S] prostheticgroups are found in virtually all organisms and provide a mech-anism for cells to sense changes in redox and respond to intra-cellular gases. The Mtb WhiB family of proteins WhiB1 (Rv3219),WhiB2 (Rv3260c), WhiB3 (Rv3416), WhiB4 (Rv3681c), WhiB5(Rv0022c), WhiB6 (Rv3862c) and WhiB7 (Rv3197A)] contain fourcysteine residues arranged in a CyseX-14-22-Cys-X2-Cys-X5-Cysmotif, with the exception of WhiB5 [164]. Studies of homologs inStreptomyces spp suggested that these proteins are Fe-S clustersproteins [164]. Indeed, Mtb WhiB3 is a 4Fe-4S cluster protein thatmaintains intracellular redox balance and responds to oxygen andNO [165]. Recently, WhiB3 was shown to regulate the productionof a redox buffer, ergothioneine, which is critical forMtb virulenceand drug susceptibility [38]. The WhiB proteins likely acts asredox sensors via their Fe-S clusters and are critical in maintainingredox homeostasis, a key determinant of Mtb virulence [19,38].The seven members of the Mtb WhiB family perform diversefunctions including cell division (WhiB2) [166], fatty acid meta-bolism and pathogenesis (WhiB3) [165,167,168], oxidative stress(WhiB6) [169,170] and antibiotic resistance (WhiB7) despite onlymoderate sequence homology among themselves [171,172](Fig. 6).

Since the discovery of aWhiB-like protein in Streptomyces spp in1992, a long-standing question has beenwhether these proteins areDNA-binding proteins that regulate gene expression [173]. Nearly20 years later, it was shown that Mtb WhiB3 binds DNA [168].Important features of its DNA binding capacity, which was

Fig. 6. Schematic overview of gasotransmitters in Mtb physiology. Mtb heme sensor kresponse regulator DosR which induces the Mtb dormancy (Dos) regulon. SenX3 of the Sengenerated O2, NO and CO. Binding of O2 to SenX3 stimulates Mtb growth during reactivatioeffects downstream of SenX3. Further, interaction of O2 or NO with members of the WhiB faas well as metabolic adaptation under conditions of host-generated stress.

extrapolated to other WhiB members, was that oxidized apo-WhiB3 binds DNA much stronger than either holo-WhiB3 orreduced apo-WhiB3. Subsequently, it was shown that Mtb apo-WhiB1, apo-WhiB2 and apo-WHiBTM4 bind DNA strongly incontrast to the holo-forms, which bind DNA weakly or not at all[164]. These findings revealed that DNA binding and transcriptionalactivity are modified by environmental conditions such as hypoxia,free radicals such as NO and oxidative stressors that influence theredox state of the 4Fe-4S cluster (of the holo-form) or the oxidationstates of the Cys residues (of the apo-form). Overall, it appears thatMtb has evolved a sophisticated family of proteins that function as aflexible redox switch in response to oxido-reductive stress thatcauses an imbalance in intracellular redox homeostasis.

Redox homeostasis in mycobacteria is distinct from otherintracellular pathogens as they lack a conventional glutathionesystem and homologues of the prototype sensor proteins in otherintracellular pathogens such as SoxR, ArcAB, FNR and OxyR (apseudogene inMtb) [172e174]. Instead,Mtb uses two distinct redoxcouples, mycothiol and ergothioneine, and harbors at least 50proteins containing Fe-S clusters that facilitate an intercellular lifecycle [37,175]. Interestingly, Mtb has a higher incidence of Fe-Scluster proteins (~6.5motifs/1000 ORFs) than other aerobic bacteria(2.8 motifs/1000 ORFs), underscoring the importance of Fe-S clus-ter proteins to pathogenic adaptations of Mtb [176,177].

6.3. Other gas-sensing systems

Apart from DosS/R/T, Mtb has other heme sensor kinase andresponse regulator protein systems that are predicted to play roles inits metabolic adaptation, including the SenX3-RegX3 system and thePhoPR systems [178,179]. Transcriptomic analysis of a senX3-regX3knockout strain of Mtb showed downregulation of several DNArepair and protein synthesis genes as well as genes important forsurvival under hypoxic conditions such as cydB and gltA1 [180].Further, this Mtb knockout strain fails to establish successful infec-tion of macrophages in vitro and in guinea pig and murine models ofTB [181e183]. More recently, SenX3was shown to sense and bind O2,NO and CO. Binding of O2 to SenX3 is associated with growth

inase proteins DosS and DosT sense host-generated NO, CO and O2 and activate theX3-RegX3 two-component system is a heme sensor kinase that also responds to host-n; however, binding of CO or NO inhibits growth. RegX3 is presumed to mediate thesemily of iron-sulfur cluster proteins regulates the intracellular redox homeostasis of Mtb

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stimulation during reactivation whereas binding of NO and CO toSenX3 inhibits growth via inhibition of kinase activity [184] (Fig. 6).

7. Future challenges and conclusions

The development of highly effective therapeutic interventionssuch as new antimycobacterial drugs and vaccines hinges on un-derstanding themechanisms bywhichMtb can persist for decades inthe human lung to ultimately cause disease. Given the ubiquitousnature of O2, NO, CO and H2S, it is not surprising that some of theseendogenous gases play critical roles in a wide range of pathophysi-ological processes. The importance of these gases in human health isunderlined by genetic deficiency and polymorphism studies. Forexample, the pathophysiological consequences of deficiencies in HO-1 (systemic inflammation, intravascular hemolysis, asplenia,vulnerability to stressful injury) [185,186], CBS, CSE or 3-MST (hyper-homocysteinemia, mercaptolactate-cysteine disulfiduria, mentalretardation and aggressive vascular disease, neuoroblastoma andhepatoblastoma) [187e189] and iNOS (susceptibility to TB, and awide range of diseases) [190] have profound effects on humanhealth. Therefore, dysregulation of the levels of these gases at the siteof infection could lead to ineffective drug treatment or impair in-flammatory responses to exacerbate disease.

A practical advantage in studying the role of NO, CO and H2S inMtb infection is that mice with deficiencies in expression of iNOS,HO-1, CBS, CSE and 3-MST are available for experimentation. Toconfirm the clinical relevance of these gases, findings from thesestudies should be expanded to the analysis of human pulmonary TBtissue samples. Unfortunately, a major impediment to these studiesis the paucity of freshly resected humanTB lung tissue. Of particularinterest would be to determine what cell populations in animal andhumanTB lung tissues express CBS, CSE, 3-MST, iNOS and HO-1, andtheir respective levels. It is important to note that in the case ofiNOS and HO-1, which are expressed at the site of infection[67,107,108], gradients of NO, CO and O2 are likely to exist in hypoxicMtb granulomas [19,191]. This presents an interesting opportunityfor studying crosstalk among the gases since CBS and iNOS containheme moieties, which represent potential interaction sites for NO,CO and O2.

Another important albeit unexplored area of investigation is therole of these gases in antimycobacterial drug susceptibility in vivo.First considered for NO, it is tempting to speculate that gaso-transmitters play a role in antimycobacterial drug efficacy [67]. Onthe other hand, since these gasotransmitters are ubiquitous, phar-macologic modulation of gene expression or the application ofiNOS, HO-1, CBS, CSE or 3-MSTendproducts has long been proposedas a therapeutic avenue based on model studies [3e7]. Under-standing how Mtb directly interacts with these gases, their corre-sponding targets, how these targets discriminate between thesegases, and the signaling responses induced by these gases will be ofsubstantial value. NO, CO and O2 have been shown to target theheme irons of Mtb DosS and DosT. However, targets other than theWhiB family proteins and the SenX3-RegX3 two-component sys-tem also exist. Further, a detailed understanding of how thesegasotransmitters target the Mtb electron transport chain, whichcontains numerous heme and Fe-S cluster prosthetic groups,should provide new information on energy metabolism, and howthe bacillus is capable of surviving under harsh intracellular con-ditions including hypoxia and gradients of NO, CO and H2S. TheMtbelectron transport chain is remarkably plastic and capable ofrapidly re-routing electrons around specific inhibition by switchingbetween terminal oxidases. Despite the many potential targets,very little is known about this ATP-generating pathway in Mtb.Many provocative questions can emerge from such studies. Forexample, do these gasotransmitters inhibits or stimulate

respiration, and do they have a predominantly bactericidal orbacteriostatic effect on Mtb?

In conclusion, it is anticipated that as more is learned about howthese gasotransmitters regulate host immunity and directly targetsMtb, many new avenues of research will open up, compelling re-searchers to reassess existing paradigms forMtb persistence, and toseek new testable hypothesis.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by NIH grants AI058131 and AI111940(to AJCS). AJCS is a Burroughs Wellcome Investigator in the Path-ogenesis of Infectious Diseases. This work was also supported bythe UAB Center for AIDS Research (CFAR) and UAB Center for FreeRadical Biology (CFRB). The research was also co-funded by theSouth African Medical Research Council. Open access publication ofthis article has beenmade possible through support from the VictorDaitz Information Gateway, an initiative of the Victor Daitz Foun-dation and the University of KwaZulu-Natal.

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