Caseum: a Niche for Mycobacterium tuberculosis Drug-Tolerant Persisters

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum.

SUMMARY

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum. Here we review widely accepted beliefs about the caseum-specific stress factors thought to trigger the shift of Mycobacterium tuberculosis to drug tolerance. Our current state of knowledge reveals that M. tuberculosis is faced with a lipid-rich diet rather than nutrient deprivation in caseum. Variable caseum pH is seen across lesions, possibly transiently acidic in young lesions but overall near neutral in most mature lesions. Oxygen tension is low in the avascular caseum of closed nodules and high at the cavity surface, and a gradient of decreasing oxygen tension likely forms toward the cavity wall. Since caseum is largely made of infected and necrotized macrophages filled with lipid droplets, the microenvironmental conditions encountered by M. tuberculosis in foamy macrophages and in caseum bear many similarities. While there remain a few knowledge gaps, these findings constitute a solid starting point to develop high-throughput drug discovery assays that combine the right balance of oxygen tension, pH, lipid abundance, and lipid species to model the profound drug tolerance of M. tuberculosis in caseum.

INTRODUCTION

In tuberculosis (TB) disease, complex lesions form in the infected organ. Typical pulmonary TB lesions are closed granulomas that start as fully cellular structures and gradually necrotize from the center outward. As they expand, damaging inflammatory processes may locally erode airways, leading to fusion with the necrotic granuloma and formation of an open cavity filled with granuloma remnants. Cavities may also begin as localized foci of lipid pneumonia in the alveolar space upon reactivation of latent infection. In both closed and open lesions, diverse microniches present the infectious agent, Mycobacterium tuberculosis, with a variety of environmental conditions to which it must adapt to thrive and/or survive. Caseous necrosis is a hallmark of pulmonary TB pathology. The soft necrotic debris that accumulates in the center of TB granulomas resembles cheese, earning its Latin name, “caseum.” The prevailing hypothesis is that the intracaseum M. tuberculosis subpopulation (here referred to as “caseum M. tuberculosis”) contributes to the persistent nature of TB infection in humans and the need for prolonged chemotherapeutic intervention. The proportion of adults with pulmonary TB who have cavitary pathology at the time of diagnosis ranges from 40 to 87% (1). In patients with uncomplicated drug-susceptible TB, a minimum of 6 months of combination therapy with up to four antibiotics is required to optimize the chances of cure (2). The caseous foci of necrotic granulomas and cavities are reservoirs of extracellular bacilli that are recalcitrant to antibiotic treatment (3–6). Direct comparisons between treatment efficacy in TB-infected C3HeB/FeJ (Kramnik) mice, which develop caseous pulmonary granulomas, and BALB/c mice, which do not, revealed that the former are much more refractory to antibiotic treatment (7–9). Lesion-centric drug efficacy studies using rabbit and cynomolgus macaque models of TB infection have been useful in highlighting (i) higher bacterial burden, (ii) deficiency in self-sterilization, and (iii) limited drug-mediated sterilization of caseous granulomas and cavities compared to cellular lesions without a necrotic core (10–12).

Persistence of caseum M. tuberculosis is caused by a set of metabolic and physiologic adaptations leading to replication arrest. Using an ex vivo assay designed to measure the activity of anti-TB drugs against M. tuberculosis present in cavity caseum from infected rabbits, we have shown that this subpopulation exists in a viable but nonreplicating state and exhibits extreme drug tolerance to many 1st- and 2nd-line drugs (13). This problem of phenotypic drug resistance is further compounded by suboptimal drug distribution in the nonvascularized caseous core of necrotic lesions, potentially creating pockets of subinhibitory drug concentrations and de facto monotherapy (9, 14, 15). Thus, both pharmacodynamic (drug potency) and pharmacokinetic (drug concentration) factors join forces to increase the chances of treatment failure, relapse, and emergence of drug resistance (16, 17).

In this review, we focus on the progression of necrotic granulomas and the specific metabolic and morphological characteristics of nonreplicating persistent M. tuberculosis in caseum. Over the years, scientists have suggested and explored numerous environmental stress factors in the caseous foci. We review four of these factors and their role in triggering M. tuberculosis “dormancy” and phenotypic drug resistance: decreased pH, hypoxia, iron deprivation, and nutrient shift or depletion. We highlight knowledge gaps that should be filled to develop predictive in vitro assays for the discovery and development of shorter treatment regimens.

HOW DOES CASEUM FORM AND CONTRIBUTE TO DISEASE?

Two schools of thoughts promote partially overlapping concepts, both highlighting the prominent role of lipids and lipid-laden (foamy) macrophages in the generation of caseous necrosis. When cellular lipid homeostasis is perturbed by pathological conditions, such as sustained inflammation, lipid droplets may accumulate in the cytoplasm of macrophages (18). One theory proposes that postprimary tuberculosis begins as localized foci of lipid pneumonia in the alveolar space, where infection of alveolar macrophages triggers their differentiation into foamy macrophages. In early postprimary TB, cavities would develop by necrosis of tuberculous lipid pneumonia rather than through erosion of caseating granulomas into bronchi (19–21). This paradigm largely emerged in the preantibiotic era, when TB-related deaths and subsequent autopsies were more frequent. Today’s prevalent views favor the thesis that the primary site of caseation is the central core of a granuloma, where multiple coordinated events lead to the necrosis of epithelioid foamy macrophages (22, 23). Subsequently, an expanding necrotic granuloma may erode into a nearby airway and coalesce with its wall, forming a cavity, the prerequisite to disease transmission. Both types of pathologies were recognized as early as 1821 by Laennec (24). They were described as “exudative,” referring to pneumonia, and “productive,” referring to nodular tubercles or granulomas. As access to untreated clinical samples declined and with the rise of animal models that reproduce primary rather than postprimary TB, the concept of lipid pneumonia as an early source of caseation has been displaced by the caseating granuloma, although the two types of pathologies are not mutually exclusive.

The induction of foam cell formation is multifactorial and only partially understood. In vitro, infection of macrophages by M. tuberculosis causes intracellular lipid accumulation that exceeds the host cell’s capacity to maintain lipid homeostasis (25). Stimulation of lipid-sensing nuclear receptors, Toll-like receptors, and other membrane-bound macrophage receptors by M. tuberculosis cell wall lipids, including oxygenated mycolic acids, is involved in foam cell biogenesis (23, 26, 27). Host lipids are sequestered in lipid droplets that form in the endoplasmic reticulum and accumulate within the cytosol, giving the cells their characteristic “foamy” appearance (22, 23, 25). Other triggers of foaminess, with potential in vivo relevance, have been demonstrated and characterized in vitro: hypoxia and exposure to very-low-density lipoproteins and to selected fatty acids (28–30). Because lipid droplets are intimately connected to immune modulation, critical macrophage immune functions such as autophagy are diminished, which could facilitate intracellular survival and persistence of M. tuberculosis in the host (18, 31).

Several experimental findings support the view that caseum is largely made of necrotized foamy macrophages. In clinical samples and animal models, foamy macrophages directly surround the caseous core of necrotic granulomas (10, 23, 25, 32), consistent with the idea that their necrotic death leads to the release of lipid droplets and cellular debris at the caseum interface. However, the mechanisms leading to foamy macrophage cell death, whether apoptotic or necrotic, are only partially understood. According to a long-held belief, in a cytotoxic delayed-type hypersensitivity reaction, activated T lymphocyte-mediated killing of infected foamy macrophages destroys local tissue, leading to expansion of the caseous center of the granuloma and release of extracellular bacilli (21, 33, 34). The paradigm that human T cell responses contribute to the lung tissue destruction underlying cavitary TB is consistent with the low frequency of cavitation among HIV-TB patients and the linear correlation between the number of circulating CD4⁺ T cells and the frequency of cavitary TB (35). However, much remains to be elucidated on the mechanisms triggering foamy macrophage death at the cellular/necrotic interface of granulomas.

Investigation of the molecular composition of caseum, on the other hand, is rapidly progressing. Using a range of biochemical and molecular techniques, Kim et al. have shown that the protein, nucleic acid, and lipid composition of caseum bears numerous similarities with that of foamy macrophages. Genes involved in lipid sequestration and metabolism were highly expressed in human caseous granulomas, and the products of these genes were similarly overrepresented in cells surrounding the caseum. Furthermore, they determined that the most abundant lipid species in caseum are also dominant in in vitro M. tuberculosis-infected foamy macrophages that acquire lipids from low-density lipoproteins (LDLs) (22). These observations suggest that induction of foam cell formation by M. tuberculosis may represent a mechanism by which the pathogen drives the progression of the granuloma toward cavitation and transmission (36). They are also consistent with the view that lipid-laden foam cells contribute to TB disease pathology.

Neutrophils have been the center of renewed and rising interest in TB disease progression and pathology (their damaging and protective roles are comprehensively and elegantly reviewed elsewhere) (37, 38). They are an important component of exudative lesions (39) and granulomatous lesions of both TB patients and animal models (10, 40). The extent of neutrophil infiltration in caseum is highly variable, even within different lesions of the same host. In caseous foci where neutrophils are found in high numbers, they are associated with high pathogen load (38). In vitro studies suggest that necrotic neutrophils may exert detrimental effects on the host response in active TB (41), an important finding given the relatively short life span of neutrophils and long residence time in caseum in the absence of an active drainage system. In sputum, bronchoalveolar lavage fluid, and pulmonary TB cavity contents, neutrophils are a dominant cell type, again implying that neutrophilic inflammation can be a manifestation of failed immunity to M. tuberculosis in humans (42). In short, M. tuberculosis appears to utilize exquisite strategies to evade neutrophil-mediated immunity and exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage such as necrotic foci (43). In the C3HeB/FeJ mouse model, massive neutrophilic infiltration correlated with liquefactive necrosis (44), a concept that has gained momentum recently but remains to be demonstrated in clinical TB. Caseum liquefaction is an in vivo phenomenon, occasionally observed in selected animal models, and thus a challenging field of study. In 1955, Canetti discussed four possible mechanisms underlying caseum liquefaction, based on microscopy, biochemical, and histological analyses of human autopsy samples: (i) the release of proteolytic enzymes during a secondary non-TB infection, (ii) the reactivation of proteolytic enzymes from necrotized host cells, (iii) the influx of polymorphonuclear cells, and (iv) robust M. tuberculosis growth (45). Caseous foci appeared to disintegrate into viscid masses of varied consistencies, and intermediary stages between solid and liquid caseum were often observed (45).

In a more recent review of the liquefaction process, the authors speculate that lesions in the upper lobes of the lungs are more prone to liquefaction because higher oxygen tension favors increased bacterial growth, which is further enhanced by reduced immunological surveillance due to reduced blood flow in this part of the lungs (46). Overall, high neutrophil numbers, liquefaction, and increased multiplication of extracellular bacilli have been found in many studies to be intertwined (46); however, causality relationships between these features remain to be clearly established. Regardless of liquefaction processes, clinical data strongly suggest that cavitary TB disease is associated with higher bacterial burdens, disease transmission, and the requirement of longer therapy duration (1, 47–50).

M. TUBERCULOSIS IN CASEUM

Detection

The acid-fast (AF) stain, also known as Ziehl-Neelsen (ZN) stain, is the traditional microscopic detection method for the diagnosis of TB infection in patient sputum. Consistent with the concept that expectorated sputum drains liquefied cavity caseum, acid-fast stains of necrotic granulomas from C3HeB/FeJ mice, guinea pigs, rabbits, and humans reveal large extracellular subpopulations within the acellular matrix of the caseous loci (3, 4, 10, 32, 51–53). The conversion of M. tuberculosis from the AF-positive form during active replication to the AF-negative form during dormancy was reviewed recently by Vilcheze and Kremer (54). Called Koch’s paradox, this phenomenon has been observed in both clinical specimens and animal models of TB infection. For instance, mycobacterial titers of lung homogenates of chronically infected mice are maintained during the later stages of infection, while AF detection of bacterial burden is significantly reduced (55). Additionally, tissue sections from patients with latent TB infections (LTBI) are positive for staining with pAbBCG, a polyclonal antibody, but remain AF negative. This is in contrast with tissue sections from patients with acute or reactivated TB, which are positive for both stains (55, 56). Although the precise cell wall components involved in AF staining remain incompletely described, mycolic acids and other cell wall-associated (glyco)lipids are known to play an important role. Changes in the composition and spatial architecture of these lipids in the cell wall lead to the AF-negative phenotype (54). However, the recent discovery that cytokinin-induced expression of Rv0077c also results in the loss of acid-fastness in M. tuberculosis without accompanying alterations in the mycolic acid profile highlights that this phenomenon is driven by several distinct mechanisms (57). Rhodamine-auramine staining of mycobacteria, which is also cell wall composition dependent, is similarly lost during dormancy in AF-negative bacilli (55).

ZN staining of the caseous foci of closed necrotic granulomas and cavities revealed that the distribution of AF-positive M. tuberculosis bacilli is highly heterogeneous, with distinctly visible regions of high and low bacterial burdens (51). Given the nonreplicating status of caseum M. tuberculosis, the standard ZN staining technique may not be sufficiently sensitive to completely visualize this subpopulation. To overcome this issue, Blanc et al. developed a matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) technique capable of detecting and mapping the two-dimensional distribution of abundant M. tuberculosis cell wall lipids (51). They successfully imaged multiple phosphatidylinositol mannoside (PIM) species and their precursors in the caseous granulomas of infected rabbits and C3HeB/FeJ mice. The precise colocation of these lipid biomarkers with bacilli in thin tissue sections indicates that these cell wall lipids are not secreted and do not spread within lesions. This technique does not allow visualization of individual bacilli due to (i) limited sensitivity and (ii) limited spatial resolution (50 by 50 μm, or the size of a pixel in MALDI MS images), which approximately corresponds to clusters of 5 to 10 bacilli where bacterial burden is high (51). Alternatively, bacteria can be labeled with fluorescent M. tuberculosis-specific antibodies and detected by confocal microscopy imaging (55, 58). The SYBR gold stain is also capable of detecting replicating and nonreplicating M. tuberculosis by intercalating with DNA and RNA regardless of the pathogen’s metabolic state (53, 59). These confocal microscopy techniques have the advantage of single-cell resolution but consume resources and time.

Bacterial Burden

Information regarding the specific bacterial burden of caseum is scarce because clinical and preclinical studies often provide bacterial counts for entire lung organs or whole dissected granulomas, at best, but not distinct lesion compartments such as the cellular rim versus caseous center. The overall burden of necrotic granulomas is often used as a surrogate to assess the burden of the caseous cores. Bacterial burden estimates date from the preantibiotic era, when autopsies of TB patients were accessible.

In humans, necrotic lesions are thought to persist for extended durations without complete healing while the central caseous compartment hardens and calcifies (45). It is believed that during this process, the majority of the M. tuberculosis burden in caseum dies over time while a small subpopulation survives in a state of limited metabolic activity. This was concluded through systematic enumeration of ZN-stained bacilli in thin sections of multiple clinical pulmonary lesions, which showed that the bacillary content of younger, nuclear debris-rich caseum is significantly higher than that of more advanced and mature caseum (45). The same study showed that (i) mature solid caseum with homogenous necrosis often yielded no or very few AF-positive bacilli and (ii) liquefied caseum can sometimes be the site of explosive M. tuberculosis multiplication. Multiple fields of view within the same human caseous nodule revealed zones of solid caseum with no visible bacilli and zones of liquefaction containing thousands (45). In 1965, Canetti determined that pulmonary TB cavities of patients harbor as much as 10⁷ to 10⁹ bacilli, in contrast to only 10² to 10⁴ bacilli in closed necrotic lesions (60). Since the high oxygen content of airways favors M. tuberculosis growth, the development of cavitary disease marks the first burst of extracellular multiplication since the onset of infection (33). In our recent study, we noted that the M. tuberculosis burden of cavity caseum excised from rabbit lung ranges from 6 × 10⁵ to 3 × 10⁸ CFU per gram of tissue (13). This range is dramatically higher than in seemingly uninvolved lung sections (0 to 1.0 × 10³ CFU/g) and in cellular lesions (0 to 3.2 × 10⁴ CFU/g) from chronically infected rabbits (10). Necrotic lesions from these rabbits harbor only up to 2.3 × 10⁵ CFU/g, confirming that M. tuberculosis replication increases dramatically upon the reintroduction of oxygen when closed nodules transform into cavities.

Differentially culturable tubercle bacilli (DCTB) constitute a subpopulation of M. tuberculosis that escapes detection by standard culture methods (61). They do not grow when plated on solid media but can do so in liquid media that has been supplemented with M. tuberculosis culture filtrate or recombinant resuscitation-promoting factors (Rpfs) (62). Importantly, DCTB often fails to respond to antibiotic treatment in the way that readily detectable M. tuberculosis does (61). Sputum from TB patients harbors a variable but significant proportion of DCTB (62–64). Since it is widely accepted that sputum M. tuberculosis originates from pulmonary cavities, this raises the possibility that caseum may also harbor a significant DCTB subpopulation. The enumeration of M. tuberculosis burden in tissues is traditionally achieved by “plating” homogenates onto agar media, but this method would not account for DCTB. Indeed, lung tissue specimens from latently infected rabbits, for instance, appear sterile on agar media until animals receive an immunosuppressive agent, which facilitates ex vivo bacillary growth of what the authors refer to as “dormant M. tuberculosis” (58). Further studies using Rpf-supplemented liquid media are required to improve bacterial detection in seemingly sterile and low-burden caseum specimens.

Replication State

In the caseous core of necrotic granulomas, M. tuberculosis bacilli are thought to exist in a slow or nonreplicating state, mostly because the avascular and necrotic nature of this microenvironment generates hypoxic conditions which, in turn, induce nonreplicating persistence in mycobacteria (56, 65–67). In addition, the sustained release of necrotizing foamy macrophages in caseum is thought to increase oxygen- and nitrogen-reactive species, also known to induce nonreplicating persistence (68, 69). In vitro models designed to mimic these environmental stressors correspondingly shift replicating M. tuberculosis to the nonreplicating state (65, 70–72). However, direct evidence of nonreplicating M. tuberculosis in caseum was lacking until recently. Using caseum excised from the lung cavities of TB-infected rabbits, we provided first proof of this phenomenon, showing that the bacterial burden of caseum samples remained constant when the samples were incubated ex vivo for up to 28 days at 37°C (Fig. 1) (13).

FIG 1.

Bacterial burden and growth kinetics of M. tuberculosis in ex vivo rabbit caseum. Bacterial burdens on day 1 and day 8, 22, or 29 after excision of caseum from 13 rabbit cavities are shown. M. tuberculosis in caseum appears to be nonreplicating over the whole incubation period. Adapted from the work of Sarathy et al. (13).

To determine whether the constant CFU profile was the result of true nonreplication versus balanced growth and death, we measured bacterial chromosome equivalents (CEQ), which reflect the cumulative burden of live and dead bacteria in the lesion, and CEQ/CFU ratios, indicative of the extent of bacterial killing over time (73). In all ex vivo caseum samples analyzed, both CEQ and CEQ/CFU ratios remained constant, indicating true nonreplication rather than a balance between growth and death. Interestingly, we have observed a wide range of CEQ/CFU ratios in rabbit caseum specimens, indicative of a large spectrum from robust immune-mediated killing to failed immunity. This observation agrees with previous studies that have noted heterogeneity in the extent of immune-mediated killing of M. tuberculosis in lesions from rabbits and cynomolgus macaques (10, 12).

Intracellular Lipophilic Inclusions

Intracellular lipophilic inclusions (ILIs) were first observed in M. tuberculosis by Burdon in 1946 (74). Since then, they have been visualized in multiple mycobacterial species using electron microscopy and a combination of confocal microscopy and auramine-O/Nile red dual staining (75). Both techniques have clearly revealed ILIs in M. tuberculosis from clinical sputum samples (76–78). The ILI-positive subpopulation in sputum samples accounts for 3% to 86% of the total quantity of M. tuberculosis organisms detected (77). Using auramine-O/Nile red dual staining, we recently showed that a large proportion of M. tuberculosis bacilli in rabbit caseum contain ILIs, the first such evidence in any caseum specimens (13). Most ILI-positive bacilli did not stain with auramine, suggesting a loss of acid-fastness of caseum M. tuberculosis as described by others (79–81).

The major lipid species found in lipid extracts from ILI-rich M. tuberculosis include triacylglycerols (TAGs), fatty acids (FAs), polar lipids, and wax esters (WEs) (79, 80, 82). TAGs, which are nonpolar and water insoluble, are a better long-term form of stored carbon-based energy than carbohydrates and proteins because they are less sensitive to oxidation and have higher calorific value (83). TAG storage and utilization are widespread among eukaryotic organisms, such as yeasts, fungi, plants, and animals, but uncommon among bacteria with the exception of the Actinomycetes group, which includes mycobacteria (83). The exhaustive lipid composition of ILIs from caseum M. tuberculosis has yet to be determined. However, we know that ILI lipid reserves are a direct reflection of the FA building blocks available in its immediate external milieu. It has been shown that FA compositions of host macrophage TAGs and intracellular M. tuberculosis TAGs are nearly identical, suggesting that the pathogen derives FA from host TAGs for incorporation into its private ILI stores. M. tuberculosis in oleic acid-induced foamy macrophages accumulate TAGs consisting of C_16:0, C_18:0, and C_18:1 FAs predominantly and C_24:0, C_26:0, and C_28:0 to a much lesser extent (80). In axenic cultures, M. tuberculosis under hypoxia accumulates TAGs and WEs with C_16:0 and C_18:0 as the major FA constituents (79). Supplemented oleic acid is specifically incorporated into C_26:0-containing TAGs, whereas acetate and palmitate are incorporated into C_16:0- to C_26:0-containing TAGs and C_14:0- to C_28:0-containing TAGs, respectively (84).

The presence of ILIs in M. tuberculosis in vivo is significant because there is overwhelming evidence of its strong association with the state of dormancy and phenotypic resistance to rifampin and isoniazid (77, 79–82). The removal of lipid supplementation and reversal of ILI accumulation leads to the resumption of mycobacterial cell division (85). In sputum samples from drug-naive patients, a higher frequency of ILI-positive M. tuberculosis correlates with longer time to positivity, consistent with the notion that they are slowly replicating or nonreplicating bacteria (77). In vitro assays using single and multiple stress nonreplication models induce accumulation of ILIs in M. tuberculosis, with corresponding drastic increases in triacylglycerol synthase (tgs1) gene expression (77, 79). Collectively, the evidence suggests that the M. tuberculosis tgs1 gene product is responsible for the re-esterification of host lipid-derived FAs into TAGs for intrabacterial storage in response to environmental stresses. The Δtgs1 deletion mutant is hence severely compromised in its ability to accumulate TAGs under various stress conditions, fails to develop tolerance to antibiotics, and is more likely to be AF positive (79, 80, 86). Fifteen putative triglyceride synthases (encoded by tgs genes), also known as diacylglycerol transferases, were identified in M. tuberculosis, and all showed TGS activity when expressed in Escherichia coli. Of these genes, tgs1 (rv3130c) is the most active and the most induced in nonreplicating persistent bacilli (84). tgs1 lies in close proximity to the dormancy transcription factor gene dosR, and disruption of the latter completely prevents the induction of tgs1 under hypoxia (87). Members of the triacylglycerol synthase family are differentially regulated at the transcription level, and some members are exclusively present in pathogenic mycobacteria (88).

Daniel et al. identified PPE15, a perilipin-like protein in M. tuberculosis that regulates lipid droplet homeostasis. The PPE15 knockout mutant is compromised in its ability to accumulate TAG and develop tolerance to rifampin (82). Furthermore, Rv2744c, an ortholog of phage shock protein A (PspA), localizes to the surface of ILIs, where it oligomerizes, and regulates ILI number and size and M. tuberculosis survival in the nonreplicating persistent state (89). Utilization of energy stored as TAGs requires hydrolytic activity to release FAs for β-oxidation. Of the 24 putative lipases identified in M. tuberculosis, the product of rv3097c (LipY) has the highest hydrolytic activity and is most induced under stress. Correspondingly, the lipY deletion mutant is unable to utilize stored TAGs as efficiently as wild-type M. tuberculosis (90) and resuscitate from dormancy (81).

Pacl et al. presented an alternative hypothesis in which hypoxia and lipid metabolism saturate the electron transport chain and this reductive stress is balanced in part by lipid synthesis in a DosR/S/T-dependent manner (91). Interestingly, strains belonging to the Beijing lineage of M. tuberculosis, which is associated with increased pathogenicity and a higher propensity for developing drug resistance, accumulate TAGs more readily than other strains (92). This has been attributed to the constitutive overexpression of genes of the dormancy regulon, including tgs1 (rv3130). The Beijing strains may be “preadapted” to stressful environmental conditions, such as hypoxia in caseous granulomas, and their abundant energy stores could confer an advantage over other lineages during latency and transmission (92). Mycobacterium avium, Mycobacterium marinum, and Mycobacterium canetti, to name a few other mycobacterial species, have been shown to develop lipid inclusions as well (85, 93, 94). However, ILI formation in intracellular M. marinum is not accompanied by growth arrest or decreased metabolic activity (93).

Drug Susceptibility

In recent studies, our group measured the susceptibility of M. tuberculosis present in rabbit cavity caseum to major first- and second-line anti-TB drugs (11, 13). This ex vivo assay involves exposing M. tuberculosis present in excised caseum to a range of concentrations of a drug or drug candidate over 7 days and determining the minimum concentration required to kill 90% of the bacteria present in the sample (casMBC₉₀) and the concentration required to fully sterilize caseum when applicable. Results clearly indicate that M. tuberculosis in caseum is profoundly tolerant to TB drugs. Depending on the drug, there was a 3- to 400-fold increase in MBC₉₀ compared to that for replicating M. tuberculosis cultures (Table 1). Only the rifamycins and fluoroquinolones were effective at killing this M. tuberculosis subpopulation (casMBC₉₀ < 10 μM). Interestingly, rifamycins are the only drug class capable of fully sterilizing caseum specimens, albeit at relatively high concentrations (50 to 100 μM). This suggests that caseum M. tuberculosis is vulnerable to RpoB inhibition, at least via the specific mechanism of action of rifamycins. Bedaquiline, linezolid, and pyrazinamide performed modestly in the assay (casMBC₉₀ of 32 to 512 μM), whereas isoniazid, clofazimine, and kanamycin showed marginal to no activity (13). We have expanded the panel of TB drugs tested in this bactericidal assay, and all results support the notion of extreme drug tolerance of caseum M. tuberculosis. Results from the casMBC assay were compared with results from the nutrient starvation (Loebel) and oxygen deprivation (Wayne) models, both of which are frequently used to test drug susceptibility of nonreplicating M. tuberculosis (65, 95) (Table 1). The general trend across all three assays is a striking loss of bactericidal activity. Overall, drug potencies were more similar in Wayne versus casMBC than in Loebel versus casMBC assays. Most notable differences between the Wayne and casMBC assays were found with the fluoroquinolones, markedly more potent in caseum, and kanamycin as a representative of aminoglycosides, much less potent in caseum (13). These two antibiotic classes are currently the mainstay of second-line therapy against multidrug-resistant TB. Thus, the TB community still lacks a potency assay that is compatible with medium- to high-throughput requirements and reproduces the drug tolerance of caseum M. tuberculosis.

TABLE 1.

Bactericidal activities of TB drugs against M. tuberculosis in ex vivo caseum and in vitro under replicating and nonreplicating conditions^a

Antibiotic	Caseum MBC90 (μM)	MBC90 (μM)b	WCC90 (μM)b	LCC90 (μM)b
Isoniazid	>128	0.31–0.63	>100	>100
Pyrazinamide	512	>80	>100	>100
Rifampin	8	0.078	1	10
Rifapentine	2	0.078	0.5	10
Rifabutin	2	0.039	0.5	10
Rifalazil	2	0.036c
Moxifloxacin	2	0.31–0.63	10	>100
Levofloxacin	8	1.25–2.5	50	>100
Gatifloxacin	2	0.63	50	>100
Linezolid	128	10	>100	>100
Radezolid	2
Pretomanid	32	0.63	20
Kanamycin	>128	5.0	20	>100
Clofazimine	>128	40	50	>100
Bedaquiline	32	10	>20	>20

^aAdapted from the work of Sarathy et al. (13). MBC₉₀, minimum concentration required to kill 90% of the bacterial population; WCC, Wayne (hypoxic culture) bactericidal concentration; LCC, Loebel (nutrient starved culture) bactericidal concentration.

^bMost MBC and all WCC and LCC data were obtained from Lakshminarayana et al. (119).

^cMBC₉₀ data for rifalazil were obtained from Mor et al. (153).

ENVIRONMENTAL STRESS FACTORS

Given the persistent nature of caseum M. tuberculosis, understanding this subpopulation is key to shortening treatment duration and improving cure rates. In this section, we examine the environmental stress factors that are often credited for triggering the shift of caseum M. tuberculosis to the nongrowing state.

Acidic pH

The acidity of caseum is important because M. tuberculosis growth in vitro—whether in nutrient-rich or minimal medium—slows at acidic pH (<6.5) and is completely halted at pH 5.0 (71, 96, 97). However, the organism remains viable since it can resist external pH as low as 4.5 and maintain neutral cytosolic pH (98, 99). The notion that the caseous core of necrotic granulomas forms an acidic microenvironment has been promoted since the 1950s, most likely as an explanation for the remarkable treatment-shortening capacity of pyrazinamide, which is more potent in vitro at acidic pH (100, 101). As summarized in Table 2, the pH of caseous foci of mice, guinea pigs, and rabbits is, on average, close to neutral. A study from 1954 with TB-infected rabbits showed that caseum pH increased from 6.4 to 7.4 as lesions matured and liquefied (102). In contrast, Kempker et al. measured the pH of caseum in the lesions of 10 pyrazinamide-treated TB patients using indicator strips and found a median pH of 5.5, with a range of 5 to 7.2, and only 2 readings exceeding pH 7.0. Both tissue samples with neutral caseum presented severe necrosis and an abundance of AF-staining bacilli (103). In older studies of human caseum specimens, more neutral conditions were observed (104, 105). These observations collectively indicate that acidity is not the primary environmental pressure contributing to caseum M. tuberculosis’s nonreplication and the drug-tolerant phenotype. It seems more likely that cytosolic pH homeostasis in M. tuberculosis evolved to combat phagosome acidification as opposed to acidification of caseum. The phagosomal compartment in which mycobacteria resides during intracellular infections has a pH range of 4.5 to 6.5 depending on the activation state of the macrophage (106–108).

TABLE 2.

Measurement of pH of caseum in various species using different techniques

Species	Nature of specimen	Measurement technique	pH range	Reference
C3HeB/FeJ mouse	In situ	16-gauge needle tip micro-pH electrode	7.2–7.5 (7.4a )	154
	Homogenate	pH indicator strips	7.4–7.6	9
Guinea pig	In situ	16-gauge needle tip micro-pH electrode	7.0–7.5 (7.2a )	150
Rabbit	3-fold diluted in water	pH indicator strips	7.0–7.5	13
	Undiluted smear	pH indicator strips	6.1–8.0 (6.7b )	10
	Homogenate in sucrose solution	Glass electrode	6.4–7.4	102
Human	In situ	pH indicator strips	5–7.1 (5.5b )	103
	Homogenate	Glass electrode	7.3–8	104
	Aspiratesc	Glass electrode	7.2–7.5	105

^aMean.

^bMedian.

^cNonpulmonary TB.

Hypoxia

Low oxygen tension is a widely accepted feature of necrotic granulomas given the lack of vascularization in the caseous core. Tsai et al. confirmed the absence of endothelial cells in the necrotic region of human TB lung granulomas by observing a lack of immunoreactivity with anti‐CD31 antibodies (109). The fibrotic cuff of more mature granulomas theoretically further reduces its ability to “breathe.” Table 3 summarizes published observations of hypoxia in TB granulomas from various animal infection models and the detection techniques used. The oncology probe pimonidazole hydrochloride (PIMO) has been useful for the visualization of hypoxic regions in mammalian and human tumors (110). Also known as Hypoxyprobe, PIMO is bioreductively activated by mammalian nitroreductases at low oxygen concentrations and binds to thiol groups on cellular proteins, forming adducts that can be detected with specific antibodies. Prolonged oxygen tensions of ≤10 mm Hg are required for thiol adduct formation. Via et al. infused TB-infected guinea pigs, rabbits, and cynomolgus macaques with PIMO prior to necropsy and showed distinct regions of adduct formation in the caseous core of pulmonary necrotic granulomas in all three species (111). Lenaerts et al. similarly used PIMO to visualize hypoxic regions of necrotic granulomas in the guinea pig model of TB infection and found that persistent M. tuberculosis in the necrotic pulmonary lesions of antibiotic-treated guinea pigs are primarily located within the hypoxic caseous foci. In this animal model, hypoxic regions of caseous granulomas are established as early as 30 days after infection, meaning that oxygen deprivation is not just a feature of persistent or latent infections (3). In both studies, PIMO staining appears as a characteristic ring encircling the caseous center because deep necrotic tissue is devoid of viable cells that reduce PIMO and facilitate adduct formation. Therefore, an assumption is made regarding anaerobicity in the deepest regions of the caseous core (3, 111). The former study also provided further evidence of hypoxia by using a fiber optic oxygen sensor inserted directly into the granulomas of infected rabbits. The mean oxygen partial pressure of TB granulomas with caseous centers was 1.6 mm Hg, 37-fold lower than that of seemingly normal sections of lung. It is worth mentioning that cavities have the highest oxygenation levels of all TB lesion types, as their surface is in contact with the airway and has an oxygen tension similar to that of the communicating bronchi (112). Therefore, M. tuberculosis residing in cavity caseum likely encounters only limited hypoxic stress that could be present in the deeper layers of cavity caseum toward the cellular and fibrotic wall.

TABLE 3.

Detection of hypoxic granulomas in various species using different techniques^a

Species	Caseous necrosis	Measurement technique	pO2	Reference
BALB/c mouse	Absent	Immunodetection of PIMO	Not hypoxic	8
		[64Cu]ATSM PET imaging	Not hypoxic	8
C57BL/6 mouse	Absent	Immunodetection of EF5	Not hypoxic	109
		Immunodetection of PIMO	Not hypoxic	115
		Catheter microelectrode	Not hypoxic	115
C3HeB/FeJ mouse	Present	Immunodetection of PIMO	<10 mm Hg	8
		Immunodetection of PIMO	<10 mm Hg	7
		[64Cu]ATSM PET imaging	<3.8 mm Hg	8
Guinea pig	Present	Immunodetection of PIMO	<10 mm Hg	111
		Immunodetection of PIMO	<10 mm Hg	3
Rabbit	Present	Immunodetection of PIMO	<10 mm Hg	111
		Fiber optic oxygen sensor	1.6 mm Hg	111
Cynomolgus macaque	Present	Immunodetection of PIMO	<10 mm Hg	111
		Immunodetection of PIMO	<10 mm Hg	122
Human	Present	[18F]FMISO PET-CT imaging	<10 mm Hg	113

^a[⁶⁴Cu] ATSM PET, ⁶⁴Cu-diacetyl-bis (N⁴-methylthiosemicarbazone) positron emission tomography; EF5, 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-[18F]pentafluoropropyl)-acetamide; PIMO, pimonidazole hydrochloride; [¹⁸F]FMISO, [¹⁸F]fluoromisonidazole.

Positron emission tomography (PET)-computed tomography (CT) scans of TB patients dosed with the hypoxia-specific tracer [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) showed distinctive accumulation of [¹⁸F]FMISO in lesions and the regions surrounding cavities. Heterogeneous degrees of hypoxia were observed within and between lesions, consistent with the concept of multiple diverse TB microenvironments within individual patients (113). However, PET-CT does not allow for the colocalization of FMISO “hot spots” with caseum. Neither BALB/c nor C57BL/6 mice develop necrotic lesions with caseous centers upon TB infection (32, 114). Using the hypoxic tracer copper(II)-diacetyl-bis(N⁴-methyl-thiosemicarbazone) ([⁶⁴Cu]ATSM), hypoxic necrotic granulomas were detected in TB-infected C3HeB/FeJ mice but not in BALB/c mice. These results were confirmed by PIMO immunostaining in the same study (8). Similarly, granulomas from C57BL/6 mice are not stained by PIMO or EF5, another hypoxia-sensitive compound which forms thiol adducts that can be immunodetected (109, 115).

As mentioned previously, bacterial burden is significantly higher in pulmonary cavities than in closed necrotic lesions (60), due to the reintroduction of oxygen and the subsequent resumption of bacterial replication. The implications of hypoxic condition in caseum are significant because an in vitro model that gradually depletes oxygen and then maintains low oxygen tension (Wayne model) triggers an adaptive dormancy response in M. tuberculosis (65, 116). In this model, M. tuberculosis arrests replication, decreases intracellular ATP levels (117), represses an array of cellular functions, including protein and DNA synthesis (118), and develops phenotypic resistance to most TB-active antibiotics (119, 120). At the molecular level, the dormancy response to hypoxia is mediated by the transcription factor Rv3133c, or DosR (for dormancy survival regulator) (87, 118, 121). Consequently, greater PIMO staining of granulomas from macaques with LTBI versus those from individuals with the active disease corresponded with higher induction of the DosR regulon in the former. In the same study, the strongest induction of the DosR regulon was observed in the most hypoxic tissue regions (i.e., caseum from LTBI granulomas) (122).

These findings highlight the value of animal models that reproduce caseous necrosis, fibrosis, and/or cavitation. These include the C3HeB/FeJ mouse, guinea pig, minipig, rabbit, and nonhuman primate models (32, 123, 124). Murine models in which the bulk of the bacterial population resides in immune cells—such as BALB/c and C57Bl6—and do not develop hypoxic necrotic lesions may be useful for the evaluation of drug efficacy against intracellular bacteria but do not reveal the complexities of a persistent extracellular subpopulation (114).

Iron Starvation

Iron is essential for life of most organisms, including mycobacteria. M. tuberculosis uses an array of metal detoxification and acquisition systems to combat phagosome-centric host responses to infection, including iron and manganese deprivation and copper and zinc overload (125). The host’s propensity to sequester iron for itself makes free iron a scarce commodity in the phagolysosomes of infected macrophages. M. tuberculosis overcomes iron deprivation via mechanisms that include siderophore-based ferric ion chelators, ABC-type heme transporters, and ferritin-based iron storage (126). Iron-starved growth-arrested M. tuberculosis is tolerant to several antibiotics (127). Whereas direct observation of depleted iron levels in caseum has not been made, iron limitation in human lesions is implied because of the following: (i) M. tuberculosis genes involved in iron homeostasis are required for virulence (128–130); (ii) “African iron overload,” a condition associated with high dietary iron intake and specific genetic defects, is significantly associated with developing pulmonary TB and TB-related mortality (131, 132); and (iii) LTBI patients can experience reactivation of disease upon receiving iron supplementation as treatment for anemia, since iron loading enhances the growth of intracellular M. tuberculosis (133, 134).

The necrotic center of a caseous granuloma is enriched in host-derived transferrin, haptoglobin, hemopexin, lactoferrin, and lipocalin, all of which collectively sequester free Fe³⁺, hemoglobin, heme, and siderophores (127, 135). Assuming that the relative abundance of iron-sequestering proteins is inversely proportional to iron bioavailability, Kurthkoti et al. concluded that caseum constitutes an iron-depleted environment and demonstrated using an in vitro iron starvation model that M. tuberculosis is capable of persisting in a nonreplicating state under these conditions (127). However, whether these proteins continue to actively sequester iron within dead cell debris as they do in nonnecrotic lesion areas is an open question. Interestingly, M. tuberculosis in an adipocyte model meant to mimic the lipid-rich environment of caseating granulomas downregulates the iron-responsive regulator IdeR, among other gene expression changes that suggest iron abundance (136). Finally, gene expression profiling in sputum M. tuberculosis did not reveal the induction of iron-scavenging genes, suggesting adequate iron supply in the cavities from which these bacilli originate (77). Collectively, these findings provide mostly indirect and at times conflicting information on the availability of iron in caseum. Direct quantitation of free iron in the caseous foci of TB granulomas is required to assess whether M. tuberculosis is iron starved in this environment.

Nutrient Deprivation or a Shift in Carbon Sources

Nutrient limitation or deprivation is thought to stimulate the nonreplicating persistent state in caseum M. tuberculosis. In 1933, Loebel et al. developed an in vitro culture model that maintains nonreplicating M. tuberculosis with a low metabolic rate in a nutrient-deprived oxygen-rich medium (Loebel model) (70), characterized by lowered intracellular ATP levels (137), decreased energy metabolism, lipid biosynthesis, and cell division, among other changes in the transcriptome (138). These changes are accompanied by decreased drug penetration and intracellular accumulation (139) and drastic losses in susceptibility to multiple anti-TB drugs (119, 137–139). However, the relevance of the Loebel culture medium—phosphate-buffered saline with Tween—to recapitulate nutrient conditions found in lesions is debatable.

Total lipid extraction from the caseous foci of human pulmonary caseous TB granulomas followed by thin-layer chromatography (TLC) and mass spectrometric analyses revealed marked accumulation of cholesterol, cholesterol esters (CEs), triacylglycerols (TAGs), and lactosylceramides (LacCer) compared to the levels in uninvolved normal lung tissue (22). Guerrini et al. subsequently conducted comprehensive liquid chromatography coupled to mass spectrometry (LC/MS) analyses of lipids from human, marmoset, and rabbit caseum specimens that were dissected by a laser capture microdissection (LCM) platform and found TAGs and CEs to be the predominant lipid classes. The TAG profiles from all three species were highly conserved, with three highly abundant clusters: TAGs containing FA chains of (i) 50 carbons and 0 to 3 double bonds, (ii) 52 carbons and 0 to 4 double bonds, and (iii) 54 carbons and 1 to 5 double bonds. The CE profiles, on the other hand, were more variable within and between species (25). These findings suggest that rather than nutrient deprivation, M. tuberculosis is faced with a lipid-rich diet in caseum.

Indeed, the M. tuberculosis genome is loaded with about 250 genes encoding enzymes involved in FA metabolism, an impressive proportion of which are dedicated to FA degradation and oxidation, strongly suggesting that M. tuberculosis is committed to using host lipids in vivo (140). Electron microscopy images of infected foamy macrophages revealed the tight apposition of M. tuberculosis-containing phagosomes to macrophage lipid bodies followed by the appearance of ILIs, implying the internalization of lipid bodies into the phagosomes, where M. tuberculosis utilizes host-derived lipids (23, 141). Given the similarities in the lipid profiles of caseum and its surrounding ring of infected foamy macrophages (22, 25), it is likely that M. tuberculosis is similarly adapted to the utilization of caseous lipids as a nutrient source. The observation of lipid accumulation in caseum M. tuberculosis supports this hypothesis (13). The upregulation of isocitrate lyase (icl1), a member of the glyoxylate cycle, in ILI-rich M. tuberculosis also suggests a shift to utilizing FAs as a source of energy (77). Icl1 plays a second role in M. tuberculosis as a methylisocitrate lyase (MCL) in the methylcitrate cycle which degrades propionate, further enabling cholesterol utilization by M. tuberculosis (142). Other studies have also shown that M. tuberculosis is well adapted to metabolizing and utilizing cholesterol and FAs during the chronic phase of infection (143–147), both of which are abundant in caseous foci (22). Since M. tuberculosis bacilli found in caseum largely come from necrotized foamy macrophages where M. tuberculosis has already adapted to a lipid-rich environment and energy is stored in the form of ILIs, it is possible that little to no further metabolic adaptation is required when the bacteria transit from an intracellular lifestyle in lipid-laden macrophages to an extracellular existence in necrotic caseum.

CONCLUSION

In 1955, Canetti regarded caseation as the most important event of tuberculosis infection. M. tuberculosis manipulates the host’s metabolism by inducing lipid accumulation in foamy macrophages (22), which subsequently necrotize and release their stored lipids, forming a CE- and TAG-rich reservoir in caseum, where the pathogen feeds and persists. M. tuberculosis is believed to remain dormant in caseous lesions for extended periods before reactivation and cavitation occur. The cavity surface is known as a microenvironment of failed immunity due to the selective absence of CD4⁺ and CD8⁺ T cells at the luminal surface (in contact with airways), preventing direct T cell-macrophage interactions at this site and allowing luminal phagocytes to remain permissive for bacillary growth (148). This provides the pathogen with a safe haven for robust replication and an outlet for transmission (149). Although plating on standard growth media has shown that the bacterial burden of caseous granulomas reaches high numbers in various animal models, we may still underestimate total counts and miss differentially culturable bacteria. New tools capable of detecting all viable M. tuberculosis in caseum are being developed to provide better information on the efficacy of antibiotic treatment and predict relapse.

Here we have revisited widely accepted concepts about loss of acid-fastness and the stress factors thought to trigger M. tuberculosis’s shift to the nongrowing state in caseum, leading to phenotypic drug resistance. (i) pH measurements in multiple animal models and in patients indicate that acidic conditions may be only transient (10, 13, 102, 150) and that caseum pH in mature lesions varies across lesions even within the same organism but remains near neutral overall. Additional pH measurements of resected human cavities using modern readouts are required to confirm caseum pH and pH variability across lesions and design in vitro potency assays that recapitulate in vivo conditions. (ii) Proof of hypoxia by immunostaining techniques and the lack of blood supply in caseum collectively point to oxygen deprivation as one major environmental pressure driving nonreplicating persistence in TB infections (151) (Fig. 2). While marked hypoxia is found in the caseum of closed granulomas, normoxic conditions are found at the cavity surface (112). It is likely that a gradient of oxygen tension exists from the surface of the cavity lumen—in direct contact with the airway—toward the deeper layers of caseum that border the cavity wall. Again, visualizing and quantifying such a gradient are critical to develop predictive in vitro potency assays. (iii) Deprivation of free iron and other trace elements in caseous foci is often implied by extension from the well-characterized iron sequestration in host cells. However, the equilibrium between bound and free iron in two oxidation states is a dynamic process that is likely not conserved in dead caseum and remains to be quantified. (iv) The abundance of lipid-based carbon sources in caseum and M. tuberculosis’s inclination for lipid catabolism (146, 147, 152) call into question the hypothesis that nutrient deprivation plays a significant role in the development of nonreplicating persistence.

FIG 2.

The caseous compartments of necrotic granulomas contain extracellular M. tuberculosis. This subpopulation adapts to local stresses such as low oxygen tension and high lipid content, resulting in the accumulation of intrabacterial lipid inclusions, a shift to a state of nonreplication, and phenotypic drug resistance. Created with BioRender.

Physiology and omics studies all point to a link between metabolic adaptations of M. tuberculosis to lipid-rich environments, ILI accumulation, and nonreplicating persistence (Fig. 2). Novel therapeutics that disrupt TAG hydrolysis, resynthesis, and storage in ILIs are likely to interfere with M. tuberculosis’s ability to survive in a lipid-rich microenvironment, inhibit the shift to nonreplication, increase susceptibility to antibiotics, and shorten treatment duration. Not surprisingly since caseum is largely made of necrotizing foamy macrophages, the microenvironmental conditions found in caseum are reminiscent of those present in infected macrophages and their phagolysosomes, suggesting that when released into caseum, M. tuberculosis is largely preadapted to the set of stressors it encounters (Fig. 2). The characteristics of caseum M. tuberculosis discussed in this review stem from observations of the pathogen at the population level but do not highlight heterogeneity in composition and physiological state of the caseous microenvironment and its residents, respectively, within each granuloma. This adds layers of complexity to understanding disease biology and treatment of this bacterial subpopulation. But advancements in the development of molecular probes and microscopy platforms should soon enable single-cell resolution while maintaining granuloma architecture and shed light on these issues. The major challenge for developing predictive and high-throughput assays that model the profound drug tolerance of caseum M. tuberculosis consists of reproducing the right balance of oxygen tension, pH, lipid abundance, and lipid species, using the ex vivo caseum bactericidal assay as a benchmark.

Biographies

Jansy P. Sarathy received her Ph.D. from the National University of Singapore in conjuction with the Novartis Institute for Tropical Diseases. She has since completed a postdoctoral research fellowship at the Public Health Research Institute, Rutgers, NJ, and now works as a Research Assistant Member at the Center for Discovery and Innovation, Hackensack Meridian Health, NJ. She has spent the past 10 years developing in vitro assays to facilitate the field of tuberculosis drug discovery. The past few years have been especially focused on exploring the pharmacokinetic and pharmacodynamic interactions of Mycobacterium tuberculosis in the caseous core of granulomas.

Véronique Dartois is a Member of the Center for Discovery and Innovation and Professor at the Hackensack School of Medicine. She received her Ph.D. in microbial genetics at the University of Louvain, Belgium. After postdoctoral fellowships at the Scripps Research and Pasteur Institutes, she joined the biotechnology industry in California, with a focus on anti-infective drug discovery. As Executive Director of Pharmacology at the Novartis Institute for Tropical Diseases from 2005 to 2012, she supported drug discovery programs in tuberculosis (TB), dengue fever, and malaria. In 2012, she joined the Public Health Research Institute of Rutgers University to focus on the pharmacological mechanisms contributing to the very long therapy duration required to cure TB and nontuberculous mycobacterial disease. Her team’s work has paved to way to rationally build new drug regimens that combine agents with complementary distribution and activity at the complex site of disease, a significant departure from current empirical approaches.