Antimicrobial Activity of Gallium(III) Compounds: Pathogen-Dependent Targeting of Multiple Iron/Heme-Dependent Biological Processes.

Metals play vital roles in biological systems, with iron/heme being essential for cellular and metabolic functions necessary for survival and/or virulence in many bacterial pathogens. Given the rise of bacterial resistance to current antibiotics, there is an urgent need for the development of non-toxic and novel antibiotics that do not contribute to resistance to other antibiotics. Gallium, which mimics iron, has emerged as a promising antimicrobial agent, offering a novel approach to combat bacterial infections. Gallium does not have any known functions in biological systems. Gallium exerts its effects primarily by replacing iron in redox enzymes, effectively inhibiting bacterial growth by targeting multiple iron/heme-dependent biological processes and suppressing the development of drug resistance. The aim of this review is to highlight recent findings on the mechanisms of action of gallium and provide further insights into the development of gallium-based compounds. Understanding the mechanisms underlying gallium's biological activities is crucial for designing drugs that enhance their therapeutic therapies while minimizing side effects, offering promising avenues for the treatment of infectious diseases.

Development of Gallium(III) as an Antimicrobial Drug Targeting Pathophysiologic Iron Metabolism of Human Pathogens.

The treatment of infections is becoming more difficult due to emerging resistance of pathogens to existing drugs. As such, alternative druggable targets, particularly those that are essential for microbe viability and thus make it harder to develop resistance, are desperately needed. In turn, once identified, safe and effective agents that disrupt these targets must be developed. Microbial acquisition and use of iron is a promising novel target for antimicrobial drug development. In this Review we look at the various facets of iron metabolism critical to human infection with pathogenic microbes and the various ways in which it can be targeted, altered, disrupted, and taken advantage of to halt or eliminate microbial infections. Although a variety of agents will be touched upon, the primary focus will be on the potential use of one or more gallium complexes as a new class of antimicrobial agents. In vitro and in vivo data on the activity of gallium complexes against a variety of pathogens including ESKAPE pathogens, mycobacteria, emerging viruses, and fungi will be discussed in detail, as well as pharmacokinetics, novel formulations and delivery approaches, and early human clinical results.

Combining Gallium Protoporphyrin and Gallium Nitrate Enhances In Vitro and In Vivo Efficacy against Pseudomonas aeruginosa: Role of Inhibition of Bacterial Antioxidant Enzymes and Resultant Increase in Cytotoxic Reactive Oxygen Species.

Pseudomonas aeruginosa is a highly antibiotic-resistant opportunistic pathogenic bacteria that is responsible for thousands of deaths each year. Infections with P. aeruginosa disproportionately impact individuals with compromised immune systems as well as cystic fibrosis patients, where P. aeruginosa lung infection is a leading cause of morbidity and mortality. In previous work, we showed that a combination of gallium (Ga) nitrate and Ga protoporphyrin worked well in several bacterial infection models but its mechanism of action (MOA) is unknown. In the current work, we have investigated the MOA of Ga combination therapy in P. aeruginosa and its analysis in the in vivo model. In P. aeruginosa treated with Ga combination therapy, we saw a decrease in catalase and superoxide dismutase (SOD) activity, key antioxidant enzymes, which could correlate with a higher potential for oxidative stress. Consistent with this hypothesis, we found that, following combination therapy, P. aeruginosa demonstrated higher levels of reactive oxygen species, as measured using the redox-sensitive fluorescent probe, H2DCFDA. We also saw that the Ga combination therapy killed phagocytosed bacteria inside macrophages in vitro. The therapy with low dose was able to fully prevent mortality in a murine model of P. aeruginosa lung infection and also significantly reduced lung damage. These results support our previous data that Ga combination therapy acts synergistically to kill P. aeruginosa, and we now show that this may occur through increasing the organism's susceptibility to oxidative stress. Ga combination therapy also showed itself to be effective at treating infection in a murine pulmonary-infection model.

Gallium Porphyrin and Gallium Nitrate Synergistically Inhibit Mycobacterial Species by Targeting Different Aspects of Iron/Heme Metabolism.

There is an urgent need for new effective and safe antibiotics active against pathogenic mycobacterial species. Gallium (Ga) nitrate (Ga(NO3)3) and Ga porphyrin (GaPP) have each been shown to inhibit the growth of a variety of mycobacterial species. The Ga(III) ion derived from Ga(NO3)3 has the potential to disrupt the mycobacterial Fe(III) uptake mechanisms and utilization, including replacing iron (Fe) in the active site of enzymes, resulting in the disruption of function. Similarly, noniron metalloporphyrins such as heme mimetics, which can be transported across the bacterial membrane via heme-uptake pathways, would potentially block the acquisition of iron-containing heme and bind to heme-utilizing proteins, making them nonfunctional. Given that they likely act on different aspects of mycobacterial Fe metabolism, the efficacy of combining Ga(NO3)3 and GaPP was studied in vitro against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium tuberculosis (M. tb). The combination was then assessed in vivo in a murine pulmonary infection model of M. abscessus. We observed that Ga(NO3)3 in combination with GaPP exhibited synergistic inhibitory activity against the growth of M. avium, M. tb, and M. abscessus, being most active against M. abscessus. Activity assays indicated that Ga(NO3)3 and GaPP inhibited both catalase and aconitase at high concentrations. However, the combination showed a synergistic effect on the aconitase activity of M. abscessus. The Ga(NO3)3/GaPP combination via intranasal administration showed significant antimicrobial activity in mice infected with M. abscessus. M. abscessus CFU from the lungs of the Ga(NO3)3/GaPP-treated mice was significantly less compared to that of nontreated or single Ga(III)-treated mice. These findings suggest that combinations of different Ga(III) compounds can synergistically target multiple iron/heme-utilizing mycobacterial enzymes. The results support the potential of combination Ga therapy for development against mycobacterial pathogens.

Neural Glyoxalase Pathway Enhancement by Morin Derivatives in an Alzheimer's Disease Model.

The glyoxalase pathway (GP) is an antioxidant defense system that detoxifies metabolic byproduct methylglyoxal (MG). Through sequential reactions, reduced glutathione (GSH), glyoxalase I (glo-1), and glyoxalase II (glo-2) convert MG into d-lactate. Spontaneous reactions involving MG alter the structure and function of cellular macromolecules through the formation of inflammatory advanced glycation endproducts (AGEs). Accumulation of MG and AGEs in neural cells contributes to oxidative stress (OS), a state of elevated inflammation commonly found in neurodegenerative diseases including Alzheimer's disease (AD). Morin is a common plant-produced flavonoid polyphenol that exhibits the ability to enhance the GP-mediated detoxification of MG. We hypothesize that structural modifications to morin will improve its inherent GP enhancing ability. Here we synthesized a morin derivative, dibromo-morin (DBM), formulated a morin encapsulated nanoparticle (MNP), and examined their efficacy in enhancing neural GP activity. Cultured mouse primary cerebellar neurons and Caenorhabditis elegans were induced to a state of OS with MG and treated with morin, DBM, and MNP. Results indicated the morin derivatives were more effective compared to the parent compound in neural GP enhancement and preventing MG-mediated OS in an AD model.

Treatment of Virulent Mycobacterium tuberculosis and HIV Coinfected Macrophages with Gallium Nanoparticles Inhibits Pathogen Growth and Modulates Macrophage Cytokine Production.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global threat. The course of TB is negatively impacted by coexistent infection with human immunodeficiency virus type 1 (HIV). Macrophage infection with these pathogens modulates their production of pro- and anti-inflammatory cytokines, which could play a crucial role in pathogenesis. Despite the important role of macrophages in containing infection by a variety of microbes, both HIV and M. tuberculosis infect and replicate within these cells during the course of HIV-M. tuberculosis coinfection. Both M. tuberculosis and HIV require iron for growth and replication. We have previously shown that gallium encapsulated in nanoparticles, which interferes with cellular iron acquisition and utilization, inhibited the growth of HIV and an attenuated strain of M. tuberculosis within human monocyte-derived macrophages (MDMs) in vitro Whether this was true for a fully virulent strain of M. tuberculosis and whether gallium treatment modulates cytokine production by HIV- and/or M. tuberculosis-infected macrophages have not been previously addressed. Therefore, coinfection of MDMs with HIV and a virulent M. tuberculosis strain (H37Rv) was studied in the presence of different gallium nanoparticles (GaNP). All GaNP were readily internalized by the MDMs, which provided sustained drug (gallium) release for 15 days. This led to significant growth inhibition of both HIV and M. tuberculosis within MDMs for up to 15 days after loading of the cells with all GaNP tested in our study. Cytokine analysis showed that HIV-M. tuberculosis coinfected macrophages secreted large amounts of interleukin 6 (IL-6) and IL-8 and smaller amounts of IL-1ß, IL-4, and tumor necrosis factor alpha (TNF-α) cytokines. However, all GaNP were able to regulate the release of cytokines significantly. GaNP interrupts iron-mediated enzymatic reactions, leading to growth inhibition of HIV-M. tuberculosis coinfection in macrophages, and also modulates release of cytokines that may contribute to HIV-TB pathogenesis.IMPORTANCE GaNP interrupts iron-mediated enzymatic reactions, leading to growth inhibition of virulent HIV-M. tuberculosis coinfection in macrophages, and also modulates release of cytokines that may contribute to HIV-TB pathogenesis. Macrophage-targeting GaNP are a promising therapeutic approach to provide sustained antimicrobial activity against HIV-M. tuberculosis coinfection.

Dual Inhibition of Klebsiella pneumoniae and Pseudomonas aeruginosa Iron Metabolism Using Gallium Porphyrin and Gallium Nitrate.

Iron- and heme-uptake pathways and metabolism are promising targets for the development of new antimicrobial agents, as their disruption would lead to nutritional iron starvation and inhibition of bacterial growth. Salts of gallium(III) (Ga), an iron mimetic metal, disrupt iron-dependent biological processes by binding iron-utilizing proteins and competing with iron for uptake by bacterial siderophore-mediated iron uptake systems. Ga porphyrins, heme mimetic complexes, disrupt heme-utilizing hemoproteins. Because Ga(NO3)3 and Ga porphyrin disrupt different pathways of bacterial ion acquisition and utilization, we hypothesized that if used in combination, they would result in enhanced antimicrobial activity. Antimicrobial activity of Ga porphyrins (Ga protoporphyrin, GaPP, or Ga mesoporphyrin, GaMP) alone and in combination with Ga(NO3)3 were evaluated against Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA) under iron-limited conditions. The Ga porphyrin/Ga(NO3)3 combination demonstrated substantial synergism against K. pneumoniae, P. aeruginosa, and MRSA. Time-kill assays revealed that the synergistic combination of GaPP/Ga(NO3)3 was bacteriostatic against K. pneumoniae and MRSA and bactericidal against P. aeruginosa. The GaPP/Ga(NO3)3 combination significantly disrupted K. pneumoniae and P. aeruginosa biofilms on plasma-coated surfaces and increased the survival of Caenorhabditis elegans infected with K. pneumoniae or P. aeruginosa. When assessing the antibacterial activity of the Ga(III)/antibiotic combinations, GaPP/colistin and Ga(NO3)3/colistin combinations also showed synergistic activity against K. pneumoniae and P. aeruginosa. Our results demonstrate that GaPP and Ga(NO3)3 have significant synergistic effects against several important human bacterial pathogens through dual inhibition of iron and heme metabolism.

Iron/Heme Metabolism-Targeted Gallium(III) Nanoparticles Are Active against Extracellular and Intracellular Pseudomonas aeruginosa and Acinetobacter baumannii.

Iron/heme acquisition systems are critical for microorganisms to acquire iron from the human host, where iron sources are limited due to the nutritional immune system and insolubility of the ferric form of iron. Prior work has shown that a variety of gallium compounds can interfere with bacterial iron acquisition. This study explored the intra- and extracellular antimicrobial activities of gallium protoporphyrin (GaPP), gallium mesoporphyrin (GaMP), and nanoparticles encapsulating GaPP or GaMP against the Gram-negative pathogens Pseudomonas aeruginosa and Acinetobacter baumannii, including clinical isolates. All P. aeruginosa and A. baumannii isolates were susceptible to GaPP and GaMP, with MICs ranging from 0.5 to ∼32 µg/ml in iron-depleted medium. Significant intra- and extracellular growth inhibition was observed against P. aeruginosa cultured in macrophages at a gallium concentration of 3.3 µg/ml (5 µM) of all Ga(III) compounds, including nanoparticles. Nanoparticle formulations showed prolonged activity against both P. aeruginosa and A. baumannii in previously infected macrophages. When the macrophages were loaded with the nanoparticles 3 days prior to infection, there was a 5-fold decrease in growth of P. aeruginosa in the presence of single emulsion F127 copolymer nanoparticles encapsulating GaMP (eFGaMP). In addition, all Ga(III) porphyrins and nanoparticles showed significant intracellular and antibiofilm activity against both pathogens, with the nanoparticles exhibiting intracellular activity for 3 days. Ga nanoparticles also increased the survival rate of Caenorhabditis elegans nematodes infected by P. aeruginosa and A. baumannii Our results demonstrate that Ga nanoparticles have prolonged in vitro and in vivo activities against both P. aeruginosa and A. baumannii, including disruption of their biofilms.

In Vitro Efficacy of Free and Nanoparticle Formulations of Gallium(III) meso-Tetraphenylporphyrine against Mycobacterium avium and Mycobacterium abscessus and Gallium Biodistribution in Mice.

The nontuberculous mycobacterial (NTM) pathogens, M. avium complex (MAC) and M. abscessus, can result in severe pulmonary infections. Current antibiotics confront significant challenges for treatment of these NTM infections due to emerging multidrug-resistance. Thus, development of new antibiotics targeted against these agents is needed. We examined the inhibitory activities of Ga(NO3)3, GaCl3, gallium meso-tetraphenylporphyrine (GaTP), and gallium nanoparticles (GaNP) against intra- and extracellular M. avium and M. abscessus. GaTP, an analogue of natural heme, inhibited growth of both M. avium and M. abscessus with MICs in Fe-free 7H9 media of 0.5 and 2 µg/mL, respectively. GaTP was more active than Ga(NO3)3 and GaCl3. Ga(NO3)3 and GaCl3 were not as active in Fe-rich media compared to Fe-free media. However, GaTP was much less impacted by exogenous Fe, with MICs against M. avium and M. abscessus of 2 and 4 µg/mL, respectively, in 7H9 OADC media (Fe rich). Confocal microscopy showed that GaNP penetrates the M. avium cell wall. As assessed by determining colony forming units, GaNP inhibited the growth of NTM growing in THP-1 macrophages up to 15 days after drug-loading of the cells, confirming a prolonged growth inhibitory activity of the GaNP. Biodistribution studies of GaNP conducted in mice showed that intraperitoneal injection is more effective than intramuscular injection in delivering Ga(III) into lung tissue. GaTP exhibits potential as a lead compound for development of anti-NTM agents that target heme-bound iron uptake mechanisms by mycobacteria and inhibit growth by disrupting mycobacterial iron acquisition/utilization.

Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages.

New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

Ga(III) Nanoparticles Inhibit Growth of both Mycobacterium tuberculosis and HIV and Release of Interleukin-6 (IL-6) and IL-8 in Coinfected Macrophages.

Treatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-ß/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections.

Novel long-chain compounds with both immunomodulatory and MenA inhibitory activities against Staphylococcus aureus and its biofilm.

Menaquinone (MK) biosynthesis pathway is a potential target for evaluating antimicrobials in gram-positive bacteria. Here, 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) was targeted to reduce methicillin-resistant Staphylococcus aureus (MRSA) growth. MenA inhibiting, long chain-based compounds were designed, synthesized and evaluated against MRSA and menaquinone utilizing bacteria in aerobic conditions. The results showed that these bacteria were susceptible to most of the compounds. Menaquinone (MK-4) supplementation rescued MRSA growth, suggesting these compounds inhibit MK biosynthesis. 3a and 7c exhibited promising inhibitory activities with MICs ranging 1-8 µg/mL against MRSA strains. The compounds did not facilitate small colony variant formation. These compounds also inhibited the biofilm growth by MRSA at high concentration. Compounds 3a, 6b and 7c displayed a promising extracellular bactericidal activity against MRSA at concentrations equal to and four-fold less than their respective MICs. We also observed cytokines released from THP-1 macrophages treated with compounds 3a, 6b and 7c and found decreases in TNF-α and IL-6 release and increase in IL-1ß. These data provide evidence that MenA inhibitors act as TNF-α and IL-6 inhibitors, raising the potential for development and application of these compounds as potential immunomodulatory agents.

Prolonged-acting, multi-targeting gallium nanoparticles potently inhibit growth of both HIV and mycobacteria in co-infected human macrophages.

Human immunodeficiency virus (HIV) infection and Mycobacterium tuberculosis (TB) are responsible for two of the major global human infectious diseases that result in significant morbidity, mortality and socioeconomic impact. Furthermore, severity and disease prevention of both infections is enhanced by co-infection. Parallel limitations also exist in access to effective drug therapy and the emergence of resistance. Furthermore, drug-drug interactions have proven problematic during treatment of co-incident HIV and TB infections. Thus, improvements in drug access and simplified treatment regimens are needed immediately. One of the key host cells infected by both HIV and TB is the mononuclear phagocyte (MP; monocyte, macrophage and dendritic cell). Therefore, we hypothesized that one way this can be achieved is through drug-targeting by a nanoformulated drug that ideally would be active against both HIV and TB. Accordingly, we validated macrophage targeted long acting (sustained drug release) gallium (Ga) nanoformulation against HIV-mycobacterium co-infection. The multi-targeted Ga nanoparticle agent inhibited growth of both HIV and TB in the macrophage. The Ga nanoparticles reduced the growth of mycobacterium and HIV for up to 15 days following single drug loading. These results provide a potential new approach to treat HIV-TB co-infection that could eventually lead to improved clinical outcomes.

Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.

Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 µg/10(6) cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 µg/10(6) cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.

Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection.

Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.

Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages.

Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was "entrapped" in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4(-) cells. The data support a significant role for exosomes as facilitators of viral infection.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non-replicating persistence.

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.