Mycobacteria, metals, and the macrophage

Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here, we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies.Microbiology and Molecular Genetic

Interplays between copper and Mycobacterium tuberculosis GroEL1

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target

Die physiologische Funktion kanalbildender Proteine der äußeren Membran in Mykobakterien

Mycobacterium tuberculosis is the leading cause of deaths resulting from a single infectious disease with 1.7 million victims annually. The exceptionally low permeability of the outer membrane contributes to the intrinsic resistance of mycobacteria to many antibiotics. Despite the well-documented importance of outer membrane proteins for nutrient uptake, secretion, and host-pathogen interactions in Gram-negative bacteria, only the porin MspA of M. smegmatis and the channel-forming protein OmpA of M. tuberculosis have been characterized as mycobacterial integral outer membrane proteins. By contrast, E. coli uses more than 60 proteins to functionalize its outer membrane, none of which has significant sequence similarity to any M. tuberculosis protein. Rv1698 of M. tuberculosis was discovered by us as an outer membrane channel protein with unknown function. Intracellular copper in an M. tuberculosis mutant lacking Rv1698 was 100-fold increased. An M. smegmatis mutant lacking the close homolog Msm_3747 accumulated 11-fold more copper than the wild-type, while uptake of glucose remained unchanged. These results demonstrated that Rv1698-like channel proteins are required for copper efflux across the mycobacterial outer membrane and that secretion of Cu+ is a mechanism by which M. tuberculosis maintains copper homeostasis to prevent copper toxicity. Rv1698 is the first identified mycobacterial channel protein that is involved in efflux across the outer membrane. In addition, Rv1698 lacks a predicted copper binding motive and there is no energy source in the outer membrane that would support efflux trough the Rv1698 channels against the concentration gradient. Thus, Rv1698 is likely recruited by Cu+ specific inner membrane translocases that determine substrate specificity and provide energy for the transport. These findings indicate that mycobacteria possess multicomponent efflux systems that are functionally similar to those of Gram-negative bacteria. We also found that M. tuberculosis did not grow at Cu2+ concentrations above 25 µM. The amount of copper in phagosomes of macrophages stimulated with interferon-gamma increases to similar concentrations after infection with M. tuberculosis. Thus, macrophages appear to utilize copper to control intracellular growth of M. tuberculosis. Uptake pathways for the essential micronutrient copper are unknown in mycobacteria. However, an M. smegmatis porin mutant did not grow with trace amounts of copper (<1 µM), but was more resistant than wild-type, demonstrating that channel proteins are required for copper uptake across the outer membrane. These outer membrane channels are essential components of a considerably revised model of copper homeostasis in M. tuberculosis. The implications of these findings for our understanding of transport mechanisms and, in particular efflux systems, in mycobacteria are profound.Mykobakterien sind von großer Bedeutung, da Tuberkulose die weltweit verbreitetste Infektionskrankheit mit 1.7 Millionen Todesopfern darstellt. Die außergewöhnlich niedrige Durchlässigkeit der mykobakteriellen äußeren Membran gilt als Hauptursache für deren Widerstandsfähigkeit gegenüber vielen Antibiotika. Äußere Membranproteine in Gram-negativen Bakterien sind wichtig für die Nahrungsaufnahme, Sekretion und deren Infektionsvermögen. Escherichia coli hat mehr als 60 verschiedene äußere Membranproteine. Keines hat Ähnlichkeit mit mykobakteriellen Proteinen. Bisher sind das Porin MspA von Mycobacterium smegmatis und das kanalbildende Protein OmpA von Mycobacterium tuberculosis die einzigen bekannten mykobakteriellen Membranproteine. Rv1698 von M. tuberculosis wurde von uns als Kanalprotein der äußeren Membran mit unbekannter Funktion entdeckt. Zellen der entsprechenden Mutante reicherten 100-mal mehr Kupfer an als der Wildtyp. Eine M. smegmatis Mutante der das Homolog Msm_3747 fehlt, zeigte einen 11-mal höheren Kupfergehalt. Das Aufnahmevermögen für Glukose blieb jedoch unverändert. Diese Ergebnisse zeigen, dass diese Kanalproteine eine wichtige Rolle für die Ausscheidung von Kupferionen über die mykobakterielle äußere Membran spielen. Damit wurde die Ausscheidung von Kupfer als ein wichtiger Mechanismus identifiziert, mit dem Mykobakterien der Ansammlung von giftigen Kupferionen in der Zelle entgegenwirken. Rv1698 ist somit das erste mykobakterielle äußere Membranprotein mit nachgewiesener Ausscheidungs-funktion. Rv1698 hat kein Kupferbindemotiv. Ebenso fehlen der äußeren Membran die Energiequellen für den Transport entgegen dem Konzentrationsgefälle. Darum ist es wahrscheinlich, dass Rv1698 mit Kupfer-spezifischen Transportproteinen der inneren Membran interagiert. Diese sind in der Regel substratspezifisch und stellen die Energie für den Transport bereit. Darum ist zu vermuten, dass die mykobakteriellen Transportsysteme prinzipiell denen in Gram-negativen Bakterien ähneln. Kupfer über einer Konzentration von 25 µM hemmt das Wachstum von M. tuberculosis auf künstlichem Nährmedium. Ähnliche Konzentrationen wurden in Phagosomen von Interferon-gamma stimulierten Makrophagen gemessen. Dies lässt vermuten, dass Makrophagen das Wachstum von M. tuberculosis mit Hilfe von Kupfer eindämmen. Aufnahmemechanismen für Kupferionen sind unbekannt. Wir haben gezeigt, dass Porinmutanten von M. smegmatis im Gegensatz zum Wildtyp auf nahezu kupferfreiem Medium kaum wachsen und eine Toleranz bei erhöhtem Kupfergehalt zeigen. Kupferaufnahme erfolgt somit über Porine. Kanäle in der mykobakteriellen äußeren Membran sind daher unverzichtbar für die Aufnahme und Ausscheidung überschüssiger Kupferionen. Diese Ergebnisse erweitern dramatisch unser Verständnis über mykobakterielle Transportprozesse

Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis▿

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown

Identification of Two Mycobacterium smegmatis Lipoproteins Exported by a SecA2-Dependent Pathway▿ †

The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and ΔsecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a ΔsecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis

The role of porins in copper acquisition by mycobacteria

Aims and objectives: Copper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis (Mtb), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. Methods: Mycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb, and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. Results: Deletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5 μM copper and eliminated growth at 15 μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb. Conclusions: It was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier (e.g., by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb

A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis

In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentrations, which seems to be the best predictors of in vivo efficacy. Production of these Mtb/macrophage aggregates could be easily scaled up to support throughput efforts. Overall, its simplicity and scalability should make this Mtb/macrophage aggregate model a valuable addition to the currently available Mtb drug discovery tools

Protein phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection

Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients