Alexander Speer

Discovery of a siderophore export system essential for virulence of Mycobacterium tuberculosis.

Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ΔmmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52–140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Significance The mechanisms that enable Mycobacterium tuberculosis, the causative agent of tuberculosis, to resist drug treatment and survive the immune response are poorly understood. In this study we discovered that M. tuberculosis produces the protein channel protein with necrosis-inducing toxin (CpnT), which forms a channel in the outer membrane and releases a toxic domain into the extracellular milieu. This toxin has no similarity to known bacterial toxins and kills eukaryotic cells by necrosis, suggesting that it is required for escape of M. tuberculosis from macrophages and for dissemination. The channel domain of CpnT is used for uptake of nutrients across the outer membrane. Taken together, CpnT is a protein with functions in two fundamental processes in M. tuberculosis physiology: nutrient acquisition and control of host cell death. The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.

Copper-Boosting Compounds: a Novel Concept for Antimycobacterial Drug Discovery

ABSTRACT We and others recently identified copper resistance as important for virulence of Mycobacterium tuberculosis. Here, we introduce a high-throughput screening assay for agents that induce a copper hypersensitivity phenotype in M. tuberculosis and demonstrate that such copper-boosting compounds are effective against replicating and nonreplicating M. tuberculosis strains.

The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD+. Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD+ glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT–IFT complex revealed a new NAD+ glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms.

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages

Sphingomyelinases secreted by pathogenic bacteria play important roles in host–pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface‐exposed C‐terminal sphingomyelinase domain and a putative N‐terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sphingomyelinase of Mycobacterium tuberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtbs hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5‐ and 100‐fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.

Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner

ABSTRACT Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategies are hampered both by the prohibitively long treatment regimen and the rise of drug-resistant strains. Significant effort has been expended in the search for new treatments, but few options have successfully emerged, and new treatment modalities are desperately needed. Recently, there has been growing interest in the synergistic antibacterial effects of copper ions (CuII/I) in combination with certain small molecular compounds, and we have previously reported development of a drug screening strategy to harness the intrinsic bactericidal properties of CuII/I. Here, we describe the copper-dependent antimycobacterial properties of disulfiram, an FDA-approved and well-tolerated sobriety aid. Disulfiram was inhibitory to mycobacteria only in the presence of CuII/I and exerted its bactericidal activity well below the active concentration of CuII/I or disulfiram alone. No other physiologically relevant bivalent transition metals (e.g., FeII, NiII, MnII, and CoII) exhibited this effect. We demonstrate that the movement of the disulfiram-copper complex across the cell envelope is porin independent and can inhibit intracellular protein functions. Additionally, the complex is able to synergistically induce intracellular copper stress responses significantly more than CuII/I alone. Our data suggest that by complexing with disulfiram, CuII/I is likely allowed unfettered access to vulnerable intracellular components, bypassing the normally sufficient copper homeostatic machinery. Overall, the synergistic antibacterial activity of CuII/I and disulfiram reveals the susceptibility of the copper homeostasis system of Mycobacterium tuberculosis to chemical attacks and establishes compounds that act in concert with copper as a new class of bacterial inhibitors.

Ectoine Biosynthesis in Mycobacterium smegmatis

ABSTRACT Mycobacterium smegmatis is a commonly used mycobacterial model system. Here, we show that M. smegmatis protects itself against elevated salinity by synthesizing ectoine and hydroxyectoine and characterize the phenotype of a nonproducing mutant. This is the first analysis of M. smegmatis halotolerance and of the molecular mechanism that supports it.

Protein phosphatase, Mg 2+ /Mn 2+ -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.

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis and is a devastating disease affecting eight million people each year. Iron is an essential nutrient for replication of M. tuberculosis in the human host. More than 70% of iron in the human body is bound in heme. Not surprisingly, many bacterial pathogens, including M. tuberculosis, are able to acquire iron from heme. However, the mechanism of heme uptake by M. tuberculosis is poorly understood. We have identified two novel surface proteins that bind heme and are required for heme utilization by M. tuberculosis. These findings constitute a major advancement of our understanding of iron acquisition by M. tuberculosis and show that M. tuberculosis has evolved heme uptake systems different from the paradigms established by other bacteria. IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis and is a devastating disease affecting eight million people each year. Iron is an essential nutrient for replication of M. tuberculosis in the human host. More than 70% of iron in the human body is bound in heme. Not surprisingly, many bacterial pathogens, including M. tuberculosis, are able to acquire iron from heme. However, the mechanism of heme uptake by M. tuberculosis is poorly understood. We have identified two novel surface proteins that bind heme and are required for heme utilization by M. tuberculosis. These findings constitute a major advancement of our understanding of iron acquisition by M. tuberculosis and show that M. tuberculosis has evolved heme uptake systems different from the paradigms established by other bacteria.