Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism.

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA.

Mycobacterium tuberculosis' success as a pathogen comes from its ability to evade degradation by macrophages. Normally macrophages clear microorganisms that activate pathogen-recognition receptors (PRRs) through a lysosomal-trafficking pathway called "LC3-associated phagocytosis" (LAP). Although Mtuberculosis activates numerous PRRs, for reasons that are poorly understood LAP does not substantially contribute to Mtuberculosis control. LAP depends upon reactive oxygen species (ROS) generated by NADPH oxidase, but Mtuberculosis fails to generate a robust oxidative response. Here, we show that CpsA, a LytR-CpsA-Psr (LCP) domain-containing protein, is required for Mtuberculosis to evade killing by NADPH oxidase and LAP. Unlike phagosomes containing wild-type bacilli, phagosomes containing the ΔcpsA mutant recruited NADPH oxidase, produced ROS, associated with LC3, and matured into antibacterial lysosomes. Moreover, CpsA was sufficient to impair NADPH oxidase recruitment to fungal particles that are normally cleared by LAP. Intracellular survival of the ΔcpsA mutant was largely restored in macrophages missing LAP components (Nox2, Rubicon, Beclin, Atg5, Atg7, or Atg16L1) but not in macrophages defective in a related, canonical autophagy pathway (Atg14, Ulk1, or cGAS). The ΔcpsA mutant was highly impaired in vivo, and its growth was partially restored in mice deficient in NADPH oxidase, Atg5, or Atg7, demonstrating that CpsA makes a significant contribution to the resistance of Mtuberculosis to NADPH oxidase and LC3 trafficking in vivo. Overall, our findings reveal an essential role of CpsA in innate immune evasion and suggest that LCP proteins have functions beyond their previously known role in cell-wall metabolism.

Ubiquilin 1 Promotes IFN-γ-Induced Xenophagy of Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen rests upon its ability to grow intracellularly in macrophages. Interferon-gamma (IFN-γ) is critical in host defense against Mtb and stimulates macrophage clearance of Mtb through an autophagy pathway. Here we show that the host protein ubiquilin 1 (UBQLN1) promotes IFN-γ-mediated autophagic clearance of Mtb. Ubiquilin family members have previously been shown to recognize proteins that aggregate in neurodegenerative disorders. We find that UBQLN1 can interact with Mtb surface proteins and associates with the bacilli in vitro. In IFN-γ activated macrophages, UBQLN1 co-localizes with Mtb and promotes the anti-mycobacterial activity of IFN-γ. The association of UBQLN1 with Mtb depends upon the secreted bacterial protein, EsxA, which is involved in permeabilizing host phagosomes. In autophagy-deficient macrophages, UBQLN1 accumulates around Mtb, consistent with the idea that it marks bacilli that traffic through the autophagy pathway. Moreover, UBQLN1 promotes ubiquitin, p62, and LC3 accumulation around Mtb, acting independently of the E3 ligase parkin. In summary, we propose a model in which UBQLN1 recognizes Mtb and in turn recruits the autophagy machinery thereby promoting intracellular control of Mtb. Thus, polymorphisms in ubiquilins, which are known to influence susceptibility to neurodegenerative illnesses, might also play a role in host defense against Mtb.

Structural basis of Na(+)-independent and cooperative substrate/product antiport in CaiT.

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-A and from Escherichia coli (EcCaiT) at 3.5-A resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na(+) or H(+) dependent, whereas EcCaiT is Na(+) and H(+) independent. The three-dimensional architecture of CaiT resembles that of the Na(+)-dependent transporters LeuT and BetP, but in CaiT a methionine sulphur takes the place of the Na(+) ion to coordinate the substrate in the central transport site, accounting for Na(+)-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.

Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking.

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.

Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

The secreted pore-forming toxin listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes is a member of the family of cholesterol-dependent cytolysins (CDC) with broad properties in pathogenesis. Its role as a virulence factor is enigmatic: it disrupts membranes and acts as an inductor of both pro- and anti-inflammatory responses in infected cells. In addition, LLO is also a potent target for immunogenicity during infection. Natively secreted LLO from a recombinant L. innocua strain was crystallized in its water-soluble monomeric form. The crystals obtained belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 26.7, b = 85.1, c = 230.0 Å, and diffracted to beyond 2.2 Šresolution. The Matthews coefficient and the solvent content were estimated to be 2.4 Å(3) Da(-1) and 49.2%, respectively. The structure with one molecule in the asymmetric unit was solved using Phaser employing the structure of the previously characterized CDC toxin perfringolysin O as a search model.

Structure of human Na+/H+ exchanger NHE1 regulatory region in complex with calmodulin and Ca2+.

The ubiquitous mammalian Na(+)/H(+) exchanger NHE1 has critical functions in regulating intracellular pH, salt concentration, and cellular volume. The regulatory C-terminal domain of NHE1 is linked to the ion-translocating N-terminal membrane domain and acts as a scaffold for signaling complexes. A major interaction partner is calmodulin (CaM), which binds to two neighboring regions of NHE1 in a strongly Ca(2+)-dependent manner. Upon CaM binding, NHE1 is activated by a shift in sensitivity toward alkaline intracellular pH. Here we report the 2.23 Å crystal structure of the NHE1 CaM binding region (NHE1(CaMBR)) in complex with CaM and Ca(2+). The C- and N-lobes of CaM bind the first and second helix of NHE1(CaMBR), respectively. Both the NHE1 helices and the Ca(2+)-bound CaM are elongated, as confirmed by small angle x-ray scattering analysis. Our x-ray structure sheds new light on the molecular mechanisms of the phosphorylation-dependent regulation of NHE1 and enables us to propose a model of how Ca(2+) regulates NHE1 activity.

Dual energy landscape: the functional state of the ß-barrel outer membrane protein G molds its unfolding energy landscape.

We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy of activation and distance of the transition state from the folded state) characterizing the energy barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from Escherichia coli. The pH-dependent functional switching of OmpG directs the protein along different regions on the unfolding energy landscape. The two functional states of OmpG take the same unfolding pathway during the sequential unfolding of ß-hairpins I-IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of ß-hairpin V in one step precedes the unfolding of ß-hairpin VI. In the closed state, ß-hairpin V and ß-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently ß-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of ß-hairpin VII. Also, the conformational change from the open to the closed state witnesses a rigidified extracellular gating loop L6. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.

Inhibition of Fatty Acid Oxidation Promotes Macrophage Control of Mycobacterium tuberculosis.

Macrophage activation involves metabolic reprogramming to support antimicrobial cellular functions. How these metabolic shifts influence the outcome of infection by intracellular pathogens remains incompletely understood. Mycobacterium tuberculosis (Mtb) modulates host metabolic pathways and utilizes host nutrients, including cholesterol and fatty acids, to survive within macrophages. We found that intracellular growth of Mtb depends on host fatty acid catabolism: when host fatty acid ß-oxidation (FAO) was blocked chemically with trimetazidine, a compound in clinical use, or genetically by deletion of the mitochondrial fatty acid transporter carnitine palmitoyltransferase 2 (CPT2), Mtb failed to grow in macrophages, and its growth was attenuated in mice. Mechanistic studies support a model in which inhibition of FAO generates mitochondrial reactive oxygen species, which enhance macrophage NADPH oxidase and xenophagy activity to better control Mtb infection. Thus, FAO inhibition promotes key antimicrobial functions of macrophages and overcomes immune evasion mechanisms of Mtb.IMPORTANCEMycobacterium tuberculosis (Mtb) is the leading infectious disease killer worldwide. We discovered that intracellular Mtb fails to grow in macrophages in which fatty acid ß-oxidation (FAO) is blocked. Macrophages treated with FAO inhibitors rapidly generate a burst of mitochondria-derived reactive oxygen species, which promotes NADPH oxidase recruitment and autophagy to limit the growth of Mtb. Furthermore, we demonstrate the ability of trimetazidine to reduce pathogen burden in mice infected with Mtb. These studies will add to the knowledge of how host metabolism modulates Mtb infection outcomes.

Purification, crystallization and preliminary X-ray diffraction analysis of the FeoB G domain from Methanococcus jannaschii.

The transmembrane protein FeoB plays a key role in ferrous iron acquisition in prokaryotes. The N-terminal domain of FeoB from Methanococcus jannaschii was overproduced, purified to homogeneity and crystallized in the presence of GTP and magnesium. The native protein crystallized in a tetragonal space group and the crystals diffracted to beyond 2.2 A resolution, with unit-cell parameters a = b = 84.77, c = 137.90 A. The Matthews coefficient and the solvent content were estimated to be 2.65 A(3) Da(-1) and 53.64%, respectively, which corresponds to the presence of two molecules per asymmetric unit. To obtain initial phases, selenomethionyl-substituted protein was overproduced, purified and crystallized.

The role of lipids for the functional integrity of porin: an FTIR study using lipid and protein reporter groups.

We have investigated the temperature-dependent interaction of the porins OmpF from Paracoccus denitrificans and OmpG from Escherichia coli with lipid molecules after reconstitution in lecithin. Effects of incubation at increased temperatures on activity were tested by functional experiments for OmpG and compared with previously published results of OmpF in order to understand the activity loss of OmpF with monomerization. Protein-lipid interaction was monitored by different reporter groups both from lipid molecules and from protein. OmpF loses its activity by approximately 90% at 50 degrees C while OmpG does not show a temperature-dependent change in activity between room temperature and 50 degrees C. The interaction between OmpF and lipid molecules is severely altered in a two-step mechanism at 55 and approximately 75 degrees C for OmpF. The first step is attributed to changes in the degree of interaction between the aromatic girdle of OmpF and the interfacial region of the lipid bilayer, leading to monomerization of this trimeric porin. The second step at 75 degrees C is attributed to the changes in lipid-porin monomer interaction. Around 90 degrees C, reconstituted porin aggregates. For OmpG, changes in lipid-protein interaction were observed starting from approximately 80 degrees C because of temperature-induced breakdown of its folding. This study provides deeper understanding of porin-lipid bilayer interaction as a function of temperature and can explain the functional breakdown by monomerization while porin secondary structure is still preserved.

Why macrophages cannot LAP up TB.

M. tuberculosis causes an enormous worldwide burden of disease. Its success depends upon subverting the antimicrobial capacity of macrophages. We have known for decades that M. tuberculosis impairs phagosomal trafficking to avoid lysosomal degradation, but the mechanism is unclear. Recent work has described a phagolysosomal pathway called LC3-associated phagocytosis (LAP), in which LC3 associates with microbe-containing phagosomes. Macrophage pathogen recognition receptors (PRRs) initiate LAP, and NADPH oxidase and RUBCN/RUBICON are required for LAP. We discovered that CpsA, an exported M. tuberculosis virulence factor, blocks LAP by interfering with recruitment of CYBB/NOX2 (cytochrome b-245, beta polypeptide) to the mycobacterial phagosome. In macrophages and in mice, M. tuberculosis mutants lacking cpsA are successfully cleared by NADPH oxidase and the ensuing LC3-associated lysosomal trafficking pathway. CpsA belongs to the LytR-CpsA-Psr family, which is found widely in Gram-positive bacilli. This family is known for its enzymatic role in cell wall assembly. However, our data suggest that CpsA inhibits CYBB oxidase independently of a cell wall function. Thus, CpsA may have evolved from an enzyme involved in cell wall integrity to an indispensable virulence factor that M. tuberculosis uses to evade the innate immune response.

Mycobacterium tuberculosis Type VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response.

Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity.IMPORTANCEMycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.

Consequence of enhanced LC3-trafficking for a live, attenuated M. tuberculosis vaccine.

Development of a new vaccine against tuberculosis is urgently needed. Recent work has demonstrated that two related LC3-associated trafficking pathways, autophagy and LC3-associated phagocytosis (LAP), enhance antigen presentation and might play a role in vaccine efficacy. Mycobacterium tuberculosis inhibits both LC3-trafficking pathways. Moreover, the vaccine strain, BCG, induces even less LC3-trafficking than M. tuberculosis, which may help explain its limited efficacy. To determine whether enhanced LC3-trafficking can improve efficacy of a live, attenuated M. tuberculosis vaccine, we took advantage of our recent finding that the bacterial virulence factor CpsA inhibits LAP. When we deleted cpsA in the mc26206 vaccine strain, it dramatically increased LC3-trafficking. We compared the protective efficacy of the strain lacking cpsA to the parent strain and to BCG in mice challenged with M. tuberculosis. We found that the strain lacking cpsA generated modestly enhanced protection in the spleen, but overall did not outperform BCG.

Influence of different intravenous lipid emulsions on hepatobiliary dysfunction in a rabbit model.

OBJECTIVES: Long-term total parenteral nutrition (TPN) in children is often complicated by the development of cholestasis, liver fibrosis, and liver failure. High doses of intravenous lipids may be involved in the pathogenesis of hepatobiliary dysfunction. The purpose of this study was to determine whether the use of 2 newly developed lipid emulsions could reduce liver damage. MATERIALS AND METHODS: Three groups of prepubescent rabbits received TPN including a lipid emulsion either based on soybean oil, olive oil, or soybean oil with n-3 fatty acids added. Enterally fed animals served as controls. After 21 d the animals were killed. Serum samples were obtained at the beginning and end of the study period. Specimens were processed for histological evaluation using a specific score to assess the severity of liver damage. RESULTS: Biochemical parameters did not predict the extent of liver damage. Hydropic degeneration as an indicator of toxic liver injury was the predominant histological alteration regardless of the type of lipids infused. The extent of fibrosis did not significantly differ among treatment groups except for animals infused with n-3 fatty acids exhibiting increased fibrotic transformation as compared with controls. CONCLUSION: In our animal model, the use of a lipid emulsion with a reduced amount of polyunsaturated fatty acids was not superior to a lipid emulsion based on soybean oil. Long-term application of n-3 fatty acids was associated with more extensive fibrosis. Therefore, intravenous n-3 fatty acids containing lipid preparations (fish oil) should not be used in patients for long-term TPN.

Purification, Refolding, and Crystallization of the Outer Membrane Protein OmpG from Escherichia coli.

OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric ß-barrel made of 14 antiparallel ß-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring ß-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.

[Adolescent psychiatric aspects of visual handicap and blindness]. / Jugendpsychiatrische Aspekte von Sehbehinderung und Blindheit.

The focus of the study was a questionnaire-based overview concerning psychiatric symptoms, self image, control beliefs and some aspects of family relations of severely visually impaired and blind adolescents and young adults. The high incidence of emotional and social problems seemed to be equal to that of a clinical sample, competence and control beliefs differed between impaired und unimpaired adolescents as well as between visually impaired und blind adolescents. Self-perceptions of bonding- vs. detachment behaviour from the viewpoint of the blind adolescents appeared somewhat "surer", nearer to the ratings of the unimpaired peers. The ratings of the visually impaired peers however seem to express more "uncertainties" in both main directions, bonding as well as detachment behaviour. Concerning global self image there seemed to exist no significant difference between our whole sample and unimpaired peers.

Enzymatic properties and substrate specificity of a bacterial phosphatidylcholine synthase.

Phosphatidylcholine (PC) is a rare membrane lipid in bacteria, but is crucial for virulence of the plant pathogen Agrobacterium tumefaciens and various other pathogens. Agrobacterium tumefaciens uses two independent PC biosynthesis pathways. One is dependent on the integral membrane protein PC synthase (Pcs), which catalyzes the conversion of cytidine diphosphate-diacylglycerol (CDP-DAG) and choline to PC, thereby releasing a cytidine monophosphate (CMP). Here, we show that Pcs consists of eight transmembrane segments with its N- and C-termini located in the cytoplasm. A cytoplasmic loop between the second and third membrane helix contains the majority of the conserved amino acids of a CDP-alcohol phosphotransferase motif (DGX2 ARX12 GX3 DX3 D). Using point mutagenesis, we provide evidence for a crucial role of this motif in choline binding and enzyme activity. To study the catalytic features of the enzyme, we established a purification protocol for recombinant Pcs. The enzyme forms stable oligomers and exhibits broad substrate specificity towards choline derivatives. The presence of CDP-DAG and manganese is a prerequisite for cooperative binding of choline. PC formation by Pcs is reversible and proceeds via two successive reactions. In a first choline- and manganese-independent reaction, CDP-DAG is hydrolyzed releasing a CMP molecule. The resulting phosphatidyl intermediate reacts with choline in a second manganese-dependent step to form PC. STRUCTURED DIGITAL ABSTRACT: Pcs and Pcs bind by molecular sieving (1, 2, 3).