Extraction and Separation of Mycobacterial Proteins.

The extraction and separation of native mycobacterial proteins remain necessary for antigen discovery, elucidation of enzymes to improve rational drug design, identification of physiologic mechanisms, use as reagents for diagnostics, and defining host immune responses. In this chapter, methods for the manipulation of whole mycobacterial cells and culture exudates are described in detail as these methods are the requisite first steps towards native protein isolation. Specifically, several methods for the inactivation of viable Mycobacterium tuberculosis along with qualification assays are provided, as this is key to safe manipulation of cell pastes for downstream processes. Next, the concentration of spent culture filtrate media in order to permit separation of soluble, secreted proteins is described followed by the separation of mycobacteria extracellular vesicles (MEV) from the remaining soluble proteins in spent media. We then describe the generation of whole-cell lysate and facile separation of lysate into subcellular fractions to afford cell wall, cell membrane, and cytosol-enriched proteins. Due to the hydrophobic nature of cell wall and cell membrane proteins, several extraction protocols to resolve protein subsets (such as extraction with urea and SDS) are also provided. Finally, methods for separation of hydrophobic and hydrophilic proteins from both whole-cell lysate and spent culture media are included. While these methods were optimized for the manipulation of Mycobacterium tuberculosis cells, they have been successfully applied to extract and isolate Mycobacterium leprae, Mycobacterium ulcerans, and Mycobacterium avium proteins.

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Metabolic adaptability shifts of cell membrane fatty acids of Komagataeibacter hansenii HDM1-3 improve acid stress resistance and survival in acidic environments.

Komagataeibacter hansenii HDM1-3 (K. hansenii HDM1-3) has been widely applied for producing bacterial cellulose (BC). The yield of BC has been frequently limited by the acidification during sugar metabolism, due to the generation of organic acids such as acetic acid. In this study, the acid resistance mechanism of K. hansenii HDM1-3 has been investigated from the aspect of metabolic adaptability of cell membrane fatty acids. Firstly, we observed that the survival rate of K. hansenii HDM1-3 was decreased with lowered pH values (adjusted with acetic acids), accompanied by increased leakage rate. Secondly, the cell membrane adaptability in response to acid stress was evaluated, including the variations of cell membrane fluidity and fatty acid composition. The proportion of unsaturated fatty acids was increased (especially, C18-1w9c and C19-Cyc), unsaturation degree and chain length of fatty acids were also increased. Thirdly, the potential molecular regulation mechanism was further elucidated. Under acid stress, the fatty acid synthesis pathway was involved in the structure and composition variations of fatty acids, which was proved by the activation of both fatty acid dehydrogenase (des) and cyclopropane fatty acid synthase (cfa) genes, as well as the addition of exogenous fatty acids. The fatty acid synthesis of K. hansenii HDM1-3 may be mediated by the activation of two-component sensor signaling pathways in response to the acid stress. The acid resistance mechanism of K. hansenii HDM1-3 adds to our knowledge of the acid stress adaptation, which may facilitate the development of new strategies for improving the industrial performance of this species under acid stress.

Saccharomyces cerevisiae accumulates GAPDH-derived peptides on its cell surface that induce death of non-Saccharomyces yeasts by cell-to-cell contact.

During wine fermentations, Saccharomyces cerevisiae starts to excrete antimicrobial peptides (AMPs) into the growth medium that induce death of non-Saccharomyces yeasts at the end of exponential growth phase (24-48 h). Those AMPs were found to derive from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). On the other hand, the early death of non-Saccharomyces yeasts during wine fermentations was also found to be mediated by a cell-to-cell contact mechanism. Since GAPDH is a cell-wall-associated protein in S. cerevisiae, we put forward the hypothesis that the GAPDH-derived AMPs could accumulate on the cell surface of S. cerevisiae, thus inducing death of non-Saccharomyces yeasts by cell-to-cell contact. Here we show that 48-h grown (stationary phase) cells of S. cerevisiae induce death of Hanseniaspora guilliermondii and Lachancea thermotolerans by direct cell-to-cell contact, while 12-h grown cells (mid-exponential phase) do not. Immunological tests performed with a specific polyclonal antibody against the GAPDH-derived AMPs revealed their presence in the cell wall of S. cerevisiae cells grown for 48 h, but not for 12 h. Taken together, our data show that accumulation of GAPDH-derived AMPs on the cell surface of S. cerevisiae is one of the factors underlying death of non-Saccharomyces yeasts by cell-to-cell contact.

Structure of the Cellulose Synthase Complex of Gluconacetobacter hansenii at 23.4 Å Resolution.

Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into ß-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.

Construction and in vitro testing of a cellulose dura mater graft.

INTRODUCTION: Cerebrospinal fluid (CSF) leaks are a common complication after cranial and spinal surgery and are associated with increased morbidity. Despite continuous research in this field, this problem is far from solved. In this paper, we describe the construction and testing of a bacterial cellulose (BC) membrane as a new dural patch. MATERIALS AND METHODS: The synthesis of BC was performed using Gluconacetobacter hansenii (ATCC 23769) and films were sterilized by autoclaving. The membranes were seeded with human dural fibroblasts. Growth, shape, and cell viability were assessed after 4 weeks. RESULTS: Normally shaped fibroblasts were seen on the BC grafts; confocal microscopy showed cells inside the structure of the mesh. Both viable and nonviable cells were present. Cellular attachment and viability were confirmed by replating of the membranes. DISCUSSION: BC membranes are used in clinical practice to improve skin healing. In the presence of water, they form an elastic, nontoxic, and resistant biogel that can accommodate collagen and growth factors within their structure, thus BC is a good candidate for dural graft construction.

Biosynthesis of cell envelope-associated phenolic glycolipids in Mycobacterium marinum.

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.

Differential trafficking of TLR1 I602S underlies host protection against pathogenic mycobacteria.

We recently identified I602S as a frequent single-nucleotide polymorphism of human TLR1 that greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Because mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHC class II, CD64, and IFN-γ responses compared with individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1, an enzyme that depletes cellular arginine stores required for the production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria.

Molecular basis of phosphatidyl-myo-inositol mannoside biosynthesis and regulation in mycobacteria.

Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway.

Evidence for leptin receptor isoforms heteromerization at the cell surface.

Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

The crystal structures of yeast Get3 suggest a mechanism for tail-anchored protein membrane insertion.

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a "finger" domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an "open" conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a "closed" conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Cutting edge: A common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy.

TLRs constitute an essential family of pattern recognition molecules that, through direct recognition of conserved microbial components, initiate inflammatory responses following infection. In this role, TLR1 enables host responses to a variety of bacteria, including pathogenic species of mycobacteria. In this study, we report that I602S, a common single nucleotide polymorphism within TLR1, is associated with aberrant trafficking of the receptor to the cell surface and diminished responses of blood monocytes to bacterial agonists. When expressed in heterologous systems, the TLR1 602S variant, but not the TLR1 602I variant, exhibits the expected deficiencies in trafficking and responsiveness. Among white Europeans, the 602S allele represents the most common single nucleotide polymorphism affecting TLR function identified to date. Surprisingly, the 602S allele is associated with a decreased incidence of leprosy, suggesting that Mycobacterium leprae subverts the TLR system as a mechanism of immune evasion.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

BACKGROUND INFORMATION: Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. RESULTS: The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. CONCLUSIONS: Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms.

Mycolic acids are major and specific constituents of the cell envelope of Corynebacterineae, a suborder of bacterial species including several important human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, or Corynebacterium diphtheriae. These long-chain fatty acids are involved in the unusual architecture and impermeability of the cell envelope of these bacteria. The condensase, the enzyme responsible for the final condensation step in mycolic acid biosynthesis, has remained an enigma for decades. By in silico analysis of various mycobacterial genomes, we identified a candidate enzyme, Pks13, that contains the four catalytic domains required for the condensation reaction. Orthologs of this enzyme were found in other Corynebacterineae species. A Corynebacterium glutamicum strain with a deletion in the pks13 gene was shown to be deficient in mycolic acid production whereas it was able to produce the fatty acids precursors. This mutant strain displayed an altered cell envelope structure. We showed that the pks13 gene was essential for the survival of Mycobacterium smegmatis. A conditional M. smegmatis mutant carrying its only copy of pks13 on a thermosensitive plasmid exhibited mycolic acid biosynthesis defect if grown at nonpermissive temperature. These results indicate that Pks13 is the condensase, a promising target for the development of new antimicrobial drugs against Corynebacterineae.

Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis.

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.

The genome of Mycobacterium leprae: a minimal mycobacterial gene set.

Comparison of the recently sequenced genome of the leprosy-causing pathogen Mycobacterium leprae with other mycobacterial genomes reveals a drastic gene reduction and decay in M. leprae affecting many metabolic areas, exemplified by the retention of a minimal set of genes required for cell-wall biosynthesis.

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy.

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.