Azido Inositol Probes Enable Metabolic Labeling of Inositol-Containing Glycans and Reveal an Inositol Importer in Mycobacteria.

Bacteria from the genus Mycobacterium include pathogens that cause serious diseases in humans and remain as difficult infectious agents to treat. Central to these challenges are the composition and organization of the mycobacterial cell envelope, which includes unique and complex glycans. Inositol is an essential metabolite for mycobacteria due to its presence in the structural core of the immunomodulatory cell envelope glycolipids phosphatidylinositol mannoside (PIM) and PIM-anchored lipomannan (LM) and lipoarabinomannan (LAM). Despite their importance to mycobacterial physiology and pathogenesis, many aspects of PIM, LM, and LAM construction and dynamics remain poorly understood. Recently, probes that allow metabolic labeling and detection of specific mycobacterial glycans have been developed to investigate cell envelope assembly and dynamics. However, these tools have been limited to peptidoglycan, arabinogalactan, and mycolic acid-containing glycolipids. Herein, we report the development of synthetic azido inositol (InoAz) analogues as probes that can metabolically label PIMs, LM, and LAM in intact mycobacteria. Additionally, we leverage an InoAz probe to discover an inositol importer and catabolic pathway in Mycobacterium smegmatis. We anticipate that in the future, InoAz probes, in combination with bioorthogonal chemistry, will provide a valuable tool for investigating PIM, LM, and LAM biosynthesis, transport, and dynamics in diverse mycobacterial organisms.

Structural and Functional Characterization of Phosphatidylinositol-Phosphate Biosynthesis in Mycobacteria.

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from naïve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

Purification and Analysis of Mycobacterial Phosphatidylinositol Mannosides, Lipomannan, and Lipoarabinomannan.

Mycobacteria and related bacteria in the Actinobacteria phylum are unusual in that they produce phosphatidylinositol (PI) as a major phospholipid species. PI can be further modified by glycan polymers, leading to the synthesis of PI mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). Small lipids such as PI and PIMs are extracted with a mixture of chloroform, methanol, and water and analyzed by thin layer chromatography. For larger glycolipids, such as LM and LAM, more hydrophilic solvent is needed for the extraction, and SDS-PAGE is better suited for the analysis. For LM, further structural characterization can be performed by MALDI-TOF mass spectrometry. Precise quantification of PIMs, LM, and LAM can be performed by quantification of glycan staining using analytical software. The metabolic radiolabeling protocol is also described.

Mycobacterium indicus pranii (Mw) inhibits invasion by reducing matrix metalloproteinase (MMP-9) via AKT/ERK-1/2 and PKCα signaling: A potential candidate in melanoma cancer therapy.

Invasion and metastasis via induction of matrix metalloproteinases are the main causes of death in melanoma cancer. In this study, we investigated the inhibitory effects of heat killed saprophytic bacterium Mycobacterium indicus pranii (Mw) on B16F10 melanoma cell invasion. Mw reported to be an immunomodulator has antitumor activity however, its effect on cancer cell invasion has not been studied. Highly invasive B16F10 melanoma was found sensitive to Mw which downregulated MMP-9 expression. Mw treatment inhibited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) transcriptional activity and respective DNA binding to MMP-9 promoter. Moreover, Mw also overcame the promoting effects of PMA on B16F10 cell invasion. Mw decreased PMA-induced transcriptional activation of NF-κB and AP-1 by inhibiting phosphorylation of AKT and ERK-1/2. Furthermore, Mw strongly suppressed PMA-induced membrane localization of protein kinase C α (PKCα) since PKCα inhibition caused a marked decrease in PMA-induced MMP-9 secretion as well as AKT/ERK-1/2 activation. These results suggest that Mw may be a promising anti-invasive agent as it blocks tumor growth and inhibits B16F10 cell invasion by reducing MMP-9 activation through inhibition of PKCα/ AKT/ ERK-1/2 phosphorylation and NF-κB/AP-1 activation.

Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria.

Mycobacteria synthesize a variety of structurally related glycolipids with major biological functions. Common themes have emerged for the biosynthesis of these glycolipids, including several families of proteins. Genes encoding these proteins are usually clustered on bacterial chromosomal islets dedicated to the synthesis of one glycolipid family. Here, we investigated the function of a cluster of five genes widely distributed across non-tuberculous mycobacteria. Using defined mutant analysis and in-depth structural characterization of glycolipids from wild-type or mutant strains of Mycobacterium smegmatis and Mycobacterium abscessus, we established that they are involved in the formation of trehalose polyphleates (TPP), a family of compounds originally described in Mycobacterium phlei. Comparative genomics and lipid analysis of strains distributed along the mycobacterial phylogenetic tree revealed that TPP is synthesized by a large number of non-tuberculous mycobacteria. This work unravels a novel glycolipid biosynthetic pathway in mycobacteria and extends the spectrum of bacteria that produce TPP.

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD.

The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin- and flavodoxin reductase-based reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.

Cytotoxic and apoptotic effects of heat killed Mycobacterium indicus pranii (MIP) on various human cancer cell lines.

Mycobacterium indicus pranii (MIP) is a non-pathogenic mycobacterium, which has been tested on several cancer types like lung and bladder where tumour regression and complete recovery was observed. In discovering the potential cytotoxic elements, a preliminary test was carried out using four different fractions consisting of live bacteria, culture supernatant, heat killed bacteria and heat killed culture supernatant of MIP against two human cancer cells A549 and CaSki by 3-(4,5-dimethyl thiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was investigated in MCF-7 and ORL-115 cancer cells by poly-(ADP-ribose) polymerase (PARP) and DNA fragmentation assays. Among four MIP fractions, only heat killed MIP fraction (HKB) showed significant cytotoxicity in various cancer cells with inhibitory concentration, IC50 in the range 5.6-35.0 µl/(1.0 × 10(6) MIP cells/ml), while cytotoxicity effects were not observed in the remaining fractions. HKB did not show cytotoxic effects on non-cancerous cells contrary to cancerous cells, suggesting its safe usage and ability to differentially recognize between these cells. Evaluation on PARP assay further suggested that cytotoxicity in cancer cells were potentially induced via caspase-mediated apoptosis. The cytotoxic and apoptotic effects of MIP HKB have indicated that this fraction can be a good candidate to further identify effective anti-cancer agents.

Mycobacterial glycolipids di-O-acylated trehalose and tri-O-acylated trehalose downregulate inducible nitric oxide synthase and nitric oxide production in macrophages.

BACKGROUND: Tuberculosis (TB) remains a serious human health problem that affects millions of people in the world. Understanding the biology of Mycobacterium tuberculosis (Mtb) is essential for tackling this devastating disease. Mtb possesses a very complex cell envelope containing a variety of lipid components that participate in the establishment of the infection. We have previously demonstrated that di-O-acylated trehalose (DAT), a non-covalently linked cell wall glycolipid, inhibits the proliferation of T lymphocytes and the production of cytokines. RESULTS: In this work we show that DAT and the closely related tri-O-acylated trehalose (TAT) inhibits nitric oxide (NO) production and the inducible nitric oxide synthase (iNOS) expression in macrophages (MØ). CONCLUSIONS: These findings show that DAT and TAT are cell-wall located virulence factors that downregulate an important effector of the immune response against mycobacteria.

A universal stress protein (USP) in mycobacteria binds cAMP.

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 µg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

Classification algorithm for subspecies identification within the Mycobacterium abscessus species, based on matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.

First report of Mycobacterium canariasense catheter-related bacteremia in the Americas.

Mycobacterium canariasense is a recently described late-pigmenting, rapidly growing mycobacterium linked to bacteremia in patients with underlying malignant diseases. We report a case of M. canariasense infection in a patient from Massachusetts with underlying diffuse B cell lymphoma, which was identified both by multilocus sequence typing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To our knowledge, this is the first description after its original identification in Spain and the first report of this opportunistic pathogen in the Americas.

Synthesis of lipid-linked arabinofuranose donors for glycosyltransferases.

Mycobacteria and corynebacteria use decaprenylphosphoryl-ß-D-arabinofuranose (DPA) as a critical cell wall building block. Arabinofuranosyltransferases that process this substrate to mediate cell wall assembly have served as drug targets, but little is known about the substrate specificity of any of these enzymes. To probe substrate recognition of DPA, we developed a general and efficient synthetic route to ß-D-arabinofuranosyl phosphodiesters. In this approach, the key glycosyl phosphodiester bond-forming reaction proceeds with high ß-selectivity. In addition to its stereoselectivity, our route provides the means to readily access a variety of different lipid analogues, including aliphatic and polyprenyl substrates.

Effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares.

The objective of the present study was to evaluate the effect of immunomodulatory therapy (glucocorticoids (GC) and mycobacterium cell wall extract (MCWE)) on the endometrial gene expression of inflammatory cytokines in susceptible mares with induced infectious endometritis. Endometrial gene expression of pro- and anti-inflammatory cytokines; interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-1 receptor antagonist (ra), acute phase protein (APP) serum amyloid A (SAA) and clinical parameters were evaluated. Five mares were classified as susceptible to persistent endometritis based on their endometrial histopathology and ability to clear an induced uterine inflammation. To investigate the effect of immunomodulatory therapy, the mares were inoculated with 10(5) colony forming units (CFU) Escherichia coli in three consecutive estrus cycles in a modified cross-over study design. Thus, each mare served as its own control and the treatment type was performed in randomized order. The effect of treatment with MCWE (1.5 mg Settle IV), dexamethasone (0.1 mg per kg IV) or no treatment was investigated. All mares were free from uterine inflammation before each E. coli inoculation. Endometrial biopsies were recovered 3, 24 and 72 h post inoculation. Relative gene-expression analyses were performed by quantitative reverse transcriptase PCR (qRT-PCR). Endometrial gene expression of inflammatory cytokines was modulated by administration of GC. Expression of proinflammatory cytokines (IL-1ß, IL-6, IL-8) and SAA was significantly lower in the GC treated group late in the study period (72 h) compared to "no treatment" and MCWE treatment. Increased expression of the anti-inflammatory cytokine IL-10 was observed 3 and 24 h after E. coli infusion and GC treatment. A significant decrease of SAA expression was observed after MCWE treatment compared to "no treatment". MCWE and GC treatment had a significant effect on the clearance of uterine pathogens and number of mares retaining fluid after E. coli infusion. The results of the current investigation suggest that GC is capable of effectively modulating the innate immune response to induced infectious endometritis in susceptible mares.

Staining for acid-fast bacilli in surgical pathology: practice patterns and variations.

Analysis of acid-fast bacilli stains on sputum smears for the diagnosis of tuberculosis has a long history, but quality control for acid-fast bacilli in histologic sections is not as well established. In tissues, necrotizing granulomas are closely linked to positive cultures for mycobacteria. However, the practices of pathologists examining acid-fast bacilli in surgical specimens are not well described in the literature. This study characterizes practice patterns related to the histologic interpretation of acid-fast bacilli stains. A survey invitation was sent to 1299 pathologists including members of the Pulmonary Pathology Society and randomly selected fellows of the College of American Pathologists. Twenty-one questions inquired about demographics, ordering and interpreting acid-fast bacilli stains, reporting, and correlation. Of the 392 responses (30.2% response rate), 363 respondents review acid-fast bacilli stains on histologic sections. Approximately half of respondents practice in an academic setting, with the other half in community practice. Most respondents examine the entire acid-fast bacilli slide with the ×40 objective; approximately half confirm the organisms under oil immersion at ×100. There was considerable variation in when an acid-fast bacilli stain is ordered, as well as possible additional workup for negative cases, reporting of results, correlation with clinical and culture findings, and training. Many respondents reported never having been taught a general approach to acid-fast bacilli interpretation. There is substantial variation in practice patterns involving all aspects of ordering, histologic examination, and reporting of acid-fast bacilli stains. Future efforts to standardize the interpretation of acid-fast bacilli stains can potentially improve the diagnosis of mycobacterial disease.

Polyclonal antibody against conserved sequences of mce1A protein blocks MTB infection in macrophages.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. It is suggested that a specific protein namely mammalian cell entry protein is involved in the pathogenesis and the specific gene for this protein mce1A has been identified in several pathogenic organisms such as Rickettsia, Shigella, Escherichia coli, Helicobacter, Streptomyces, Klebsiella, Vibrio, Neisseria, Rhodococcus, Nocardioides, Saccharopolyspora erthyrae, and Pseudomonas. Analysis of mce1 operons in the above mentioned organisms through bioinformatics tools has revealed the presence of unique sequences (conserved regions) suggesting that these sequences may be involved in the process of infection. Presently, the mce1A full-length (1,365 bp) region from Mycobacterium bovis and its conserved regions (303 bp) were cloned in to an expression vector and the purified expressed proteins of molecular weight ~47 and ~11 kDa, respectively, were injected to rabbits to raise the polyclonal antibodies. The purified polyclonal antibodies were checked for their ability to inhibit the Mycobacterium infection in cultured human macrophages. In macrophage invasion assay, when antibody added at high concentration, decrease in viable counts was observed in all cell cultures within the first 5 days after infection, where the intracellular bacterial CFU obtained from the infected MTB increased by the 3rd day at low concentration of antibody. The macrophage invasion assay has indicated that the purified antibodies of mce1A conserved region can inhibit the infection of Mycobacterium.

Prediction of residues involved in inhibitor specificity in the dihydrofolate reductase family.

Dihydrofolate reductase (DHFR) is of significant recent interest as a target for drugs against parasitic and opportunistic infections. Understanding factors which influence DHFR homolog inhibitor specificity is critical for the design of compounds that selectively target DHFRs from pathogenic organisms over the human homolog. This paper presents a novel approach for predicting residues involved in ligand discrimination in a protein family using DHFR as a model system. In this approach, the relationship between inhibitor specificity and amino acid composition for sets of protein homolog pairs is examined. Similar inhibitor specificity profiles correlate with increased sequence homology at specific alignment positions. Residue positions that exhibit the strongest correlations are predicted as specificity determinants. Correlation analysis requires a quantitative measure of similarity in inhibitor specificity (S(lig)) for a pair of homologs. To this end, a method of calculating S(lig) values using K(I) values for the two homologs against a set of inhibitors as input was developed. Correlation analysis of S(lig) values to amino acid sequence similarity scores - obtained via multiple sequence alignments - was performed for individual residue alignment positions and sets of residues on 13 DHFRs. Eighteen alignment positions were identified with a strong correlation of S(lig) to sequence similarity. Of these, three lie in the active site; four are located proximal to the active site, four are clustered together in the adenosine binding domain and five on the ßFßG loop. The validity of the method is supported by agreement between experimental findings and current predictions involving active site residues.

Identification of the regulator gene responsible for the acetone-responsive expression of the binuclear iron monooxygenase gene cluster in mycobacteria.

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ(54) factor, a gene encoding the σ(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.