Interaction between Polyketide Synthase and Transporter Suggests Coupled Synthesis and Export of Virulence Lipid in M. tuberculosis

Virulent mycobacteria utilize surface-exposed polyketides to interact with host cells, but the mechanism by which these hydrophobic molecules are transported across the cell envelope to the surface of the bacteria is poorly understood. Phthiocerol dimycocerosate (PDIM), a surface-exposed polyketide lipid necessary for Mycobacterium tuberculosis virulence, is the product of several polyketide synthases including PpsE. Transport of PDIM requires MmpL7, a member of the MmpL family of RND permeases. Here we show that a domain of MmpL7 biochemically interacts with PpsE, the first report of an interaction between a biosynthetic enzyme and its cognate transporter. Overexpression of the interaction domain of MmpL7 acts as a dominant negative to PDIM synthesis by poisoning the interaction between synthase and transporter. This suggests that MmpL7 acts in complex with the synthesis machinery to efficiently transport PDIM across the cell membrane. Coordination of synthesis and transport may not only be a feature of MmpL-mediated transport in M. tuberculosis, but may also represent a general mechanism of polyketide export in many different microorganisms

Sulfolipid-1 Biosynthesis Restricts Mycobacterium tuberculosis Growth in Human Macrophages

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages

Prevalence of 22q11.2 microdeletion in 146 patients with cardiac malformation in a referral hospital of North India

<p>Abstract</p> <p>Background</p> <p>The 22q11.2 microdeletion syndrome is a common condition that is associated with cardiac as well as extra-cardiac manifestations. Its prevalence and manifestations from north India has not been reported. This study was designed to determine the prevalence and ability of clinical criteria to predict 22q11.2 microdeletion.</p> <p>Methods</p> <p>A total of 146 cases of cardiac malformation requiring tertiary care at a teaching hospital were prospectively screened for 22q11.2 microdeletion using fluorescence in situ hybridization test. Detailed clinical information was obtained as per guidelines of Tobias, <it>et al </it>(1999).</p> <p>Results</p> <p>Nine out of 146 patients (6.16%) was found to have 22q11.2 microdeletion. All the positive patients showed the presence of extra-cardiac features of 22q11.2 microdeletion syndrome. None of the cases with isolated cardiac defect were positive for microdeletion.</p> <p>Conclusions</p> <p>It seems that 22q11.2 microdeletion syndrome is over-suspected in children with isolated congenital heart defects. Screening for 22q11.2 microdeletion should be considered in those cardiac malformation cases which have extra-cardiac manifestations in the form of facial dysmorphism and hypocalcaemia.</p

Combining Cellulose and Cyclodextrins: Fascinating Designs for Materials and Pharmaceutics

Cellulose and cyclodextrins possess unique properties that can be tailored, combined, and used in a considerable number of applications, including textiles, coatings, sensors, and drug delivery systems. Successfully structuring and applying cellulose and cyclodextrins conjugates requires a deep understanding of the relation between structural, and soft matter behavior, materials, energy, and function. This review focuses on the key advances in developing materials based on these conjugates. Relevant aspects regarding structural variations, methods of synthesis, processing and functionalization, and corresponding supramolecular properties are presented. The use of cellulose/cyclodextrin conjugates as intelligent platforms for applications in materials science and pharmaceutical technology is also outlined, focusing on drug delivery, textiles, and sensors

Interaction between polyketide synthase and transporter suggests coupled synthesis and export of virulence lipid in M. tuberculosis.

Virulent mycobacteria utilize surface-exposed polyketides to interact with host cells, but the mechanism by which these hydrophobic molecules are transported across the cell envelope to the surface of the bacteria is poorly understood. Phthiocerol dimycocerosate (PDIM), a surface-exposed polyketide lipid necessary for Mycobacterium tuberculosis virulence, is the product of several polyketide synthases including PpsE. Transport of PDIM requires MmpL7, a member of the MmpL family of RND permeases. Here we show that a domain of MmpL7 biochemically interacts with PpsE, the first report of an interaction between a biosynthetic enzyme and its cognate transporter. Overexpression of the interaction domain of MmpL7 acts as a dominant negative to PDIM synthesis by poisoning the interaction between synthase and transporter. This suggests that MmpL7 acts in complex with the synthesis machinery to efficiently transport PDIM across the cell membrane. Coordination of synthesis and transport may not only be a feature of MmpL-mediated transport in M. tuberculosis, but may also represent a general mechanism of polyketide export in many different microorganisms

Mosaic 22q11.2 microdeletion syndrome: diagnosis and clinical manifestations of two cases

Abstract Chromosome 22q11.2 microdeletion syndrome is due to microdeletion of 22q11.2 region of chromosome 22. It is a common microdeletion syndrome however mosaic cases are very rare and reported only few previous occasions. In this report we describe two unrelated male children with clinical features consistent with 22q11.2 microdeletion syndrome characterized by cardiac defect, facial dysmorphism and developmental deficiency. One of the cases also had trigonocephaly. Interphase & metaphase FISH with 22q11.2 probe demonstrated mosaicism for hemizygous deletion of 22q11.2 region. Mosaicism is also observed in buccal cells as well as urine cells. Parents were without any deletion. These two cases represent rare cases of mosaic 22q11.2 microdeletion syndrome.</p

Identification of Residues in MmpL7 Domain 2 Required for PpsE Interaction

<div><p>(A) Twelve MmpL7 domain 2 mutants defective for PpsE binding were isolated in a reverse two-hybrid screen. These amino acid substitutions are displayed on a linear map of MmpL7 domain 2, with changes to amino acids other than proline or glycine in bold. Amino acid numbers correspond to positions in full-length MmpL7. TM domains 7 and 8 are denoted by hatched bars.</p><p>(B) Yeast strains expressing the PpsE prey construct and various MmpL7 domain 2 bait plasmids were transferred onto X-gal indicator plates (inset), and reporter activity was quantified from liquid cultures by monitoring β-galactosidase activity.</p><p>(C) Beads containing equal amounts of MmpL7 domain 2 and the I611S mutant were incubated with protein extracts containing myc-tagged PpsE and washed. Bound proteins were eluted and separated by SDS-PAGE, and PpsE was visualized by Western blot using anti-myc antibodies. GST-coated beads served as a negative control, and 1% of the protein extract added to the pulldown was loaded as a positive control (“input”).</p></div

MmpL7 Domain 2 Acts as a Dominant Negative Inhibitor of PDIM Synthesis In Vivo

<div><p>(A) The indicated strains carrying either no plasmid (−), control vector (vector), or a plasmid with MmpL7 domain 2 under the control of the constitutive <i>groEL2</i> promoter (MmpL7^d2) were labeled with ¹⁴C-propionate. Surface-exposed lipids (S) were extracted by resuspension in hexanes, and cell pellets (P) were harvested by centrifugation. Lipids from both fractions were extracted and separated by TLC under solvent conditions to separate PDIM (upper panel, keto and methoxy forms) and SL-1 (lower panel).</p><p>(B) Top: lipids were extracted as in (A) from pellets of wild-type cells carrying either no plasmid (−), the MmpL7 domain 2 expression plasmid (d2), or the MmpL7 domain 2 expression plasmid with the I611S mutation (d2-I611S). Bottom: Western blot analysis was performed to confirm equivalent expression of wild-type MmpL7 domain 2 and the I611S mutant by using antibodies against the hemagglutinin epitope tag.</p><p>(C) Complementation of an <i>mmpL7⁻ M. tuberculosis</i> strain with the wild-type (<i>mmpL7^wt</i>) or the I611S mutant <i>mmpL7</i> (<i>mmpL7^I611S</i>). Surface-exposed lipids (S) and lipids associated with the remaining cell pellet (P) were extracted and separated by TLC to resolve PDIM as in (A).</p></div