Metabolic tagging reveals surface-associated lipoproteins in mycobacteria.

Mycobacteria such as the causative agent of tuberculosis, , encode over 100 bioinformatically predicted lipoproteins. Despite the importance of these post-translationally modified proteins for mycobacterial survival, many remain experimentally unconfirmed. Here we characterized metabolic incorporation of diverse fatty acid analogues as a facile method of adding chemical groups that enable downstream applications such as detection, crosslinking and enrichment, of not only lipid-modified proteins, but also their protein interactors.

A Loss of Function in LprG-Rv1410c Homologues Attenuates Growth during Biofilm Formation in .

MmpL (mycobacterial membrane protein large) proteins are integral membrane proteins that have been implicated in the biosynthesis and/or transport of mycobacterial cell wall lipids. Given the cellular location of these proteins, however, it is unclear how cell wall lipids are transported beyond the inner membrane. Moreover, given that mycobacteria grow at the poles, we also do not understand how new cell wall is added in a highly localized and presumably coordinated manner.

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

() is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for .

Cell wall proteomics in live uncovers exposure of ESX substrates to the periplasm.

The cell wall of mycobacteria plays a key role in interactions with the environment and its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites from ions to lipids to proteins. Accurately identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues.

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria.

Unlabelled: Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition.

Confusion.

Can we do better when it comes to the "other-race effect"?

Optimized APEX2 peroxidase-mediated proximity labeling in fast- and slow-growing mycobacteria.

Proximity labeling is a technology for tagging proteins and other biomolecules in living cells. These methods use enzymes that generate reactive species whose properties afford high spatial resolution for the localization of proteins to subcellular compartments and the identification of endogenous interaction partners. Here we present the adaptation of the engineered peroxidase APEX2 to proximity labeling in mycobacteria, including the human pathogen Mycobacterium tuberculosis.

Recent advances in the mass spectrometric profiling of bacterial lipids.

Exploring the lipids of bacteria presents a predicament that may not be broadly recognized in a field dominated by the biology and biochemistry of eukaryotic - and especially, mammalian - lipids. Bacteria make multifarious metabolites that contain fatty acyl chains of unusual length and unsaturation attached to assorted headgroups, including sugars and fatty alcohols. Lipid profiling approaches developed for eukaryotic lipids often fail to detect, resolve, or identify bacterial lipids due to their wide range of polarities (including very hydrophobic species) and diverse positional and stereochemical variations.

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling.

The identification and validation of a small molecule's targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule's targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored.

Dimethylaminophenyl Hydrazides as Inhibitors of the Lipid Transport Protein LprG in Mycobacteria.

Assembly of the bacterial cell wall requires not only the biosynthesis of cell wall components but also the transport of these metabolites to the cell exterior for assembly into polymers and membranes required for bacterial viability and virulence. LprG is a cell wall protein that is required for the virulence of and is associated with lipid transport to the outer lipid layer or mycomembrane. Motivated by available cocrystal structures of LprG with lipids, we searched for potential inhibitors of LprG by performing a computational docking screen of ∼250 000 commercially available small molecules.

Opportunities and Challenges in Activity-Based Protein Profiling of Mycobacteria.

Mycobacteria, from saprophytic to pathogenic species, encounter diverse environments that demand metabolic versatility and rapid adaptation from these bacteria for their survival. The human pathogen Mycobacterium tuberculosis, for example, can enter a reversible state of dormancy in which it is metabolically active, but does not increase in number, and which is believed to enable its survival in the human host for years, with attendant risk for reactivation to active tuberculosis. Driven by the need to combat mycobacterial diseases like tuberculosis, efforts to understand such adaptations have benefitted in recent years from application of activity-based probes.

Compartment-Specific Labeling of Bacterial Periplasmic Proteins by Peroxidase-Mediated Biotinylation.

The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this subcellular compartment. Peroxidase-mediated biotinylation has enabled targeted labeling of proteins within subcellular compartments of mammalian cells. We investigated whether this methodology could be applied to the bacterial periplasm.

A Screen for Protein-Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A.

Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein-protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria.

Transport of outer membrane lipids in mycobacteria.

The complex organization of the mycobacterial cell wall poses unique challenges for the study of its assembly. Although mycobacteria are classified evolutionarily as Gram-positive bacteria, their cell wall architecture more closely resembles that of Gram-negative organisms. They possess not only an inner cytoplasmic membrane, but also a bilayer outer membrane that encloses an aqueous periplasm and includes diverse lipids that are required for the survival and virulence of pathogenic species.

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria.

We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Using structure-based design, we engineered three preQ1 riboswitches from , , and toward achieving higher response ratios and increased repression.

Monstrous Mycobacterial Lipids.

When it comes to lipid diversity, no bacterial genus approaches Mycobacterium. In this issue of Cell Chemical Biology, Burbaud et al. (2016) provide a multi-genic working model for the biosynthesis of trehalose polyphleate (TPP), one of the largest known lipids in mycobacteria.

Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels, Growth Rate and Virulence in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) mutants lacking rv1411c, which encodes the lipoprotein LprG, and rv1410c, which encodes a putative efflux pump, are dramatically attenuated for growth in mice. Here we show that loss of LprG-Rv1410 in Mtb leads to intracellular triacylglyceride (TAG) accumulation, and overexpression of the locus increases the levels of TAG in the culture medium, demonstrating a role of this locus in TAG transport. LprG binds TAG within a large hydrophobic cleft and is sufficient to transfer TAG from donor to acceptor membranes.

Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids.

Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections.

Using riboswitches to regulate gene expression and define gene function in mycobacteria.

Mycobacteria include both environmental species and many pathogenic species such as Mycobacterium tuberculosis, an intracellular pathogen that is the causative agent of tuberculosis in humans. Inducible gene expression is a powerful tool for examining gene function and essentiality, both in in vitro culture and in host cell infections. The theophylline-inducible artificial riboswitch has recently emerged as an alternative to protein repressor-based systems.

The rv1184c locus encodes Chp2, an acyltransferase in Mycobacterium tuberculosis polyacyltrehalose lipid biosynthesis.

Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified.