Dataset Information

Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis.

ABSTRACT: Mycobacterium tuberculosis (Mtb) is an exclusively human pathogen that proliferates within phagosomes of host phagocytes. Host lipids are believed to provide the major carbon and energy sources for Mtb, with only limited availability of carbohydrates. There is an apparent paradox because five putative carbohydrate uptake permeases are present in Mtb, but there are essentially no host carbohydrates inside phagosomes. Nevertheless, carbohydrate transporters have been implicated in Mtb pathogenesis, suggesting that acquisition of host sugars is important during some stages of infection. Here we show, however, that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose, a sugar not present in mammals, thus refuting a role in nutrient acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by Mtb's antigen 85 complex. The antigen 85 complex constitutes a group of extracellular mycolyl transferases, which transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions also lead to the concomitant extracellular release of the trehalose moiety of TMM. We found that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is a recycling system mediating the retrograde transport of released trehalose. Perturbations in trehalose recycling strongly impaired virulence of Mtb. This study reveals an unexpected accessory component involved in the formation of the mycolic acid cell envelope in mycobacteria and provides a previously unknown role for sugar transporters in bacterial pathogenesis.

SUBMITTER: Kalscheuer R

PROVIDER: S-EPMC3003129 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis.

Kalscheuer Rainer R Weinrick Brian B Veeraraghavan Usha U Besra Gurdyal S GS Jacobs William R WR

Proceedings of the National Academy of Sciences of the United States of America 20101130 50

Mycobacterium tuberculosis (Mtb) is an exclusively human pathogen that proliferates within phagosomes of host phagocytes. Host lipids are believed to provide the major carbon and energy sources for Mtb, with only limited availability of carbohydrates. There is an apparent paradox because five putative carbohydrate uptake permeases are present in Mtb, but there are essentially no host carbohydrates inside phagosomes. Nevertheless, carbohydrate transporters have been implicated in Mtb pathogenesis ...[more]

PMID: 21118978

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Project description:Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ?mmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52-140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.

Project description:BackgroundMycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions.ResultsThrough the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge.ConclusionsThe presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.

Project description:Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

Dataset Information

Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis.

Publications

Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis.

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