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      Inhibition of Host Vacuolar H +-ATPase Activity by a Legionella pneumophila Effector

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          Abstract

          Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae.

          Author Summary

          One hallmark of the lysosome is a low luminal pH that is important for its maturation as well as the activity of many hydrolyzing enzymes responsible for efficient digestion of phagocytosed contents. To survive and replicate in phagocytes, successful intracellular pathogens have evolved various mechanisms to circumvent the challenges posed by lysosomal killing. One salient feature associated with infection of the intracellular bacterial pathogen Legionella pneumophila is the maintenance of a neutral pH of the Legionella containing vacuoles (LCVs) that supports its intracellular growth in the early phase of infection, while the nonpathogenic mutants are believed to be immediately trafficked to an acidic compartment. In eukaryotic cells, organelle acidification is mediated by the vacuolar H +-ATPase that translocates protons into target compartments in a process energized by ATP hydrolysis. The recent discovery of the association of v-ATPase with LCVs points to the necessity for active modulation of v-ATPase activity by the bacterium. By screening L. pneumophila proteins that cause a yeast phenotype similar to its v-ATPase mutants, we have identified a substrate of the L. pneumophila Dot/Icm type IV secretion system that specifically inhibits the activity of the proton transporter. This protein, termed SidK, inhibits the activity of v-ATPase by directly interacting with the VatA subunit that is responsible for hydrolyzing ATP. Moreover, macrophages harboring SidK display defects in phagosomal acidification and lysosomal killing of non-pathogenic bacteria. We also found that expression of sidK is highly induced right after stationary bacteria are diluted into fresh medium, suggesting that SidK plays an important role in the early phase of infection. Our results reveal a mechanism by which an intravacuolar pathogen engages the v-ATPase protein and inhibits its activity, rather than actively avoiding its association with the pathogen's vacuolar membrane.

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          Most cited references 52

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          Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

          The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
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            Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.

            An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.
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              Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.

              An expression system for Saccharomyces cerevisiae (Sc) has been developed which, depending on the chosen vector, allows the constitutive expression of proteins at different levels over a range of three orders of magnitude and in different genetic backgrounds. The expression system is comprised of cassettes composed of a weak CYC1 promoter, the ADH promoter or the stronger TEF and GPD promoters, flanked by a cloning array and the CYC1 terminator. The multiple cloning array based on pBIISK (Stratagene) provides six to nine unique restriction sites, which facilitates the cloning of genes and allows for the directed cloning of cDNAs by the widely used ZAP system (Stratagene). Expression cassettes were placed into both the centromeric and 2 mu plasmids of the pRS series [Sikorski and Hieter, Genetics 122 (1989) 19-27; Christianson et al., Gene 110 (1992) 119-122] containing HIS3, TRP1, LEU2 or URA3 markers. The 32 expression vectors created by this strategy provide a powerful tool for the convenient cloning and the controlled expression of genes or cDNAs in nearly every genetic background of the currently used Sc strains.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2010
                March 2010
                19 March 2010
                : 6
                : 3
                Affiliations
                [1 ]Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America
                [2 ]Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
                Yale University School of Medicine, United States of America
                Author notes
                [¤]

                Current address: Institute of Virology, Chinese Academy of Sciences, Wuhan, China

                Conceived and designed the experiments: LX XS AB ZQL. Performed the experiments: LX XS AB SB. Analyzed the data: LX XS AB SB MSS ZQL. Contributed reagents/materials/analysis tools: MSS. Wrote the paper: ZQL.

                Article
                09-PLPA-RA-0869R3
                10.1371/journal.ppat.1000822
                2841630
                20333253
                Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 16
                Categories
                Research Article
                Biochemistry
                Cell Biology
                Infectious Diseases/Bacterial Infections
                Microbiology
                Molecular Biology

                Infectious disease & Microbiology

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