Sruti DebRoy

Bio: Sruti DebRoy is an academic researcher from Northwestern University. The author has contributed to research in topics: Legionella pneumophila & Mutant. The author has an hindex of 3, co-authored 4 publications receiving 284 citations.

Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

Abstract: Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70–75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had ≈75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.

Legionella pneumophila Type II Secretion Dampens the Cytokine Response of Infected Macrophages and Epithelia

Abstract: The type II secretion (T2S) system of Legionella pneumophila is required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages, lsp mutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-10, IL-8, IL-1β, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complemented proA mutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system of L. pneumophila dampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, an lspF mutant, but not the proA mutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.

Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants.

Abstract: The type II secretion system of Legionella pneumophila promotes pathogenesis. Among the Legionella type II-dependent exoenzymes is a p-nitrophenol phosphorylcholine (p-NPPC) hydrolase whose activity is only partially explained by the PlcA phospholipase C. In a screen to identify other factors that promote secreted hydrolase activity, we isolated a mip mutant. L. pneumophila Mip is a surface-exposed, FK506-binding protein that is needed for optimal infection and has peptidylproline cis-trans-isomerase (PPIase) activity. Since the molecular target of Mip was undefined, we investigated a possible relationship between Mip and the secreted p-NPPC hydrolase activity. In the mip mutant there was a 40 to 70% reduction in secreted activity that was successfully complemented by providing mip on a plasmid. A similar phenotype was observed when we examined four other independently derived mip mutants, and in all cases the defect was complemented by reintroduction of mip. Thus, mip promotes the presence of a p-NPPC hydrolase activity in culture supernatants. We also found that the C terminus of Mip is required for this effect. When supernatants were examined by anion-exchange chromatography, the p-NPPC hydrolase activity associated with Mip proved to be type II dependent but distinct from PlcA. This conclusion was supported by the phenotype of a newly constructed mip plcA double mutant. Thus, Mip promotes the elaboration of a new type II exoprotein. These data provide both the first evidence for a target for Mip and the first indication that a surface PPIase is involved in the secretion or activation of proteins beyond the outer membrane.

Legionella pneumophila Mip: New Function for an Old Protein?

Abstract: The macrophage infectivity potentiator (Mip) protein of Legionella pneumophila is one of the most studied Legionella proteins and has long been known to promote virulence. Mip protein has been purified and shown to possess peptidyl-prolyl cis/trans isomerase (PPI-ase) activity. Comparison of primary structures and crystallographic studies shows that Mip proteins have an N-terminal and a C-terminal domain. Type II secretion is one of five protein secretion systems that can mediate the export of proteins across the gram-negative outer membrane into the extracellular milieu and/or into target cells, and the authors have shown that the type II protein secretion system of L. pneumophila is required for optimal replication in macrophages, amoebae, and mice. Interestingly, one of transposon mutants (previously designated NU247) proved to contain a single transposon insertion in the mip gene, suggesting, for the first time, that Mip might influence protein secretion. This chapter talks about the NU247 reproducibly that showed a 40 to 70% reduction in p-NPPC hydrolase activity in its culture supernatants in comparison to the wild-type strain 130b. Since Mip is highly conserved in the Legionella genus, and surface and secreted Mip-like proteins are present in other pathogenic microorganisms, Mip and Mip-like proteins might promote the secretion of other important effectors.

Molecular Pathogenesis of Infections Caused by Legionella pneumophila

Abstract: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.

The type II secretion system: biogenesis, molecular architecture and mechanism.

Abstract: Many Gram-negative bacteria use type II secretion systems (T2SSs) to translocate a range of proteins across the outer membrane from the periplasm. In this Review, Hol and colleagues describe how recent structural and biochemical studies have provided insights into the biogenesis and architecture of T2SSs and the mechanism by which they function.

Host proteasomal degradation generates amino acids essential for intracellular bacterial growth.

Abstract: Legionella pneumophila proliferates in environmental amoeba and human cells within the Legionella-containing vacuole (LCV). The exported AnkB F-box effector of L. pneumophila is anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys48-linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of the ankB null mutant in proliferation within amoeba and human cells is rescued by supplementation of a mixture of amino acids or cysteine, serine, pyruvate, or citrate, similar to rescue by genetic complementation. Defect of the ankB mutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore, Legionella promotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.

Comparative Genomics Reveal Extensive Transposon-Mediated Genomic Plasticity and Diversity among Potential Effector Proteins within the Genus Coxiella

Abstract: Genetically distinct isolates of Coxiella burnetii, the cause of human Q fever, display different phenotypes with respect to in vitro infectivity/cytopathology and pathogenicity for laboratory animals. Moreover, correlations between C. burnetii genomic groups and human disease presentation (acute versus chronic) have been described, suggesting that isolates have distinct virulence characteristics. To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogenic potential, we deciphered the whole-genome sequences of the K (Q154) and G (Q212) human chronic endocarditis isolates and the naturally attenuated Dugway (5J108-111) rodent isolate. Cross-genome comparisons that included the previously sequenced Nine Mile (NM) reference isolate (RSA493) revealed both novel gene content and disparate collections of pseudogenes that may contribute to isolate virulence and other phenotypes. While C. burnetii genomes are highly syntenous, recombination between abundant insertion sequence (IS) elements has resulted in genome plasticity manifested as chromosomal rearrangement of syntenic blocks and DNA insertions/deletions. The numerous IS elements, genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structures of other bacterial pathogens that have recently emerged from nonpathogens with expanded niches. The observation that the attenuated Dugway isolate has the largest genome with the fewest pseudogenes and IS elements suggests that this isolate's lineage is at an earlier stage of pathoadaptation than the NM, K, and G lineages.

Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung

Abstract: Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70–75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had ≈75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.