tractable-mammalian-cell-infections-with-protozoan-primed-bacteria

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Pathogenic Yersinia spp. secrete Yops (Yersinia outer proteins) via the type III pathway. The expression of yop genes is regulated in response to environmental cues, which results in a cascade of type III secretion reactions. yscM1 and yscM2 negatively regulate the expression of Yersinia enterocolitica yop genes. It is demonstrated that yopD and lcrH are required for yscM1 and yscM2 function and that all four genes act synergistically at the same regulatory step. Further, SycH binding to the protein products of yscM1 and yscM2 can activate yop gene expression even without promoting type III transport of YscM1 and YscM2. Reverse transcription-PCR analysis of yopQ mRNA as well as yopQ and yopE gene fusion experiments with the npt (neomycin phosphotransferase) reporter suggest that yscM1 and yscM2 regulate expression at a posttranscriptional step. The 178-nucleotide 5' untranslated region (UTR) of yopQ mRNA was sufficient to confer yscM1 and yscM2-mediated regulation on the fused reporter, as was the 28-nucleotide UTR of yopE. The sequence 5'-AUAAA-3' is located in the 5' yop UTRs, and mutations that alter the sequence motif either reduced or abolished yscM1- and yscM2-mediated regulation. A model is proposed whereby YopD, LcrH, YscM1, YscM2, and SycH regulate yop expression in response to specific environmental cues and by a mechanism that may involve binding of some of these factors to a specific target sequence within the UTR of yop mRNAs.

Yersinia enterocolitica type III secretion: yscM1 and yscM2 regulate yop gene expression by a posttranscriptional mechanism that targets the 5' untranslated region of yop mRNA.

Yersinia enterocolitica transports YscM1 and YscM2 via the type III pathway, a mechanism that is required for the establishment of bacterial infections. Prior to host cell contact, YscM1 and YscM2 exert posttranscriptional regulation to inhibit expression of effector yop genes, which encode virulence factors that travel the type III pathway into the cytoplasm of macrophages. Relief from repression has been predicted to occur via the type III secretion of YscM1 and YscM2 into the extracellular medium, resulting in the depletion of regulatory molecules from the bacterial cytoplasm. Using digitonin fractionation and fluorescence microscopy of FlAsH-labeled polypeptides in Yersinia-infected cells, we have localized YscM1 and YscM2 within the host cell cytoplasm. Type III injection of YscM1 and YscM2 required the SycH chaperone. Expression of C-terminal fusions of YscM1 and YscM2 to the neomycin phosphotransferase reporter revealed sequences required for regulatory activity and for secretion in the absence of SycH. Coexpression of SycH and glutathione S-transferase (GST)-YscM1 or GST-YscM2, hybrid GST variants that cannot be transported by the type III apparatus, also relieved repression of Yop synthesis. GST-SycH bound to YscM1 and YscM2 and activated effector yop expression without initiation of the bound regulatory molecules into the type III pathway. Further, regulation of yop expression by YscM1, YscM2, and SycH is shown to act independently of factors that regulate secretion, and gel filtration chromotography revealed populations of YscM1 and YscM2 that are not bound to SycH under conditions where Yop synthesis is repressed. Taken together, these results suggest that YscM1- and YscM2-mediated repression may be relieved through binding to the cytoplasmic chaperone SycH prior to their type III injection into host cells.

Binding of SycH chaperone to YscM1 and YscM2 activates effector yop expression in Yersinia enterocolitica.

Bacterial invasins: molecular systems dedicated to the invasion of host tissues.

The Legionella pneumophila Dot/Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot/Icm system. In this study, the mechanism of Dot/Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot/Icm system. A leucine (L372) residue at the -3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot/Icm system, most other Dot/Icm substrates were found to have amino acid residues with similar physical properties at their -3 or -4 C-terminal positions. These data demonstrate that the Dot/Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells.

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.

The direct transport of virulence proteins from bacterium to host has emerged as a common strategy employed by Gram-negative pathogens to establish infections. Specialized secretion systems function to facilitate this process. The delivery of 'effector' proteins by these secretion systems is currently confined to two functionally similar but mechanistically distinct pathways, termed type III and type IV secretion. The type III secretion pathway is ancestrally related to the multiprotein complexes that assemble flagella, whereas the type IV mechanism probably emerged from the protein complexes that support conjugal transfer of DNA. Although both pathways serve to transport proteins from the bacterium to host, the recognition of the effector protein substrates and the secretion information contained in these proteins appear highly distinct. Here, we review the mechanisms involved in the selection of substrates by each of these transport systems and secretion signal information required for substrate transport.

Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems.

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation.

In this review, we provide an overview of the methods employed in four recent studies that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. We present the results of these studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the Web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVE server (http://www.sysbep.org/sieve). Finally, we assess the accuracies of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general, arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

Article	Year
Yersinia enterocolitica type III secretion: yscM1 and yscM2 regulate yop gene expression by a posttranscriptional mechanism that targets the 5' untranslated region of yop mRNA. Journal of bacteriology\| PubMed ID: 12374821	2002
Binding of SycH chaperone to YscM1 and YscM2 activates effector yop expression in Yersinia enterocolitica. Journal of bacteriology\| PubMed ID: 14729710	2004
Bacterial invasins: molecular systems dedicated to the invasion of host tissues. Contributions to microbiology\| PubMed ID: 15496781	2005
A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15613486	2005
The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation. Molecular microbiology\| PubMed ID: 15661013	2005
Recognition and delivery of effector proteins into eukaryotic cells by bacterial secretion systems. Traffic (Copenhagen, Denmark)\| PubMed ID: 16734660	2006
The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation. PLoS pathogens\| PubMed ID: 18069892	2007
Computational prediction of type III and IV secreted effectors in gram-negative bacteria. Infection and immunity\| PubMed ID: 20974833	2011

Eric D. Cambronne

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