Unhealthy Sugars in Bacterial Cytotoxicity
Authors: D. Isui Aguilar-Salvador, Guy Tran Van Nhieu
Journal: Cell Host & Microbe (2016)

Bacterial type three secretion systems (T3SSs) are central to virulence, but the processes stimulating their activation are unclear. In this issue of Cell Host & Microbe, Blondel et al., 2016Blondel C.J. Park J.S. Hubbard T.P. Pacheco A.R. Kuehl C.J. Walsh M.J. Davis B.M. Gewurz B.E. Doench J.G. Waldor M.K. Cell Host Microbe. 2016; 20 (this issue): 226-237Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar perform a genome-wide screen using CRISPR/Cas technology to identify a role for cell surface sulfation and fucosylation in cytotoxicity mediated by Vibrio parahaemolyticus T3SSs. Bacterial type three secretion systems (T3SSs) are central to virulence, but the processes stimulating their activation are unclear. In this issue of Cell Host & Microbe, Blondel et al., 2016Blondel C.J. Park J.S. Hubbard T.P. Pacheco A.R. Kuehl C.J. Walsh M.J. Davis B.M. Gewurz B.E. Doench J.G. Waldor M.K. Cell Host Microbe. 2016; 20 (this issue): 226-237Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar perform a genome-wide screen using CRISPR/Cas technology to identify a role for cell surface sulfation and fucosylation in cytotoxicity mediated by Vibrio parahaemolyticus T3SSs. Bacterial pathogens have evolved several mechanisms to infect eukaryotic cells. Among them, type three secretion systems (T3SSs) are major virulence factors of Gram-negative bacterial pathogens, including Vibrio parahaemolyticus, a major cause of seafood-related illnesses. These macromolecular structures allow the injection of effector proteins from the bacterial cytosol into the cytoplasm of the host cell. Type III effectors then divert various host cell processes, including cytoskeleton dynamics, trafficking, cell signaling pathways regulating cell death or survival, and inflammatory responses in order to promote infection (Galán et al., 2014Galán J.E. Lara-Tejero M. Marlovits T.C. Wagner S. Annu. Rev. Microbiol. 2014; 68: 415-438Crossref PubMed Scopus (362) Google Scholar). T3SSs are evolutionarily and structurally related to the flagellum, and while there may be some minor variations, they are believed to show conserved functional features (Portaliou et al., 2016Portaliou A.G. Tsolis K.C. Loos M.S. Zorzini V. Economou A. Trends Biochem. Sci. 2016; 41: 175-189Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). T3SSs are inactive at the basal state and activated by contact with host cell membranes. Upon activation, two hydrophobic T3SS substrates are secreted and inserted into the host cell membranes, to form a so-called “translocon,” required for the subsequent injection of type III effectors into host cells (Portaliou et al., 2016Portaliou A.G. Tsolis K.C. Loos M.S. Zorzini V. Economou A. Trends Biochem. Sci. 2016; 41: 175-189Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The precise mechanism underlying the injection of these effectors is still not fully understood and has been the subject of controversy (Akopyan et al., 2011Akopyan K. Edgren T. Wang-Edgren H. Rosqvist R. Fahlgren A. Wolf-Watz H. Fallman M. Proc. Natl. Acad. Sci. USA. 2011; 108: 1639-1644Crossref PubMed Scopus (85) Google Scholar). While lipid membranes can stimulate type III secretion in vitro, “naked” lipids are unlikely to be accessible at the cell plasma membranes, and T3SS activation likely involves the recognition of host cell receptors. Biochemical and molecular approaches have revealed the functions of T3SSs of a myriad of type III effectors from various species, as well as effects on host cell molecular targets. However, genome-wide analyses have been seldom performed to identify host targets. Recently, a whole-genome siRNA screen identified the filament protein, Vimentin, as a key element for Shigella flexneri T3SS interaction with the cytoskeleton (Russo et al., 2016Russo B.C. Stamm L.M. Raaben M. Kim C.M. Kahoud E. Robinson L.R. Bose S. Queiroz A.L. Herrera B.B. Baxt L.A. et al.Nature Microbiology. 2016; 1: 16025Crossref Scopus (34) Google Scholar). In other studies, haploid cell technology revealed a chemokine receptor needed for the translocation activity of T3SS complex in Yersina pseudotuberculosis (Sheahan and Isberg, 2015Sheahan K.-L. Isberg R.R. MBio. 2015; 6 (e02023–e14)Crossref PubMed Scopus (25) Google Scholar), which remained elusive using other approaches. In this issue, Blondel et al., 2016Blondel C.J. Park J.S. Hubbard T.P. Pacheco A.R. Kuehl C.J. Walsh M.J. Davis B.M. Gewurz B.E. Doench J.G. Waldor M.K. Cell Host Microbe. 2016; 20 (this issue): 226-237Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar report the use of a CRISPR/Cas9 genome-wide screen to identify host targets for the T3SSs of V. parahaemolyticus. This bacterium harbors in its genome two T3SSs, termed T3SS1 and T3SS2. The former is found in both pathogenic and non-pathogenic strains, while the latter has been linked to toxic effects in animal models and virulent strains. By infecting a genome-wide library of mutant cells obtained using CRISPR/Cas9 technology, Blondel et al. identified specific sets of genes required for cytotoxic effects associated with each T3SS. After several rounds of enrichment for cells surviving infection with a strain expressing only the T3SS1, the group identified guide RNAs targeting genes for cell surface sulfation. Various glycosaminoglycans (GAGs) are sulfated at the cell surface, including chondroitin, heparin, and dermatan sulfate proteoglycans. Rather than specific receptors, however, the screen pointed to general sulfation of proteoglycans as a requirement for T3SS1-mediated cytotoxicity, findings that were confirmed by phenotypic complementation by coating sulfation-deficient cell lines with the various sulfated groups. Proteoglycan sulfation was further shown to enhance bacterial adhesion mediated by the T3SS1 (Figure 1). Bacterial mutant analysis indicated that all three previously characterized adhesins, Mam7, MshA1, and VpadF, contribute to binding to sulfated groups on the host cell surface (Figure 1). Following a similar strategy using a strain expressing only the T3SS2, the authors identified target genes involved in the synthesis of GDP-fucose, as well as in the fucosylation of glycans exported to the cell surface (Figure 1). As with sulfation and T3SS1-mediated cytotoxicity, no specific fucosylated glycan was identified in the screen, suggesting that multiple fucosylated surface glycans contribute to T3SS2-mediated killing of host cells. CHO (Chinese hamster ovary) cells that only express the FUT8 fucosyltransferase and are devoid of terminally fucosylated glucans are fully resistant to T3SS-mediated cytotoxicity. Transduction of these cells with FUT genes leading to different α-terminal and α-fucose linkage indicated that various terminal fucosylation forms conferred susceptibility to bacterial killing. Remarkably, and as opposed to sulfation, fucosylation did not increase bacterial adhesion to host cells, but appeared to favor insertion of the T3SS2 translocon in host cell membranes (Figure 1). Cell surface sulfation and fucosylation are processes conserved in eukaryotic cells, leading the authors to speculate that their findings could explain the wide spectrum of cells that V. parahaemolyticus can invade, and perhaps its ability to infect other organisms such as mollusks. Also, the promiscuous nature of these modifications could potentially account for the T3SSs’ activities during anti-predator defense against protists (Erken et al., 2013Erken M. Lutz C. McDougald D. Microb. Ecol. 2013; 65: 860-868Crossref PubMed Scopus (58) Google Scholar). Given the high degree of conservation among T3SSs, these results could shed light on mechanisms in other bacterial species. Of note, T3SSs have not been directly involved in mediating bacterial adhesion to host cells, and this study does not formally rule out the possibility that the V. parahaemolyticus T3SS1 recognition of sulfated GAGs indirectly enhances adhesion. For example, in S. flexneri, the bacterial surface autotransporter IcsA is most characterized for its role in intracellular actin-based motility, but has also been shown to promote bacterial adhesion to host cells in a T3SS-dependent manner (Brotcke Zumsteg et al., 2014Brotcke Zumsteg A. Goosmann C. Brinkmann V. Morona R. Zychlinsky A. Cell Host Microbe. 2014; 15: 435-445Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). On the other hand, many bacterial ligands act as lectins recognizing sugar moieties of the cell surface receptors, a common and general theme that T3SSs components involved in the initial recognition of cell surface components may also follow. The specific nature of the interactions between the V. parahaemolyticus T3SS1 and T3SS2 and sulfated and fucosylated compounds points to intriguing variations around this theme. Additionally, two recent papers by Marceau et al., 2016Marceau C.D. Puschnik A.S. Majzoub K. Ooi Y.S. Brewer S.M. Fuchs G. Swaminathan K. Mata M.A. Elias J.E. Sarnow P. Carette J.E. Nature. 2016; 535 (Published online June 17, 2016): 159-163https://doi.org/10.1038/nature18631Crossref PubMed Scopus (267) Google Scholar and Zhang et al., 2016Zhang R. Miner J.J. Gorman M.J. Rausch K. Ramage H. White J.P. Zuiani A. Zhang P. Fernandez E. Zhang Q. et al.Nature. 2016; 535 (Published online June 17, 2016): 164-168https://doi.org/10.1038/nature18625Crossref PubMed Scopus (249) Google Scholar also utilized the CRISPR/Cas9 technology to examine pathways targeted by flaviviruses, identifying both generic and specific requirements for different viruses within the family. The flaviviruses analyzed showed a strong dependency on a signal peptide processing pathway. Hepatitis C virus replication, however, showed a dependence on miRNA processing and, unexpectedly, on the metabolic cofactor, flavin adenine dinucleotide (FAD). Importantly, the genome-wide aspect of these CRISPR/Cas applications allows us to screen not only protein-coding genes, but also non-coding RNAs, metabolic factors, or other regulatory elements in DNA relevant for bacterial and viral infections. However, even this technology has limitations. For example, the number of target alleles present in cells introduces bias in the selection procedure. Nonetheless, the relative simplicity of CRISPR/Cas9 gene-editing tools allows this technology to become widely used in host-pathogen interaction studies. The advent of engineered Cas proteins with improved specificity and spatiotemporal control opens up exciting perspectives to identify host components involved at different stages of the infectious process. G.T.V.N. received suport from ANR-10-IDEX-0001-02 PSL ∗. D.I.A.-S. is a recipient of an M2 grant from Memolife Labex. CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated CytotoxicityBlondel et al.Cell Host & MicrobeJuly 21, 2016In BriefType III secretion systems (T3SSs) underlie the virulence of many bacterial pathogens, but the required host factors remain largely undefined. Blondel et al. harness CRISPR/Cas9 technology to perform genome-wide screens and identify requirements for host cell sulfation and fucosylation in conferring susceptibility to T3SS-mediated killing by Vibrio parahaemolyticus. Full-Text PDF Open Archive