Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1).

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection.

The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.

Systems-level overview of host protein phosphorylation during Shigella flexneri infection revealed by phosphoproteomics.

The enteroinvasive bacterium Shigella flexneri invades the intestinal epithelium of humans. During infection, several injected effector proteins promote bacterial internalization, and interfere with multiple host cell responses. To obtain a systems-level overview of host signaling during infection, we analyzed the global dynamics of protein phosphorylation by liquid chromatography-tandem MS and identified several hundred of proteins undergoing a phosphorylation change during the first hours of infection. Functional bioinformatic analysis revealed that they were mostly related to the cytoskeleton, transcription, signal transduction, and cell cycle. Fuzzy c-means clustering identified six temporal profiles of phosphorylation and a functional module composed of ATM-phosphorylated proteins related to genotoxic stress. Pathway enrichment analysis defined mTOR as the most overrepresented pathway. We showed that mTOR complex 1 and 2 were required for S6 kinase and AKT activation, respectively. Comparison with a published phosphoproteome of Salmonella typhimurium-infected cells revealed a large subset of coregulated phosphoproteins. Finally, we showed that S. flexneri effector OspF affected the phosphorylation of several hundred proteins, thereby demonstrating the wide-reaching impact of a single bacterial effector on the host signaling network.

Deciphering the assembly of the Yersinia type III secretion injectisome.

The assembly of the Yersinia enterocolitica type III secretion injectisome was investigated by grafting fluorescent proteins onto several components, YscC (outer-membrane (OM) ring), YscD (forms the inner-membrane (IM) ring together with YscJ), YscN (ATPase), and YscQ (putative C ring). The recombinant injectisomes were functional and appeared as fluorescent spots at the cell periphery. Epistasis experiments with the hybrid alleles in an array of injectisome mutants revealed a novel outside-in assembly order: whereas YscC formed spots in the absence of any other structural protein, formation of YscD foci required YscC, but not YscJ. We therefore propose that the assembly starts with YscC and proceeds through the connector YscD to YscJ, which was further corroborated by co-immunoprecipitation experiments. Completion of the membrane rings allowed the subsequent assembly of cytosolic components. YscN and YscQ attached synchronously, requiring each other, the interacting proteins YscK and YscL, but no further injectisome component for their assembly. These results show that assembly is initiated by the formation of the OM ring and progresses inwards to the IM ring and, finally, to a large cytosolic complex.

The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome.

The length of the Yersinia injectisome needle is determined by the protein YscP, which could act as a molecular ruler. The analysis of the correlation between the size of YscP and the needle length in seven wild-type strains of Yersinia enterocolitica reinforced this hypothesis but hinted that the secondary structure of YscP might influence needle length. Hence, 11 variants of YscP(515) were generated by multiple Pro or Gly substitutions. The needle length changed in inverse function of the helical content, indicating that not only the number of residues but also their structure controls length. Taking the secondary motifs into account, Pro/Gly-variants were subjected to in silico modelling to simulate the extension of YscP upon needle growth. The calculated lengths when the helical content is preserved correlated strikingly with the measured needle length, with a constant difference of approximately 29 nm, which corresponds approximately to the size of the basal body. These data support the ruler model and show that the functional ruler has a helical structure.

A Bartonella Effector Acts as Signaling Hub for Intrinsic STAT3 Activation to Trigger Anti-inflammatory Responses.

Chronically infecting pathogens avoid clearance by the innate immune system by promoting premature transition from an initial pro-inflammatory response toward an anti-inflammatory tissue-repair response. STAT3, a central regulator of inflammation, controls this transition and thus is targeted by numerous chronic pathogens. Here, we show that BepD, an effector of the chronic bacterial pathogen Bartonella henselae targeted to infected host cells, establishes an exceptional pathway for canonical STAT3 activation, thereby impairing secretion of pro-inflammatory TNF-α and stimulating secretion of anti-inflammatory IL-10. Tyrosine phosphorylation of EPIYA-related motifs in BepD facilitates STAT3 binding and activation via c-Abl-dependent phosphorylation of Y705. The tyrosine-phosphorylated scaffold of BepD thus represents a signaling hub for intrinsic STAT3 activation that is independent from canonical STAT3 activation via transmembrane receptor-associated Janus kinases. We anticipate that our findings on a molecular shortcut to STAT3 activation will inspire new treatment options for chronic infections and inflammatory diseases.

Multi-omic measurements of heterogeneity in HeLa cells across laboratories.

Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.

Length control of extended protein structures in bacteria and bacteriophages.

The length of the tail of bacteriophages is controlled by a protein which acts as a molecular ruler. The needle of the injectisome, which is assembled by the polymerization of subunits that are exported through the nascent injectisome, is functionally related to the tail of bacteriophages. Interestingly, its length is controlled by a protein, which is itself exported and acts as a molecular ruler that is coupled to a substrate specificity switch. The bacterial flagellum is evolutionarily related to the injectisome. The length of the hook is also controlled by a secreted protein. This protein acts as a substrate specificity switch and, possibly, also as a ruler.

Type III secretion: the bacteria-eukaryotic cell express.

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future.

Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype Yersinia enterocolitica which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different Yersinia mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, Clostridium difficile toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal GILZ promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the GILZ promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of GILZ promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.

Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation.

BACKGROUND: Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells. METHODOLOGY: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the µ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa. CONCLUSIONS: We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.

Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection.

Shigella flexneri type III secreted effector OspF harbors a phosphothreonine lyase activity that irreversibly dephosphorylates MAP kinases (MAPKs) p38 and ERK in infected epithelial cells and thereby, dampens innate immunity. Whereas this activity has been well characterized, the impact of OspF on other host signaling pathways that control inflammation was unknown. Here we report that OspF potentiates the activation of the MAPK JNK and the transcription factor NF-κB during S. flexneri infection. This unexpected effect of OspF was dependent on the phosphothreonine lyase activity of OspF on p38, and resulted from the disruption of a negative feedback loop regulation between p38 and TGF-beta activated kinase 1 (TAK1), mediated via the phosphorylation of TAK1-binding protein 1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the impact of OspF on the activation of NF-κB, JNK and c-Jun, and demonstrate the existence of a negative feedback loop regulation between p38 and TAK1 during S. flexneri infection. Furthermore, this study validates the use of bacterial effectors as molecular tools to identify the crosstalks that connect important host signaling pathways induced upon bacterial infection.

Structure of the type III secretion recognition protein YscU from Yersinia enterocolitica.

The inner-membrane protein YscU has an important role during the assembly of the Yersinia enterocolitica type III secretion injectisome. Its cytoplasmic domain (YscU(C)) recognizes translocators as individual substrates in the export hierarchy. Activation of YscU entails autocleavage at a conserved NPTH motif. Modification of this motif markedly changes the properties of YscU, including translocator export cessation and production of longer injectisome needles. We determined the crystal structures of the uncleaved variants N263A and N263D of YscU(C) at 2.05 A and 1.55 A resolution, respectively. The globular domain is found to consist of a central, mixed beta-sheet surrounded by alpha-helices. The NPTH motif forms a type II beta-turn connecting two beta-strands. NMR analysis of cleaved and uncleaved YscU(C) indicates that the global structure of the protein is retained in cleaved YscU(C). The structure of YscU(C) variant N263D reveals that wild type YscU(C) is poised for cleavage due to an optimal reaction geometry for nucleophilic attack of the scissile bond by the side chain of Asn263. In vivo analysis of N263Q and H266A/R314A YscU variants showed a phenotype that combines the absence of translocator secretion with normal needle-length control. Comparing the structure of YscU to those of related proteins reveals that the linker domain between the N-terminal transmembrane domain and the autocleavage domain can switch from an extended to a largely alpha-helical conformation, allowing for optimal positioning of the autocleavage domain during injectisome assembly.

Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD.

Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 A resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely alpha-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.

Function and molecular architecture of the Yersinia injectisome tip complex.

By quantitative immunoblot analyses and scanning transmission electron microscopy (STEM), we determined that the needle of the Yersinia enterocolitica E40 injectisome consists of 139 +/- 19 YscF subunits and that the tip complex is formed by three to five LcrV monomers. A pentamer represented the best fit for an atomic model of this complex. The N-terminal globular domain of LcrV forms the base of the tip complex, while the central globular domain forms the head. Hybrids between LcrV and its orthologues PcrV (Pseudomonas aeruginosa) or AcrV (Aeromonas salmonicida) were engineered and recombinant Y. enterocolitica expressing the different hybrids were tested for their capacity to form the translocation pore by a haemolysis assay. There was a good correlation between haemolysis, insertion of YopB into erythrocyte membranes and interaction between YopB and the N-terminal globular domain of the tip complex subunit. Hence, the base of the tip complex appears to be critical for the functional insertion of YopB into the host cell membrane.

YscU recognizes translocators as export substrates of the Yersinia injectisome.

YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.

The discovery of SycO highlights a new function for type III secretion effector chaperones.

Bacterial injectisomes deliver effector proteins straight into the cytosol of eukaryotic cells (type III secretion, T3S). Many effectors are associated with a specific chaperone that remains inside the bacterium when the effector is delivered. The structure of such chaperones and the way they interact with their substrate is well characterized but their main function remains elusive. Here, we describe and characterize SycO, a new chaperone for the Yersinia effector kinase YopO. The chaperone-binding domain (CBD) within YopO coincides with the membrane localization domain (MLD) targeting YopO to the host cell membrane. The CBD/MLD causes intrabacterial YopO insolubility and the binding of SycO prevents this insolubility but not folding and activity of the kinase. Similarly, SycE masks the MLD of YopE and SycT covers an aggregation-prone domain of YopT, presumably corresponding to its MLD. Thus, SycO, SycE and most likely SycT mask, inside the bacterium, a domain needed for proper localization of their cognate effector in the host cell. We propose that covering an MLD might be an essential function of T3S effector chaperones.

Secretion of YscP from Yersinia enterocolitica is essential to control the length of the injectisome needle but not to change the type III secretion substrate specificity.

The length of the needle of the Yersinia Ysc injectisome is determined by a protein called YscP. This protein, which acts both as a molecular ruler and as a substrate-specificity switch for type III secretion is itself secreted by the injectisome. In this report, we address the question why YscP is secreted. By a systematic deletion analysis and by fusing different parts of the molecule to the adenylate cyclase reporter, we identified two independent secretion signals. One of them is encompassed within the 35 N-terminal residues while the second one spans residues 97-137. These two signals are functionally different from Yop secretion signals. When both secretion signals were removed, Yops could still be secreted but the needle length control was lost. YscP possessing only one signal did not control needle length properly but the control was improved when more YscP was produced and secreted. YscP deprived of both signals could not control length, even when overproduced. We conclude from this that YscP needs to be secreted to exert its length control function but not its substrate-specificity switch function.

Bacterial injectisomes: needle length does matter.

Many pathogenic bacteria use a type III secretion nanomachine (an injectisome) to deliver virulence proteins into the cytosol of their eukaryotic host cells. Most injectisomes possess a stiff needlelike structure of a genetically defined length. We found that a minimal needle length was required for efficient functioning of the Yersinia enterocolitica injectisome. This minimal needle length correlated with the length of the major adhesin at the bacterial surface. The needle may be required for triggering type III secretion, and its length may have evolved to match specific structures at the bacterial and host cell surfaces.

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica.

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C-terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original alpha/beta fold, a P-x-L-G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.