Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadAV98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions

Assembly and arrangement of the type three secretion system of "yersinia enterocolitica"

The type three secretion system (T3SS) is a bacterial weapon found in many Gram-negative bacteria. It consists out of a needle like structure called injectisome, which enables bacteria to inject effector proteins into eukaryotic cells. The injectisome is a very complex molecular machine, inserted in the inner and outer bacteria membrane, passing the periplasm with the peptidoglycan layer. We used an approach with fluorescent hybrid proteins to study the assembly order of the injectisome in Yersinia enterocolitica. First the outer membrane protein YscC was genetically fused to the red fluorescent protein mCherry. This construct was able to build fluorescent foci in the bacterial membrane by itself, without any other components of the T3SS. Then we engineered fusions between the green fluorescent protein EGFP and the inner membrane protein YscD, the putative C-ring protein YscQ or the ATPase YscN. All three constructs showed fluorescent foci at the bacterial membrane. Comparison of the different EGFP constructs with the YscC-mCherry construct in double mutants showed that the proteins co-localize. Thus we considered the foci to be a read out for injectisome assembly. By combining the EGFP constructs with different deletion mutants we found that the assembly occurs from the outside to the inside. Starting with the outer membrane protein YscC to the inner membrane. Then the ATPase and C-ring assemble together and finally the needle is formed. Different single particle structures of T3SS, needle complexes, purified from Shigella flexneri and Salmonella enterica are available. But in the course of purification the inner membrane export apparatus, the ATPase complex and the C-ring were lost. As well no such complex has been purified from Y. enterocolitica. Thus we investigated the Y. enterocolitica injectisomes in situ by cryo electron tomography (cryo-ET). Unfortunately, the 1-?m diameter of Y. enterocolitica is too large to obtain optimal resolution with cryo-ET. Thus we engineered a minD mutant that forms so called minicells, due to asymmetric septum placement. We collected tomograms of particles from wild type and minicells and constructed an average structure with a resolution of 3.7 nm. In addition the 6 nm resolution in situ structure of the injectisome of S. flexneri was made. We saw significant stretching of the in situ structure compared to the isolated particles. Moreover we saw flexibility of the basal body. We can only speculate that such flexibility might increase the stability of the structure and protect it of mechanical forces. In addition for the first time we could visualize a mass in cytoplasm just below the middle of the injectisome. Due to homology to the flagellum we can assume, that this is the ATPase. But to conclusively assign proteins to masses seen in the in situ structure we would need to label them. A question mark in the Y. enterocolitica injectisome assembly is, how the structure can pass the peptidoglycan layer. The flagellum, which is evolutionary closely related to the injectisome, as well as other types of injectisomes have a muramidase or more specifically lytic transglycosylase (LT) encoded within their loci. No gene encoding for a LT, can be found on the pYV plasmid that encodes otherwise for the entire T3SS. Thus we tested several genomic LT for their involvement in the assembly of the T3SS. It is possible that the injectisome assembles through temporary gaps generated during the synthesis of new peptidoglycan strands. This theory is very intriguing, as the pattern of the fluorescent foci resembled closely the arrangement of the bacterial cytoskeleton protein MreB, which is assumed to be involved in placing the peptidoglycan synthesis machinery. Comparing the localization of MreB and the injectisome showed that both constructs seem to be arranged on two different helical paths. This convinced us that injectisome arrangement is not stochastic but rather controlled. To find the underlying structure responsible for this arrangement, we compared the localization of other proteins with similar arrangement as MreB. In Bacillus subtilis different membrane compositions were shown to be helically distributed. Unfortunately staining of the inner membrane is difficult in Gram-negative bacteria. Therefore we compared injectisome location with a potential bacterial lipid raft marker, which showed injectisomes do not insert into the lipid rafts. Thus although our knowledge about the assembly, the structure and the function of the T3SS is improving enormously, the question of how injectisomes are localized remains to be answered

Assembly of the Yersinia injectisome: the missing pieces

The assembly of the type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. We found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co-purified with the export apparatus protein YscV, even in the absence of any other protein. YscY-EGFP formed fluorescent spots, suggesting its presence in multiple copies. YscO and YscX were required for export of the early substrates YscF, YscI and YscP, but were only exported themselves after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for the assembly of the ATPase YscN. Finally, we investigated the role of the small proteins in export across the inner membrane. No export of the reporter substrate YscP(1-137) -PhoA into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP(1-137) -PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC

Yersinia enterocolitica Type III secretion injectisomes form regularly spaced clusters which incorporate new machines upon activation

Bacterial type III secretion systems or injectisomes are multi protein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca(2+) ), the intensity of single spots significantly increased compared to non-secreting conditions (presence of Ca(2+) ), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells

Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca2+), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca2+), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Structure of the Dodecameric Yersinia enterocolitica Secretin YscC and Its Trypsin-Resistant Core

The type III secretion system machinery, also known as the injectisome, delivers bacterial effector proteins into eukaryotic cells during infection. The outer membrane YscC secretin is a major part of Yersinia enterocolitica's injectisome and is among the first components to assemble, solely assisted by its pilotin, YscW. We have determined the three-dimensional structures of the native complex and its protease-resistant core to 12 Å resolution by cryo-electron microscopy (cryo-EM) and show that YscC forms a dodecameric complex. Cryo-EM of YscC reconstituted into proteoliposomes defines the secretin's membrane-spanning region. Native YscC consists of an outer membrane ring connected via a thin cylindrical wall to a conical, periplasmic region that exposes N-terminal petals connected by flexible linkers. These petals harbor the binding site of YscD, a component of the inner membrane ring. A change in their orientation adapts the length of the YscC secretin and facilitates its interaction with YscD

YadA renders <i>Y. enterocolitica</i> sensitive to NET-dependent killing.

<p>(<b>A</b>) <i>Y. enterocolitica</i> induces DNA release upon infection of PMNs. PMNs were infected for 120 min with <i>Y. enterocolitica</i> E40 (wt or ΔYadA) at an moi of 1. DNA release was quantified by Sytox staining. Untreated PMNs were used as negative control and NET formation was induced by PMA as positive control. Mean values from three or more experiments and standard deviation are shown including statistical significance in comparison to untreated PMNs with ** p<0.01 and * p<0.05 using one-way ANOVA. (<b>B</b>) % of <i>Y. enterocolitica</i> E40 (wt, ΔYadA and ΔYadA endowed with pSAM16 encoding YadA_Ψtb) killed by PMA-triggered NETs (120 min infection at a moi of 1). Phagocytosis was prevented by the addition of Cytochalasin D (CytD). Mean values from three or more experiments and standard deviation are shown. Statistical significance is shown in comparison to <i>Y. enterocolitica</i> wt with *** p<0.001 using one-way ANOVA. (<b>C</b>) Scanning electron micrograph (SEM) of untreated human PMNs and (<b>D</b>) of NETs formed by human PMNs treated with PMA. (<b>E</b>) SEM of <i>Y. enterocolitica</i> E40 wt bacteria (expressing YadA) trapped in NETs after 120 min infection at an moi of 1. (<b>F</b>) <i>Y. enterocolitica</i> E40 ΔYadA bacteria induce NET formation but are not trapped (same conditions as in A) (SEM). NET structure covers the whole bottom.</p

Collagen binding contributes to the sensitivity of <i>Y. enterocolitica</i> to NETs.

<p>(<b>A</b>) Mutation of the collagen binding motif has no effect on serum resistance. % survival of <i>Y. enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pFR1) expressing YadA_ent, or E40 ΔYadA (pFR2) expressing YadA_entV98D,N99A, incubated for 1 hour in the presence of 10% normal human serum (NHS) or heat-inactivated human serum (HI NHS). (<b>B</b>) % Survival to NET-dependent killing of <i>Y. enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pMA1) expressing YadA_ent, or E40 ΔYadA (pSAM23) expressing YadA_entV98D,N99A. (<b>C</b>) YadA_entV98D,N99A forms trimers. SDS PAGE analysis of total cells from <i>Y</i>. <i>enterocolitica</i> E40 ΔYadA, E40 ΔYadA (pFR1) expressing YadA_ent, or E40 ΔYadA (pFR2) expressing YadA_entV98D,N99A. The band corresponding to trimeric YadA is boxed.</p