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We assembled a collection of 73 Capnocytophaga canimorsus isolates obtained from blood cultures taken from patients treated at Helsinki University Hospital (Helsinki, Finland) during 2000-2017. We serotyped these isolates by PCR and Western blot and attempted to correlate pathogen serovar with patient characteristics. Our analyses showed, in agreement with previous research, that 3 C. canimorsus serovars (A-C) caused most (91.8%) human infections, despite constituting only 7.6% of isolates found in dogs. The 3 fatalities that occurred in our cohort were equally represented by these serovars. We found 2 untypeable isolates, which we designated serovars J and K. We did not detect an association between serovar and disease severity, immune status, alcohol abuse, or smoking status, but dog bites occurred more frequently among patients infected with non-A-C serovars. Future research is needed to confirm serovar virulence and develop strategies to reduce risk for these infections in humans. [ABSTRACT FROM AUTHOR]

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 Y ersinia 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 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. [ABSTRACT FROM AUTHOR]

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 YscP1-137-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, YscP1-137-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. [ABSTRACT FROM AUTHOR]

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections. [ABSTRACT FROM AUTHOR]

Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a "hybrid backbone" lacking the 49 phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2- deoxy-D-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 -- lipid A complex in case the 49 phosphate is not present. [ABSTRACT FROM AUTHOR]

Summary YscV (FlhA in the flagellum) is an essential component of the inner membrane (IM) export apparatus of the type III secretion injectisome. It contains eight transmembrane helices and a large C-terminal cytosolic domain. YscV was expressed at a significantly higher level than the other export apparatus components YscR, YscS, YscT, and YscU, and YscV-EGFP formed bright fluorescent spots at the bacterial periphery, colocalizing in most cases with YscC-mCherry. This suggested that YscV is the only protein of the export apparatus that oligomerizes. Oligomerization required the cytosolic domain of YscV, as well as YscR, -S, -T, but no other Ysc protein, indicating that an IM platform can assemble independently from the membrane-ring forming proteins YscC, -D, -J. However, in the absence of YscC, -D, -J, this IM platform moved laterally at the bacterial surface. YscJ, but not YscD could be recruited to the IM platform in the absence of the secretin YscC. As YscJ was shown earlier to be also recruited by the outer membrane (OM) platform made of YscC and YscD, we infer that assembly of the injectisome proceeds through the independent assembly of an IM and an OM platform that merge through YscJ. [ABSTRACT FROM AUTHOR]

Summary Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection. [ABSTRACT FROM AUTHOR]

C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases. [ABSTRACT FROM AUTHOR]

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 foodborne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions. [ABSTRACT FROM AUTHOR]

The type III secretion injectisome is a nanomachine that delivers bacterial proteins into the cytosol of eukaryotic target cells. It consists of a cylindrical basal structure spanning the two bacterial membranes and the peptidoglycan, connected to a hollow needle, eventually followed by a filament (animal pathogens) or to a long pilus (plant pathogens). Export employs a type III pathway. During assembly, all the protein subunits of external elements are sequentially exported by the basal structure itself, implying that the export apparatus can switch its substrate specificity over time. The length of the needle is controlled by a protein that it also secreted during assembly and presumably acts as a molecular ruler. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 YscP515 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 ∼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. [ABSTRACT FROM AUTHOR]

Capnocytophaga canimorsus, a commensal bacterium of the canine oral flora, has been repeatedly isolated since 1976 from severe human infections transmitted by dog bites. Here, we show that C. canimorsus exhibits robust growth when it is in direct contact with mammalian cells, including phagocytes. This property was found to be dependent on a surface-exposed sialidase allowing C. canimorsus to utilize internal aminosugars of glycan chains from host cell glycoproteins. Although sialidase probably evolved to sustain commensalism, by releasing carbohydrates from mucosal surfaces, it also contributed to bacterial persistence in a murine infection model: the wild type, but not the sialidase-deficient mutant, grew and persisted, both when infected singly or in competition. This study reveals an example of pathogenic bacteria feeding on mammalian cells, including phagocytes by deglycosylation of host glycans, and it illustrates how the adaptation of a commensal to its ecological niche in the host, here the dog's oral cavity, contributes to being a potential pathogen. [ABSTRACT FROM AUTHOR]

Bacteria from the genus Yersinia deliver a number of effectors into host cells via type III secretion (T3S). Injected Yop effectors interfere and prevent pro-inflammatory warning signals by hijacking the host's intracellular machinery. While macrophages infected by wild-type Yersinia enterocolitica did not release mature IL-1β, macrophages infected by Y. enterocolitica deprived of all effectors released mature IL-1β. Surprisingly, macrophages infected by Y. enterocolitica deficient for secretion of all T3S proteins, including effectors and translocators, did not release mature IL-1β. Using different genetic constructs, we show that insertion of T3S translocation pores trigger activation of caspase-1, maturation of proIL-1β and release of mature IL-1β, which occurs independently of cell osmotic lysis. These data show that T3S translocation is intrinsically a pro-inflammatory phenomenon. However, in the case of Yersinia, this effect is neutralized by the action of effectors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, yscUN263A 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, yscUN263A 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 yscUN263A mutation on needle length was the consequence of a reduced YscP export. [ABSTRACT FROM AUTHOR]

Capnocytophaga canimorsus, a commensal bacterium from dogs' mouths, can cause septicemia or meningitis in humans through bites or scratches. Here, we describe and characterize the inflammatory response of human and mouse macrophages on C. canimorsus infection. Macrophages infected with 10 different strains failed to release tumor necrosis factor (TNF)-α and interleukin (IL)-1α. Macrophages infected with live and heat-killed (HK) C. canimorsus 5 (Cc5), a strain isolated from a patient with fatal septicemia, did not release IL-6, IL-8, interferon-γ, macrophage inflammatory protein-lβ, and nitric oxide (NO). This absence of a proinflammatory response was characterized by the inability of Toll-like receptor (TLR) 4 to respond to Cc5. Moreover, live but not HK Cc5 blocked the release of TNF-α and NO induced by HK Yersinia enterocohtica. In addition, live Cc5 down-regulated the expression of TLR4 and dephosphorylated p38 mitogen-activated protein kinase. These results highlight passive and active mechanisms of immune evasion by c. can imorsus, which may explain its capacity to escape from the host immune system. [ABSTRACT FROM AUTHOR]

The type III secretion injectisome is a complex nanomachine that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. The principal structural components of the injectisome, from the base located in the bacterial cytosol to the tip of the needle protruding from the cell surface, have been investigated in detail. The structures of several constituent proteins were solved at the atomic level and important insights into the assembly process have been gained. However, despite the ongoing concerted efforts of molecular and structural biologists, the role of many of the constituent components of this nanomachine remain unknown. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel β-sheet flanked by three α-helices, SycT lacks the dimerization α-helix and has an additional β-strand capable of undergoing a conformational change. The dimer interface consists of two β-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group. [ABSTRACT FROM AUTHOR]

Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (in) P. aeruginosa. wt Y en terocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN (ΔHOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of ΔHOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopBID proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon. [ABSTRACT FROM AUTHOR]

Type III secretion (T3S) systems are widespread among Gram-negative bacteria pathogenic for animals and plants, including Yersinia spp., Salmonella spp., Shigella spp., enteropathogenic Escherichia coli , enterohaemorrhagic E. coli , or Pseudomonas spp. T3S systems allow bacteria to inject virulence proteins, called T3S effectors, into the cytosol of their eukaryotic host cells. These virulence factors will paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules such as small guanosine triphosphate (GTP)-binding proteins, mitogen-activated protein kinases (MAPKs), nuclear factor (NF)-?B, or phosphoinositides. The activity of T3S effectors allows bacteria, for example, to invade non-phagocytic cells or to inhibit phagocytosis, to downregulate or promote pro-inflammatory responses, to induce apoptosis, to prevent autophagy, or to modulate intracellular trafficking. In this review, we present the most recent advances in the understanding of the mode of action of T3S effectors. We highlight the biochemical activities of these eukaryotic-like bacterial proteins that are shared among pathogens carrying T3S systems and the sequence, structural and functional resemblances between T3S effectors and eukaryotic proteins. [ABSTRACT FROM AUTHOR]

The length of the needle ending theYersiniaYsc 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 originalα/β 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 forypeecretionubstratepecificitywitch. 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. [ABSTRACT FROM AUTHOR]

‘Type III secretion’ (T3S) refers to a secretion pathway that is common to the flagellae of eubacteria and the injectisomes of some Gram-negative bacteria. Flagellae are rotary nanomachines allowing motility but they contain a built-in secretion apparatus that exports their own distal components to the distal end of the growing structure where they polymerize. In some cases they have been shown to export non-flagellar proteins. Injectisomes are transkingdom communication apparatuses allowing bacteria docked at the surface of a eukaryotic cell membrane to inject effector proteins across the two bacterial membranes and the eukaryotic cell membrane. Both nanomachines share a similar basal body embedded in the two bacterial membranes, topped either by a hook and a filament or by a stiff short needle. Both appear to be assembled in the same fashion. They recognize their substrate by a loose N-terminal peptide signal and the help of individual chaperones of a new type. [ABSTRACT FROM AUTHOR]

Determines the role of the effector proteins in the cytoskeletal dynamics. Composition of the cocktail of effectors; Assessment on the symbiotic relations between bacteria and animals; Concerns over the recognition of the proteins that are secreted by the type III injectisome.

Despite the fact that type III chaperones were discovered approximately 10 years ago, the precise role of most of them is still mysterious. A panoply of functions has been proposed for the members of this family of proteins. Type III chaperones have been suggested to act as anti-aggregation and stabilizing factors. They have also been proposed to keep their substrates in unfolded or partially folded structures, set a hierarchy on secretion, and participate in the regulation of the transcription of the type III substrates. Here, we review this enigmatic family of proteins, and discuss the experimental data supporting the roles proposed for type III chaperones. [Copyright &y& Elsevier]

Summary The Ysc type III secretion system allows Yersinia enterocolitica to translocate virulence proteins, called Yop effectors, into the cytosol of eukaryotic cells. Some of the Yop effectors possess an individual chaperone called a Syc protein. The first 15 amino acids of the YopE effector constitute a secretion signal that is sufficient to promote secretion of several reporter proteins. Residues 15–50 of YopE comprise the minimal binding domain for the SycE chaperone. In this study, we investigated the secretion by the Ysc system of several YopE–DHFR hybrid proteins with different folding properties, and evaluated the role of SycE, the cognate chaperone of YopE, in this context. We have analysed the secretion of hybrids containing 16 (YopE 16 ), 52 (YopE 52 ) and 80 (the complete region covered by the chaperone, YopE 80 ) amino acids of YopE or full-length YopE (YopE FL ) with wild-type DHFR and two mutants with altered folding properties. The hybrids containing DHFRΔ77, the mutant whose folding properties are the most drastically affected, could be secreted in all the conditions tested, even in the absence of the chaperone SycE. In contrast, DHFRwt could only be secreted fused to the first 52 amino acids of YopE, and its secretion was strictly dependent on SycE. The hybrids YopE 80 –DHFRwt and YopE FL –DHFRwt were not secreted. YopE FL –DHFRwt completely jammed the channel in an SycE-dependent fashion. Our experiments indicate that, in order to be secreted, proteins must be unfolded or only partially folded, and that TSS chaperones could keep their substrates in a secretion-competent conformation, probably by preventing their folding. In addition, they show that the secretion apparatus can reject folded proteins if they are not deeply engaged into the injectisome. [ABSTRACT FROM AUTHOR]

'Type III secretion' — the mechanism by which some pathogenic bacteria inject proteins straight into the cytosol of eukaryotic cells to 'anaesthetize' or 'enslave' them — was discovered in 1994. Important progress has been made in this area during the past few years: the bacterial organelles responsible for this secretion — called 'injectisomes' — have been visualized, the structures of some of the bacterial protein 'effectors' have been determined, and considerable progress has been made in understanding the intracellular action of the effectors. Type III secretion is key to the pathogenesis of bacteria from the Yersinia genus. [ABSTRACT FROM AUTHOR]

Summary Phosphatidylinositol 3-kinase (PI 3-kinase) and its target protein kinase B (Akt) are involved in various processes including internalization, chemotaxis and proliferation. We analysed the activation of Akt in J774 macrophages infected with virulent (pYV+ ) or avirulent (pYV - ) Yersinia enterocolitica . During the early stage of infection with pYV + and pYV - bacteria, Akt and its targets, glycogen synthase kinase 3 (GSK-3) and forkhead transcription factor (FKHRL1), became phosphorylated. This phosphorylation induction was inhibited by wortmannin and thus dependent on PI 3-kinase. When infection was carried out with pYV + bacteria but not with pYV - bacteria, Akt and its targets became dephosphorylated at later time points. Using single knock-out mutants in bacterial effector genes, we have determined that the tyrosine phosphatase YopH was responsible for the inactivation of the PI 3-kinase cascade. In macrophages, this inactivation correlated with the downregulation of mRNA coding for monocyte chemoattractant protein 1 (MCP-1), suggesting that YopH inhibits recruitment of macrophages to lymph nodes. We also analysed the effects of Y. enterocolitica infection on the proliferation of T lymphocytes. Consistent with the observation that YopH inactivated the Akt pathway, YopH inhibited PI 3-kinase-dependent secretion of interleukin 2 and proliferation. These data reveal a new effect of YopH in Yersinia pathogenesis. [ABSTRACT FROM AUTHOR]

Several Gram negative bacteria use a complex system called "type III secretion system" (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded "Yop virulon" shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into five different clusters. The phylogenetic tree of these TTSSs is completely different from the evolutionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer. [ABSTRACT FROM AUTHOR]

Discusses the assembly and function of type III secretory systems of bacterial cells. Device to inject bacterial proteins across eukaryotic cell membranes; Translocation of effectors across animal cell membranes; Recognition of the transported proteins; Modulation of inflammation and signaling interference; Groups of hrp gene clusters in plant pathogens.

Discusses the molecular and cell biology aspects of plague. Device to inject bacterial proteins across eukaryotic cell membranes; Translocation of effectors across animal cell membranes; Cytosolic chaperones; Recognition of the transported proteins; Control of the injection.

A correction to the article "The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga Canimorsus Deglycosylates Human IqG" by Francesco Renzi, Pablo Manfredi and Manuel Mally is presented.

“Type III secretion” allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electro-physiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors. [ABSTRACT FROM AUTHOR]

The Yersinia survival strategy is based on its ability to inject effector Yops into the cytosol of host cells. Translocation of these effectors across the eukaryotic cell membrane requires YopB, YopD and LcrG, but the mechanism is unclear. An effector polymutant of Y. pseudotuberculosis has a YopB-dependent contact haemolytic activity, indicating that YopB participates in the formation of a pore in the cell membrane. Here, we have investigated the formation of such a pore in the plasma membrane of macrophages. Infection of PU5-1.8 macrophages with an effector polymutant Y. enterocolitica led to complete flattening of the cells, similar to treatment with the pore-forming streptolysin O from Streptococcus pyogenes . Upon infection, cells released the low-molecular-weight marker BCECF (623 Da) but not the high-molecular-weight lactate dehydrogenase, indicating that there was no membrane lysis but, rather, insertion of a pore of small size into the macrophage plasma membrane. Permeation to lucifer yellow CH (443 Da) but not to Texas red-X phalloidin (1490 Da) supported this hypothesis. All these events were found to be dependent not only on translocator YopB as expected but also on YopD, which was required equally. In contrast, LcrG was not necessary. Consistently, lysis of sheep erythrocytes was also dependent on YopB and YopD, but not on LcrG. [ABSTRACT FROM AUTHOR]

Extracellular Yersinia spp. disarm the immune system by injecting the effector Yersinia outer proteins (Yops) into the target cell. Yop secretion is triggered by contact with eukaryotic cells or by Ca2+ chelation. Two proteins, YopN and LcrG, are known to be involved in Yop-secretion control. Here we describe TyeA, a third protein involved in the control of Yop release. Like YopN, TyeA is localized at the bacterial surface. A tyeA knock-out mutant secreted Yops in the presence of Ca2+ and in the absence of eukaryotic cells. Unlike a yopN null mutant, the tyeA mutant was defective for translocation of YopE and YopH, but not YopM, YopO and YopP, into eukaryotic cells. This is the first observation suggesting that Yop effectors can be divided into two sets for delivery into eukaryotic cells. TyeA was found to interact with the translocator YopD and with residues 242–293 of YopN. In contrast with a yopN null mutant, a yopN.248–272 mutant was also unable to translocate YopE and YopH. Our results suggest that TyeA forms part of the translocation-control apparatus together with YopD and YopN, and that the interaction of these proteins is required for selective translocation of Yops inside eukaryotic cells. [ABSTRACT FROM AUTHOR]

Extracellular Yersinia adhering at the surface of a eukaryotic cell translocate effector Yops across the plasma membrane of the cell by a mechanism requiring YopD and YopB, the latter probably mediating pore formation. We studied the role of SycD, the intrabacterial chaperone of YopD. By producing GST–YopB hybrid proteins and SycD in Escherichia coli, we observed that SycD also binds specifically to YopB and that this binding reduces the toxicity of GST–YopB in E. coli. By analysis of a series of truncated GST–YopB proteins, we observed that SycD does not bind to a discrete segment of YopB. Using the same approach, we observed that YopD can also bind to YopB. Binding between YopB and YopD occurred even in the presence of SycD, and a complex composed of these three proteins could be immunoprecipitated from the cytoplasm of Yersinia. In a sycD mutant, the intracellular pool of YopB and YopD was greatly reduced unless the lcrV gene was also deleted. As LcrV is known to interact with YopB and YopD and to promote their secretion, we speculate that SycD prevents a premature association between YopB–YopD and LcrV. [ABSTRACT FROM AUTHOR]

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5 kDa). Its secretion required an intact Ysc apparatus and SycT (15.0 kDa, pI 4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia, producing a hybrid YopT–adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (ΔHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells. [ABSTRACT FROM AUTHOR]

Examines the interference of heparin with the translocation of Yop proteins into HeLa cells. Binding of LcrG to HeLa cells; Inhibition of binding by modification of HeLa cells; Competitive inhibition of binding by glycosaminoglycans; Direct binding of LcrG to heparin; Protein sequence analysis of LcrG; Effect of heparin on Yop secretion in brain-heart infusion.

Synthesis of the Yop proteins by yersiniae is down-regulated when secretion is prevented by closure or destruction of the contact (type III) secretion channel. In Yersinia pseudotuberculosis, a mutation in the IcrQ gene, encoding a secreted protein, reduces this feedback inhibition mechanism. Surprisingly, mutation In the yscM gene, the IcrQ homologue in Y. enterocolitica, does not lead to the same deregulated phenotype. In this paper, we addressed the question of this discrepancy. We found a new gene on the Y. enterocolitica pYV plasmid that encodes a protein with 57% identity to YscM (now called YscM1). Overexpression of this gene, called yscM2, like overexpression of IcrQ and yscMI, blocked Yop secretion. A double yscM1, yscM2 mutant had the same phenotype as that of the IcrQ mutant. The discrepancy can thus be explained by the existence of two functionally equivalent copies of yscM in Y. enterocolitica. Overexpression of yscM1 drastically reduced the expression of a yopH-cat reporter gene when tested in a pYV+ background. However, no effect could be observed in the absence of a pYV plasmid, indicating that YscM1 does not act directly as a transcriptional repressor or as an anti-VirF factor. We have also ruled out that YscM acts by obstructing the secretion channel. [ABSTRACT FROM AUTHOR]

The YscC protein of Yersinia enterocolitica is essential for the secretion of anti-host factors, called Yops, into the extracellular environment. It belongs to a family of outer membrane proteins, collectively designated secretins, that participate in a variety of transport processes. YscC has been shown to exist as a stable oligomeric complex in the outer membrane. The production of the YscC complex is regulated by temperature and is reduced in strains carrying mutations in the yscN-U operon or in the virG gene. The VirG lipoproteln was shown to be required for efficient targeting of the complex to the outer membrane. Electron microscopy revealed that purified YscC complexes form ring-shaped structures of ≈ 20 nm with an apparent central pore. Because of the architecture of the multimer, YscC appears to represent a novel type of channel-forming proteins In the bacterial outer membrane. [ABSTRACT FROM AUTHOR]

The Yop virulon enables Yersinia spp. (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host, it is an integrated system allowing extracellular bacteria to communicate with the host cell's cytosol by the injection of effector proteins. It is composed of the following four elements. (i) A contact or type III secretion system called Ysc, which is devoted to the secretion of Yop proteins. This secretion apparatus, comprising some 22 proteins recognizes the Yops by a short N-terminal signal that is not cleaved off during secretion, (ii) A system designed to deliver bacterial proteins into eukaryotic target cells. This system is made of YopB, YopD and possibly other Yops such as LcrV. (iii) A control element (YopN). (iv) A set of effector Yop proteins designed to disarm these cells or disrupt their communications (YopE, YopH, YpkA/YopO, and YopM). The whole virulon is encoded by a 70 kb plasmid designated pYV. Transcription of the genes is controlled both by temperature and by contact with a eukaryotic cell. [ABSTRACT FROM AUTHOR]

Yersinia adhering at the surface of eukaryotic cells secrete a set of proteins called Yops. This secretion which occurs via a type Ill secretion pathway is immediately followed by the injection of some Yops into the cytosol of eukaryotic cells. Translocation of YopE and YopH across the eukaryotic cell membranes requires the presence of the translocators YopB and YopD. YopE and YopH are modular proteins composed of an N-terminal secretion signal, an internalization domain, and an effector domain. Secretion of YopE and YopH requires the presence of the specific cytosolic chaperones SycE and SycH, respectively. In this work, we have mapped the regions of YopE and YopH that are involved in binding of their cognate chaperone. There is only one Syc-binding domain in YopE (residues 15-50) and YopH (residues 20-70). This domain is localized immediately after the secretion signal and it corresponds to the internalization domain. Removal of this bifunctional domain did not affect secretion of YopE and YopH and even suppressed the need for the chaperone in the secretion process. Thus SycE and SycH are not secretion pilots. Instead, we propose that they prevent intrabacterial interaction of YopE and YopH with proteins involved in translocation of these Yops across eukaryotic cell membranes. [ABSTRACT FROM AUTHOR]

Pathogenic yersiniae secrete about a dozen anti-host proteins, the Yops, by a pathway which does not involve cleavage of a classical signal peptide. The Yop secretory apparatus, called Ysc, for Y̲op s̲ec̲retion, is the archetype of type III secretion systems (which serve for the secretion of virulence proteins by several animal and plant pathogens) and is related to the flagellar assembly apparatus. The Yop secretion signal is N-terminal but has not been defined to date. Apart from the Ysc machinery, secretion of at least four Yops requires cytoplasmic proteins called Syc (for s̲pecific Y̲op c̲haperone). Each Syc protein binds to its cognate Yop. Unlike most cytoplasmic chaperones, these proteins do not have an ATP-binding domain, and are presumably devoid of ATPase activity. They share a few common properties: an acidic pt, a size in the range of 15-20 kDa, and a putative amphipathic α-helix in the C-terminal portion. They were recently shown to have counterparts in other pathogenic bacteria, where they appear to have a similar function. [ABSTRACT FROM AUTHOR]

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA , virB, and virC. In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (Cl) for their growth at 37°C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a Cl phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multi-copy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH-LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis. [ABSTRACT FROM AUTHOR]

The chromosome of Yersinia enterocolitica encodes an enterotoxin called Yst. We analysed transcription of chromosomal yst'-luxAB and plasmid-borne yst'-lacZ operon fusions and we observed that regulation of yst expression occurs at transcriptional level. In a wild-type strain, yst was transcribed from at least two major promoters, yst transcription reached a maximum at the entry to the stationary phase and significantly varied in different Y. enterocolitica strains. In some strains, it gradually decreased during the course of our work, suggesting the existence of a mechanism switching the expression of yst to a silent state. Changes in the status of bacterial host factors rather than modifications in the yst gene are responsible for this silencing. Negative regulator YmoA participates in yst silencing and temperature regulation of yst. YmoA was also required for proper growth-phase regulation of yst, although it is not the only factor involved in this regulation. Physicochemical parameters of the environment play an important role in yst transcription. In usual culture media (e.g. tryptic soy broth), the enterotoxin gene was transcribed only at temperatures below 30°C, which argued against the role of Yst in a prolonged diarrhoea at body temperatures. However, yst transcription could be induced at 37°C by increasing osmolarity and pH to the values normally present in the ileum lumen. This finding reconciles the observations concerning yst expression in a host environment and in bacterial cultures, thus supporting the idea that enterotoxin Yst is a virulence factor of Y. enterocolitica. [ABSTRACT FROM AUTHOR]