Conserved type III secretion system exerts important roles in Chlamydia trachomatis

Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis.     

Functional mapping of YadA- and Ail-mediated binding of human factor H to Yersinia enterocolitica serotype O:3

Yersinia enterocolitica is an enteric pathogen that exploits diverse means to survive in the human host. Upon Y. enterocolitica entry into the human host, bacteria sense and respond to variety of signals, one of which is the temperature. Temperature in particular has a profound impact on Y. enterocolitica gene expression, as most of its virulence factors are expressed exclusively at 37°C. These include two outer membrane proteins, YadA and Ail, that function as adhesins and complement resistance (CR) factors. Both YadA and Ail bind the functionally active complement alternative pathway regulator factor H (FH). In this study, we characterized regions on both proteins involved in CR and the interaction with FH. Twenty-eight mutants having short (7 to 41 amino acids) internal deletions within the neck and stalk of YadA and two complement-sensitive site-directed Ail mutants were constructed to map the CR and FH binding regions of YadA and Ail. Functional analysis of the YadA mutants revealed that the stalk of YadA is required for both CR and FH binding and that FH appears to target several conformational and discontinuous sites of the YadA stalk. On the other hand, the complement-sensitive Ail mutants were not affected in FH binding. Our results also suggested that Ail- and YadA-mediated CR does not depend solely on FH binding.     

LcrG is Required for Efficient Translocation of Yersinia Yop Effector Proteins into Eukaryotic Cells

Extracellular Yersinia disables the immune system of its host by injecting effector Yop proteins into host cells. We show that a Yersinia enterocolitica nonpolar lcrG mutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.     

Biogenesis and function of the autotransporter adhesins YadA,intimin and invasin

Bacteria often express numerous virulence factors. These virulence factors make them successful pathogens, by e.g. mediating attachment to host cells and thereby facilitating persistence or invasion, or by contributing to the evasion of the host immune system to allow proliferation and spread within the host and in the environment. The site of first contact of Gram negative bacteria with the host is the bacterial outer membrane (OM). Consisting of an asymmetrical lipid bilayer with phospholipids forming the inner, and lipopolysaccharides forming the outer leaflet, the OM harbors numerous integral membrane proteins that are almost exclusively β-barrel proteins. One distinct family of OM β-barrel proteins strongly linked to bacterial virulence are the autotransporter (AT) proteins. During the last years huge progress has been made to better understand the mechanisms underlying the insertion of AT proteins into the OM and also AT function for interaction with the host. This review shortly summarizes our current knowledge about outer membrane protein (OMP) and more specifically AT biogenesis and function. We focused on the AT proteins that we haved studied in most detail: i.e. the Yersinia adhesin A (YadA) and invasin of Yersinia enterocolitica (Ye) as well as its homolog intimin (Int) expressed by enteropathogenic Escherichia coli. In addition, this review provides a short outlook about how we could possibly use this knowledge to fight infection.     

Bacterial Cell Surface Structures in Yersinia enterocolitica

Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.     

Mannan-Binding Lectin, L-Ficolin and H-Ficolin Selectively Binds to Different Bacteria

Complement attack is one of the host strategies leading to elimination of pathogens. Yersinia enterocolitica , resistant to human serum, expresses several factors that may contribute to this resistance, the outer membrane proteins YadA and Ail, and lipopolysaccharide (LPS). To study relative roles of these factors for the survival of Y. enterocolitica serotype, O:3 (Ye O:3) in nonimmune human serum, we constructed 23 mutant strains of Ye O:3 expressing different combinations of YadA, Ail, LPS O-antigen and LPS outer core. Survival of bacteria was analysed in normal serum (with functional classical and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single serum resistance factor needed for long-term survival was YadA. Ail was important for short-term survival and thus delayed the bacterial killing. On the contrary, the LPS O-antigen and outer core when in combination with YadA or Ail, or both, had a minor and often negative effect on serum resistance.     

Demarcating SurA Activities Required for Outer Membrane Targeting of Yersinia pseudotuberculosis Adhesins

SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. We have previously reported that in enteropathogenic Yersinia pseudotuberculosis, SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization, and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting and a potentially compromised BAM complex assembly platform in the outer membrane. To various degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA, also depends on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process.     

Attenuated recombinant Yersinia as live oral vaccine carrier to protect against amoebiasis

Various attenuated Yersinia enterocolitica strains expressing different sections of the Entamoeba histolytica surface lectin via the type III protein secretion system (T3SS) were assessed for their use to orally vaccinate rodents against invasive amoebiasis. The T3SS was found to efficiently express and secrete or translocate subfragments as well as the entire heavy subunit of the lectin. Oral vaccination with recombinant Yersinia conferred significant protection against amoebic liver abscess formation when the antigen was expressed as a fusion molecule with the translocation domain of Yersinia outer protein E. However, effectiveness of vaccination was dependent on gender and the rodent species used. Protection was mediated primarily by cellular immune mechanisms as it was independent from the antibody titre against the amoeba lectin but correlated with an antigen-specific Th1-cytokine response. The results suggest that Gram-negative bacteria expressing E. histolytica antigens via T3SS may constitute a suitable oral vaccine carrier against amoebiasis and that an effective IFN-γ response is required for protection against invasive amoebiasis.     

Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3

Complement attack is a host strategy leading to elimination of pathogens. Yersinia enterocolitica expresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival of Y. enterocolitica serotype O:3 in nonimmune human serum, we constructed 23 mutant strains of Y. enterocolitica O:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed.     

Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection

The human-pathogenic species of the Gram-negative genus Yersinia preferentially target and inactivate cells of the innate immune defense, suggesting that this is a critical step by which these bacteria avoid elimination and cause disease. In this study, bacterial interactions with dendritic cells, macrophages, and polymorphonuclear neutrophils (PMNs) in intestinal lymphoid tissues during early Yersinia pseudotuberculosis infection were analyzed. Wild-type bacteria were shown to interact mainly with dendritic cells, but not with PMNs, on day 1 postinfection, while avirulent yopH and yopE mutants interacted with PMNs as well as with dendritic cells. To unravel the role of PMNs during the early phase of infection, we depleted mice of PMNs by using an anti-Ly6G antibody, after which we could see more-efficient initial colonization by the wild-type strain as well as by yopH, yopE, and yopK mutants on day 1 postinfection. Dissemination of yopH, yopE, and yopK mutants from the intestinal compartments to mesenteric lymph nodes was faster in PMN-depleted mice than in undepleted mice, emphasizing the importance of effective targeting of PMNs by these Yersinia outer proteins (Yops). In conclusion, escape from interaction with PMNs due to the action of YopH, YopE, and YopK is a key feature of pathogenic Yersinia species that allows colonization and effective dissemination.     

Type III secretion system-dependent translocation of ectopically expressed Yop effectors into macrophages by intracellular Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.     

Trimeric autotransporter adhesin-dependent adherence of Bartonella henselae, Bartonella quintana, and Yersinia enterocolitica to matrix components and endothelial cells under static and dynamic flow conditions

Trimeric autotransporter adhesins (TAAs) are important virulence factors of Gram-negative bacteria responsible for adherence to extracellular matrix (ECM) and host cells. Here, we analyzed three different TAAs (Bartonella adhesin A [BadA] of Bartonella henselae, variably expressed outer membrane proteins [Vomps] of Bartonella quintana, and Yersinia adhesin A [YadA] of Yersinia enterocolitica) for mediating bacterial adherence to ECM and endothelial cells. Using static (cell culture vials) and dynamic (capillary flow chambers) experimental settings, adherence of wild-type bacteria and of the respective TAA-negative strains was analyzed. Under static conditions, ECM adherence of B. henselae, B. quintana, and Y. enterocolitica was strongly dependent on the expression of their particular TAAs. YadA of Y. enterocolitica did not mediate bacterial binding to plasma or cellular fibronectin under either static or dynamic conditions. TAA-dependent host cell adherence appeared more significant under dynamic conditions although the total number of bound bacteria was diminished compared to the number under static conditions. Dynamic models expand the methodology to perform bacterial adherence experiments under more realistic, bloodstream-like conditions and allow dissection of the biological role of TAAs in ECM and host cell adherence under static and dynamic conditions.     

Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein

Yersinia pestis, the causative agent of plague, must survive in blood in order to cause disease and to be transmitted from host to host by fleas. Members of the Ail/Lom family of outer membrane proteins provide protection from complement-dependent killing for a number of pathogenic bacteria. The Y. pestis KIM genome is predicted to encode four Ail/Lom family proteins. Y. pestis mutants specifically deficient in expression of each of these proteins were constructed using lambda Red-mediated recombination. The Ail outer membrane protein was essential for Y. pestis to resist complement-mediated killing at 26 and 37°C. Ail was expressed at high levels at both 26 and 37°C, but not at 6°C. Expression of Ail in Escherichia coli provided protection from the bactericidal activity of complement. High-level expression of the three other Y. pestis Ail/Lom family proteins (the y1682, y2034, and y2446 proteins) provided no protection against complement-mediated bacterial killing. A Y. pestis ail deletion mutant was rapidly killed by sera obtained from all mammals tested except mouse serum. The role of Ail in infection of mice, Caenorhabditis elegans, and fleas was investigated.     

Two Functional Type VI Secretion Systems in Avian Pathogenic Escherichia coli Are Involved in Different Pathogenic Pathways

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenic Escherichia coli (APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucial clpV clusters of these two T6SS loci and their vgrG genes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines cultured in vitro, decreased pathogenicity in duck and mouse infection models in vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.     

On the role of specific chaperones, the specific ATPase, and the proton motive force in type III secretion

Type III secretion systems (T3SSs) are engaged by a broad number of Gram-negative bacteria to inject proteins into host cells. The simultaneous crossing of three biological membranes by these proteins is a phenomenon which is far from being understood mechanistically. However, recent work from several groups has substantially enriched our conception of how proteins travel from the bacterial into the host cell cytosol. Here, we focus on the role of specific T3SS chaperones, the specific ATPase, and the proton motive force in type III secretion.     

Edwardsiella tarda-Induced Cytotoxicity Depends on Its Type III Secretion System and Flagellin

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection. Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS of E. tarda facilitates its survival and replication in murine bone marrow-derived macrophages, and E. tarda infection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1β in a T3SS-dependent manner. Deletion of the flagellin gene fliC of E. tarda results in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in the fliC mutant strain reduces its virulence. We propose that the host controls E. tarda infection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity.     

Salmonella type III-mediated heterologous antigen delivery: a versatile oral vaccination strategy to induce cellular immunity against infectious agents and tumors

Salmonella type III secretion system (T3SS)-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of antigen-presenting cells leading to prominent CD8 T-cell priming in orally immunized mice. The time point and duration of hybrid protein translocation during the Salmonella infection cycle can be modulated by employing various type III carrier molecules. The p60 protein of Listeria monocytogenes was used as model antigen to construct chimeric SspH2/p60. SspH2 is a “Salmonella pathogenicity island 2” (SPI2) protein that is known to be translocated by Salmonella during intracellular survival and replication in macrophages. This SPI2 carrier molecule is sufficient to induce a concomitant p60-specific CD4 and CD8 T-cell response in Salmonella-vaccinated mice. Moreover, T3SS-mediated antigen delivery results in an efficient priming of central and effector memory CD8 T cells in spleens of these animals. This vaccination strategy can also be employed to efficiently protect mice from an aggressive fibrosarcoma transfected with p60 in a prophylactic setting.     

The Yersinia pestis Ail Protein Mediates Binding and Yop Delivery to Host Cells Required for Plague Virulence

Although adhesion to host cells is a critical step in the delivery of cytotoxic Yop proteins by Yersinia pestis, the mechanism has not been defined. To identify adhesins critical for Yop delivery, we initiated two transposon mutagenesis screens using the mariner transposon. To avoid redundant cell binding activities, we initiated the screen with a strain deleted for two known adhesins, pH 6 antigen and the autotransporter, YapC, as well as the Caf1 capsule, which is known to obscure some adhesins. The mutants that emerged contained insertions within the ail (attachment and invasion locus) gene of Y. pestis. A reconstructed mutant with a single deletion in the ail locus (y1324) was severely defective for delivery of Yops to HEp-2 human epithelial cells and significantly defective for delivery of Yops to THP-1 human monocytes. Specifically, the Yop delivery defect was apparent when cell rounding and translocation of an ELK-tagged YopE derivative into host cells were monitored. Although the ail mutant showed only a modest decrease in cell binding capacity in vitro, the KIM5 Δail mutant exhibited a >3,000-fold-increased 50% lethal dose in mice. Mice infected with the Δail mutant also had 1,000-fold fewer bacteria in their spleens, livers, and lungs 3 days after infection than did those infected with the parental strain, KIM5. Thus, the Ail protein is critical for both Y. pestis type III secretion in vitro and infection in mice.     

The NleE/OspZ family of effector proteins is required for polymorphonuclear transepithelial migration, a characteristic shared by enteropathogenic Escherichia coli and Shigella flexneri infections

Enteropathogenic Escherichia coli (EPEC) and Shigella flexneri are human host-specific pathogens that infect intestinal epithelial cells. However, each bacterial species employs a different infection strategy within this environmental niche. EPEC attaches to the apical surface of small intestine enterocytes, causing microvillus effacement and rearrangement of the host cell cytoskeleton beneath adherent bacteria. In contrast, S. flexneri invades the large intestine epithelium at the basolateral membrane, replicates, and spreads cell to cell. Both EPEC and S. flexneri rely on type three secretion systems (T3SS) to secrete effectors into host cells, and both pathogens recruit polymorphonuclear leukocytes (PMNs) from the submucosa to the lumen of the intestine. In this report, we compared the virulence functions of the EPEC T3SS effector NleE and the homologous Shigella protein Orf212. We discovered that Orf212 was secreted by the S. flexneri T3SS and renamed this protein OspZ. Infection of polarized T84 intestinal epithelial cells with an ospZ deletion mutant of S. flexneri resulted in reduced PMN transepithelial migration compared to infection by the wild type. An nleE deletion mutant of EPEC showed a similar reduction of PMN migration. The ability to induce PMN migration was restored in both mutants when either ospZ or nleE was expressed from a plasmid. An infection of T84 cells with the ΔospZ mutant resulted in reduced extracellular signal-related kinase phosphorylation and NF-κB activation compared to infection with the wild type. Therefore, we conclude that OspZ and NleE have similar roles in the upstream induction of host signaling pathways required for PMN transepithelial migration in Shigella and EPEC infections.