Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN

The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A lcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of lcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.     

Regulation of Salmonella pathogenicity island 2 genes by independent environmental signals

The function of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) is essential for the intracellular lifestyle of Salmonella enterica serovar Typhimurium. Expression of SPI2 genes is induced within the Salmonella-containing vacuole (SCV) inside host cells and is controlled by the SsrAB two-component system. However, the nature of the signals leading to expression of SPI2 genes is controversial. Here we report that expression of SPI2 genes can be induced independently by two different environmental stimuli. Exposure of bacteria to slightly acidic pH was sufficient to induce a rapid up-regulation of SPI2 genes. In contrast, limitation of inorganic phosphate (P(i)) in the growth media led to activation of SPI2 genes in the late exponential growth phase and was independent of the media pH. Limitation of P(i) induced an over-expression of sensor protein SsrB. Response to both environmental stimuli required a functional SsrAB system. In vivo analyses indicated that limitation of P(i) is also encountered by intracellular Salmonella. Our observations allow the integration of previous disparate reports on the induction of SPI2 genes.     

Polarisation of type III translocation by Pseudomonas aeruginosa requires PcrG, PcrV and PopN

Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell. We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PcrV, PopB and PopD. To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants. In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased. When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium. This confirms that ExoS translocation by P. aeruginosa occurs by a polarised mechanism. In contrast, high levels of ExoS were recovered in the tissue culture medium when cells were infected with pcrG, pcrV and popN mutants. Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions. This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells.     

Investigating the assembly of the bacterial type III secretion system injectisome by in vivo photocrosslinking

Virulence-associated type III secretion systems serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection, hence type III secretion systems are often essential bacterial pathogenicity factors. The core of type III secretion systems is a cell envelope-spanning macromolecular machine called injectisome. It consists of almost twenty different components in a stoichiometry of one to more than one hundred. Assembly of this 6 MDa complex requires the coordinated integration of components from the cytoplasm, the inner membrane, the periplasm, the outer membrane and even the extracellular space of Gram-negative bacteria. Here, we review injectisome assembly with an emphasis on the techniques that were employed towards its investigation. In particular, we focus on in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways.     

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.     

Biotechnological applications of bacterial protein secretion: from therapeutics to biofuel production

Recent years have witnessed significant progresses in engineering of recombinant protein secretion. The relatively simple secretion mechanisms, Type I and Type V (autotransporters), are increasingly used for secretion of recombinant proteins. The secretion level of target proteins varied from milligrams to grams per liter. The range of proteins was significantly expanded beyond medical application. Notable additions include biofuel productions from renewable feedstock. Despite the progress, almost all successes in the engineering efforts come with significant trials and errors, highlighting the need for a better understanding of secretion systems and rational based methods.     

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.     

Bacterial type III translocation: a unique mechanism for cytosolic display of heterologous antigens by attenuated Salmonella

Upon infection, Gram-negative animal and plant pathogens evade the host immune response by utilizing a specialized protein secretion machinery, known as type III secretion system, for the export of bacterial virulence factors delivered directly into the cytosol of target cells. This unique translocation mechanism can be used for the delivery of large protein fragments derived from immunodominant viral and bacterial heterologous antigens into the MHC class I-restricted antigen-processing pathway by attenuated Salmonella carrier vaccines. In orally immunized mice, this novel vaccination strategy results in the induction of pronounced peptide-specific cytotoxic CD8 T cell responses.     

Induction of type III secretion by cell-free Chlamydia trachomatis elementary bodies

Chlamydiae secrete type III effector proteins at two distinct stages of their developmental cycle. Elementary bodies (EBs) secrete at least one pre-formed effector protein, Tarp, across the host plasma membrane from an extracellular location. Once internalized, a set of newly transcribed proteins are secreted to modify the inclusion membrane. In an effort to better understand the triggers for chlamydial type III secretion and develop means to identify new effectors, we investigated various inducers of T3SS in other Gram-negative bacterial systems to determine if they were able to activate chlamydial type III secretion from EBs using Tarp secretion as an indicator of activation. Chlamydial EBs are induced to secrete Tarp by exposure to FBS, BSA, or sphingolipid and cholesterol-rich liposomes (SCRLs). The induction by FBS and BSA, but not SCRL, is enhanced in the presence of the calcium-chelator, EGTA. This secretion was temperature dependent and inhibited by paraformaldehyde fixation of the EBs.     

Delivery of dangerous goods: type III secretion in enteric pathogens

Type III secretion systems (TTSSs) of Gram-negative pathogens are molecular syringes that inject bacterial virulence factors directly into host cells. These virulence factors manipulate host cell pathways to aid bacterial survival within the host. Four important enteric pathogens use TTSSs to colonize and persist within the intestinal environment. The following is a brief review of the way in which TTSSs and their effectors contribute to the pathogenic nature of the prototypic diarrheal pathogens Salmonella, Shigella, Yersinia and enteropathogenic Escherichia coli (EPEC).     

Structure-based functional analysis of effector protein SifA in living cells reveals motifs important for Salmonella intracellular proliferation

The facultative intracellular pathogen Salmonella enterica survives and replicates inside the Salmonella-containing vacuole (SCV) of mammalian host cells. SifA is a key effector protein translocated by a type III secretion system and involved in formation of Salmonella-induced filaments (SIF), extensive tubular endosomal compartments. Recruitment of LAMP1 (lysosomal-associated membrane protein 1)-positive membranes to SIF ensures integrity and dynamics of the membrane network. The binding of SifA to the host protein SKIP (SifA and kinesin interacting protein) was proposed as crucial for this function. Due to structural mimicry SifA has further been proposed to interact with G-proteins. We conducted a mutational study of SifA to identify domains and amino acid residues specifically relevant for intracellular replication and SIF formation. Mutations were designed based on the available structural data of SifA and its interface with SKIP, or modeled for SifA as putative guanine nucleotide exchange factor. We developed a live cell imaging-based approach for volume quantification of the SIF network that allowed determination of subtle changes in SIF network and performed a comprehensive analysis of mutant forms of SifA by this approach. We found that the SifA catalytic loop of WxxxE effectors is as important for SIF formation and intracellular proliferation as the SKIP interaction motif, or the CAAX motif for membrane anchoring of SifA.     

Type IV pili are not specifically required for contact dependent translocation of exoenzymes by Pseudomonas aeruginosa

The type III secretion system (TTSS) of the opportunistic pathogen Pseudomonas aeruginosa enables the bacterium to deliver exoenzymes directly into the eukaryotic cell. In this study we have investigated the role of key factors involved in this process. We could demonstrate that the translocators PopB, PopD and PcrV are absolutely required for delivery of Exoenzyme S into host cells. By analyzing different Tfp (type IV pili) mutants we could establish a correlation between the frequency of bacteria binding to the host cell and the levels of translocated ExoS, thereby verifying that the process is contact dependent. However, there was no absolute requirement for the Tfp per se, since the pilus could be substituted with a different type of adhesin, the non-fimbrial adhesin pH6 antigen of Yersinia pestis. Taken together, our results demonstrate that binding to establish close contact between the type III secretion organelle and the host cell is essential for translocation, while the additional activities of Tfp are not essential for the delivery of TTSS proteins.     

The role of the inflammasome in cellular responses to toxins and bacterial effectors

Invading pathogens are recognized by mammalian cells through dedicated receptors found either at the cell surface or in the cytoplasm. These receptors, like the trans-membrane Toll-like Receptors (TLR) or the cytosolic Nod-like Receptors (NLR), initiate innate immunity after recognition of molecular patterns found in bacteria or viruses, such as LPS, flagellin, or double-stranded RNA. Recognition of molecules produced only by a specific pathogen, such as a viral envelop protein or a bacterial adhesin does not appear to occur. Bacterial protein toxins, however, might compose an intermediate class. Considering the diversity of toxins in terms of structure, it is unlikely that cells respond to them via specific molecular recognition. It rather appears that different classes of toxins trigger cellular changes that are sensed by the cells as danger signals, such as changes in cellular ion composition after membrane perforation by pore-forming toxins or type III secretion systems. The signaling pathways triggered through toxin-induced cell alterations will likely play a role in modulating host responses to virulent bacteria. We will here describe the few studied cases in which detection of the toxin by the host cell was addressed. The review will include not only toxins but also bacteria effectors secreted by the bacterium in to the host cell cytoplasm.     

A genotypic and phenotypic comparison of type III secretion profiles of Pseudomonas aeruginosa cystic fibrosis and bacteremia isolates

The type III secretion system (TTSS) of Pseudomonas aeruginosa enables delivery of a number of toxins involved in the disruption of eukaryotic epithelial surfaces. Whilst the ability to secrete ExoS facilitates invasion and internalization, the secretion of ExoU mediates acute cytotoxicity. In order to determine any association with the ability to secrete these toxins with the nature and severity of human infection, the TTSS genotypes and phenotypes of 163 clinical isolates were determined by multiplex PCR and Western blotting. An exoS+/exoU- genotype was associated with chronic infection in patients with cystic fibrosis whilst an exoS-/exoU+ genotype was associated with strains isolated from blood. Secretion of the ExoU protein was more commonly seen in isolates obtained from blood, suggesting this ability may be important in the development of acute invasive infection. Detection of TTSS toxins in clinical material may be useful in targeting antimicrobial therapy or identifying individuals infected with aggressive strains of P. aeruginosa.     

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.     

The Aeromonas dsbA mutation decreased their virulence by triggering type III secretion system but not flagella production

Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Dsb (Disulfide bond) proteins play an important role in catalyzing disulfide bond formation in proteins within the periplasmic space. An A. hydrophila dsbA mutant with attenuated virulence using Dictyostelium amoebae as an alternative host model was identified. The attenuated virulence was tested in other animal models (by intraperitoneal injection in fish and mice) and was correlated with the presence of a defective type III secretion system for the first time in non enteric bacteria. The dsbA mutation was shown in several enteric bacteria to involve the outer membrane secretin. The defect in Aeromonas also seems to involve the outer membrane secretin homologue named AscC. However, unlike what happen in Escherichia coli, no changes in motility or flagella expression were observed for A. hydrophila dsbA mutants. The loss of E. coli motility caused by deletion of dsbA is likely due to defective disulfide bond formation in FlgI, a component of the flagella. No disulfide bond formation in FlgI homologues in Aeromonas flagella biogenesis, either polar or lateral, could be expected according to their amino acid residues sequences.