Innate Immune Recognition of Yersinia pseudotuberculosis Type III Secretion

Specialized protein translocation systems are used by many bacterial pathogens to deliver effector proteins into host cells that interfere with normal cellular functions. How the host immune system recognizes and responds to this intrusive event is not understood. To address these questions, we determined the mammalian cellular response to the virulence-associated type III secretion system (T3SS) of the human pathogen Yersinia pseudotuberculosis. We found that macrophages devoid of Toll-like receptor (TLR) signaling regulate expression of 266 genes following recognition of the Y. pseudotuberculosis T3SS. This analysis revealed two temporally distinct responses that could be separated into activation of NFκB- and type I IFN-regulated genes. Extracellular bacteria were capable of triggering these signaling events, as inhibition of bacterial uptake had no effect on the ensuing innate immune response. The cytosolic peptidoglycan sensors Nod1 and Nod2 and the inflammasome component caspase-1 were not involved in NFκB activation following recognition of the Y. pseudotuberculosis T3SS. However, caspase-1 was required for secretion of the inflammatory cytokine IL-1β in response to T3SS-positive Y. pseudotuberculosis. In order to characterize the bacterial requirements for induction of this novel TLR-, Nod1/2-, and caspase-1-independent response, we used Y. pseudotuberculosis strains lacking specific components of the T3SS. Formation of a functional T3SS pore was required, as bacteria expressing a secretion needle, but lacking the pore-forming proteins YopB or YopD, did not trigger these signaling events. However, nonspecific membrane disruption could not recapitulate the NFκB signaling triggered by Y. pseudotuberculosis expressing a functional T3SS pore. Although host cell recognition of the T3SS did not require known translocated substrates, the ensuing response could be modulated by effectors such as YopJ and YopT, as YopT amplified the response, while YopJ dampened it. Collectively, these data suggest that combined recognition of the T3SS pore and YopBD-mediated delivery of immune activating ligands into the host cytosol informs the host cell of pathogenic challenge. This leads to a unique, multifactorial response distinct from the canonical immune response to a bacterium lacking a T3SS.     

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscU_CC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca²⁺-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscU_CC encompasses a specific C-terminal T3S signal within the 15 last residues (U₁₅). U₁₅ promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the “classical” N-terminal secretion signal, U₁₅ interacted with the ATPase YscN. Although U₁₅ is critical for YscU_CC secretion, deletion of the C-terminal secretion signal of YscU_CC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion.     

YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an α-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.     

Expression of a Yersinia pseudotuberculosis Type VI Secretion System Is Responsive to Envelope Stresses through the OmpR Transcriptional Activator

The Type VI secretion system (T6SS) is a macromolecular complex widespread in Gram-negative bacteria. Although several T6SS are required for virulence towards host models, most are necessary to eliminate competitor bacteria. Other functions, such as resistance to amoeba predation, biofilm formation or adaptation to environmental conditions have also been reported. This multitude of functions is reflected by the large repertoire of regulatory mechanisms shown to control T6SS expression, production or activation. Here, we demonstrate that one T6SS gene cluster encoded within the Yersinia pseudotuberculosis genome, T6SS-4, is regulated by OmpR, the response regulator of the two-component system EnvZ-OmpR. We first identified OmpR in a transposon mutagenesis screen. OmpR does not control the expression of the four other Y. pseudotuberculosis T6SS gene clusters and of an isolated vgrG gene, and responds to osmotic stresses to bind to and activate the T6SS-4 promoter. Finally, we show that T6SS-4 promotes Y. pseudotuberculosis survival in high osmolarity conditions and resistance to deoxycholate.     

Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU_CC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca²⁺ conditions) and calcium-depleted medium (−Ca²⁺ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca²⁺-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca²⁺ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca²⁺ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab''s group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhB_TM. The hydrophilic C-terminal domain (FlhB_C) is predicted to protrude into the cytosol. In addition, FlhB_C can be further divided into two subdomains, FlhB_CN (amino acids 211 to 269) and FlhB_CC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhB_C is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscU_CC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscU_C) can be divided into the YscU_CN and YscU_CC subdomains (Fig. ​(Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscU_CC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.Open in a separate windowFIG. 1.Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed at the site http://www.cbs.dtu.dk/services/TMHMM. IM, inner membrane. (B) E. coli expressing C-terminally His-tagged YscU_C was induced with IPTG, which was followed by sonication and solubilization and denaturation of the protein in binding buffer (8 M urea and 10 mM imidazole). The lysate (lane L) was flushed over the Ni column, and the flowthrough (lane FT) was collected. The column was washed five times with binding buffer, and the wash fractions (lanes W1 to W5) were collected. Elution buffer (8 M urea and 300 mM imidazole) was flushed over the column to release proteins bound to the column, resulting in the eluate (lane E). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain a urea concentration of 0.2 M and incubated at 21°C overnight. The resulting overnight eluate fraction (lane E/ON) was TCA precipitated and taken up in binding buffer. Samples were analyzed by 15% Tris-Tricine SDS-PAGE. The cleavage of YscU_C-His₆ to YscU_CC-His₆ and YscU_CN was verified by N-terminal sequencing. All fractions were volume corrected. Lane ST contained a protein standard.     

PCR Detection of Virulence Genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and Investigation of Virulence Gene Distribution

PCR-based assays were developed for the detection of plasmid- and chromosome-borne virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis, to investigate the distribution of these genes in isolates from various sources. The results of PCR genotyping, based on 5 virulence-associated genes of 140 strains of Y. enterocolitica, were compared to phenotypic tests, such as biotyping and serotyping, and to virulence plasmid-associated properties such as calcium-dependent growth at 37°C and Congo red uptake. The specificity of the PCR results was validated by hybridization. Genotyping data correlated well with biotype data, and most biotypes resulted in (nearly) homogeneous genotypes for the chromosomal virulence genes (ystA, ystB, and ail); however, plasmid-borne genes (yadA and virF) were detected with variable efficiency, due to heterogeneity within the bacterial population for the presence of the virulence plasmid. Of the virulence genes, only ystB was present in biotype 1A; however, within this biotype, pathogenic and apathogenic isolates could not be distinguished based on the detection of virulence genes. Forty Y. pseudotuberculosis isolates were tested by PCR for the presence of inv, yadA, and lcrF. All isolates were inv positive, and 88% of the isolates contained the virulence plasmid genes yadA and lcrF. In conclusion, this study shows that genotyping of Yersinia spp., based on both chromosome- and plasmid-borne virulence genes, is feasible and informative and can provide a rapid and reliable genotypic characterization of field isolates.     

Adhesion of Yersinia pseudotuberculosis serotype III

The use of the erythrocyte agglutination test for characterizing the properties of Y. pseudotuberculosis led to the detection of a highly adhesive strain belonging to serotype III and capable of forming pili at 37 degrees C. The adhesion of the cells provided with pili was completely inhibited by the mixture of gangliosides, specific antibodies, and the preliminary treatment of erythrocytes with neuraminidases sharply enhanced the effectiveness of adhesion. The adhesion pili consisted of protein subunits with a molecular weight of 16 800 daltons and had the isoionic point at pH 4.1.     

The Resveratrol Tetramer (-)-Hopeaphenol Inhibits Type III Secretion in the Gram-Negative Pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa

Society faces huge challenges, as a large number of bacteria have developed resistance towards many or all of the antibiotics currently available. Novel strategies that can help solve this problem are urgently needed. One such strategy is to target bacterial virulence, the ability to cause disease e.g., by inhibition of type III secretion systems (T3SSs) utilized by many clinically relevant gram-negative pathogens. Many of the antibiotics used today originate from natural sources. In contrast, most virulence-blocking compounds towards the T3SS identified so far are small organic molecules. A recent high-throughput screening of a prefractionated natural product library identified the resveratrol tetramer (-)-hopeaphenol as an inhibitor of the T3SS in Yersinia pseudotuberculosis. In this study we have investigated the virulence blocking properties of (-)-hopeaphenol in three different gram-negative bacteria. (-)-Hopeaphenol was found to have micromolar activity towards the T3SSs in Yersinia pseudotuberculosis and Pseudomonas aeruginosa in cell-based infection models. In addition (-)-hopeaphenol reduced cell entry and subsequent intracellular growth of Chlamydia trachomatis.     

Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains

Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.The genus Pectobacterium (formerly Erwinia) contains both narrow- and broad-host-range bacterial plant pathogens that cause soft rot, stem rot, wilt, and blackleg in species belonging to over 35% of plant orders (20). Four Pectobacterium species have been described: Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium carotovorum, and Pectobacterium wasabiae (9). The recently described organism P. carotovorum subsp. brasiliensis is genetically distinct from previously described Pectobacterium taxa; approximately 82% of its genes are shared with P. atrosepticum, and 84% of its genes are shared with P. carotovorum subsp. carotovorum, while 13% of its genes are found in neither P. atrosepticum nor P. carotovorum subsp. carotovorum (7, 10, 20). To date, only P. carotovorum subsp. carotovorum and P. atrosepticum have been reported to occur in the same field (14, 21). P. carotovorum subsp. carotovorum is found worldwide, and P. atrosepticum is found in cool climates; while P. carotovorum subsp. brasiliensis has been found only in Brazil, Israel, and the United States, it is likely to have a wider distribution (20). Compared to the ecology and genetics of P. carotovorum subsp. carotovorum and P. atrosepticum, little is known about the ecology and genetics of P. betavasculorum, P. wasabiae, or P. carotovorum subsp. brasiliensis.Pectobacterium strains isolated from potato are diverse based on serology, genome structure, and fatty acid composition (5, 35). Previous epidemiological studies of pectolytic Enterobacteriaceae were complicated by the diversity of this group and the lack of tools capable of placing all isolates into clades. For example, Gross et al. (14) were unable to classify over 50% of Pectobacterium isolates obtained from potato, and Pitman et al. (23) were unable to type 13% of their isolates. Novel PCR-based methods potentially capable of classifying all Pectobacterium isolates have been described, but they were developed prior to the recognition of P. carotovorum subsp. brasiliensis (1, 34).The main virulence determinants of Pectobacterium are the pectolytic enzymes secreted through the type II secretion system. Although these enzymes are required for development of symptoms, many other virulence genes have been shown to contribute to Pectobacterium pathogenicity, including the type III secretion system (T3SS) genes, the cfa gene cluster, and the type IV secretion system genes (3, 15, 19). Recent genomic analysis showed that some of these gene clusters, such as the cfa and type IV secretion system cluster genes, as well as genes important for interactions with insects, are present in only some Pectobacterium species (10). Thus, Pectobacterium species appear to use different genetic tools to overcome plant host barriers and to interact with insect vectors.Many gram-negative pathogenic bacteria secrete virulence proteins, known as effectors, through the T3SS into host cells. Once inside host cells, the effectors manipulate host defenses and promote bacterial growth (13). Unlike many other gram-negative plant pathogens, Pectobacterium does not require the T3SS for pathogenicity. Rather, this secretion system makes a small, but measurable, contribution to the early stages of P. carotovorum growth in leaves of the model plant Arabidopsis thaliana (26) and contributes to the virulence of P. atrosepticum on potato (15). Recently, we isolated Pectobacterium strains that lack the T3SS from potatoes and also found P. wasabiae and P. carotovorum subsp. brasiliensis on potatoes in Wisconsin (35). The first goal of this study was to determine if P. wasabiae and P. carotovorum subsp. brasiliensis are common in agricultural fields or if soft rot disease is typically caused by P. carotovorum subsp. carotovorum and P. atrosepticum, which have been the focus of nearly all previous studies of potato soft rot, stem rot, and blackleg disease. Second, since we recently isolated a strain lacking the T3SS (35), we also aimed to determine if strains lacking the T3SS are common in infected potatoes and if these strains tend to be less virulent on potato stems and tubers than strains encoding a T3SS.     

Type 3 Secretion System Cluster 3 Is a Critical Virulence Determinant for Lung-Specific Melioidosis

Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD₅₀ of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD₅₀ using IMIT. We subsequently examined the LD₅₀ of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system uniquely capable to characterize the fate of lung-delivered pathogen.     

The Type IV Fimbrial Subunit Gene (fimA) of Dichelobacter nodosus Is Essential for Virulence, Protease Secretion, and Natural Competence

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Virulence of Yersinia pseudotuberculosis isolated from pork and from the throats of swine

Yersinia pseudotuberculosis was isolated from retail pork and from healthy swine throats. These wild-type strains and their representative cured isogenic strains were tested for the presence of plasmids and several virulence factors, and these characteristics were compared with those of virulent strains from humans. Two pork isolates (serotype IVB) and four swine isolates (serotypes IIB, IIC, III, and IVB) harbored a 42- to 48-megadalton plasmid which had similar fragmentation patterns resulting from digestion with restriction endonuclease. These six strains were lethal for mice via oral challenge and were positive in autoagglutination and calcium dependency tests. They also invaded HeLa cells and induced cytotoxicity. Histopathological examination and indirect fluorescent-antibody staining provided definite evidence of the pathogenicity of these strains when tissue sections from orally infected mice were used. The virulence factors of wild-type pork and swine isolates with the 42- to 48-megadalton plasmid were identical to those of two human isolates (serotypes IVB and VB). Hence, these pork and swine isolates should be considered potentially pathogenic for humans. The finding suggests that retail pork and swine may play an important role in the epidemiology of human infections caused by Y. pseudotuberculosis.     

Random Mutagenesis Identifies a C-Terminal Region of YopD Important for Yersinia Type III Secretion Function

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168–203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.     

Diversity of Virulence Phenotypes among Type III Secretion Negative Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease.