Interleukin-10 induction is an important virulence function of the Yersinia pseudotuberculosis type III effector YopM

Pathogenic Yersinia species modulate host immune responses through the activity of a plasmid-encoded type III secretion system and its associated effector proteins. One effector, YopM, is a leucine-rich-repeat-containing protein that is important for virulence in murine models of Yersinia infection. Although the mechanism by which YopM promotes virulence is unknown, we previously demonstrated that YopM was required for the induction of high levels of the immunosuppressive cytokine interleukin-10 (IL-10) in sera of C57BL/6J mice infected with Yersinia pseudotuberculosis. To determine if IL-10 production is important for the virulence function of YopM, C57BL/6J or congenic IL-10^−/− mice were infected intravenously with wild-type or yopM mutant Y. pseudotuberculosis strains. Analysis of cytokine levels in serum and bacterial colonization in the spleen and liver showed that YopM is required for IL-10 induction in C57BL/6J mice infected with either the IP32953 or the 32777 strain of Y. pseudotuberculosis, demonstrating that the phenotype is conserved in the species. In single-strain infections, the ability of the 32777ΔyopM mutant to colonize the liver was significantly increased by the delivery of exogenous IL-10 to C57BL/6J mice. In mixed infections, the competitive advantage of a yopM⁺ 32777 strain over an isogenic yopM mutant to colonize spleen and liver, as observed for C57BL/6J mice, was significantly reduced in IL-10^−/− animals. Thus, by experimentally controlling IL-10 levels in a mouse infection model, we obtained evidence that the induction of this cytokine is an important mechanism by which YopM contributes to Y. pseudotuberculosis virulence.     

Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

Pathogenic Yersinia species utilize a type III secretion system to translocate Yop effectors into infected host cells. Yop effectors inhibit innate immune responses in infected macrophages to promote Yersinia pathogenesis. In turn, Yersinia-infected macrophages respond to translocation of Yops by activating caspase-1, but different mechanisms of caspase-1 activation occur, depending on the bacterial genotype and the state of phagocyte activation. In macrophages activated with lipopolysaccharide (LPS) prior to Yersinia pseudotuberculosis infection, caspase-1 is activated by a rapid inflammasome-dependent mechanism that is inhibited by translocated YopM. The possibility that other effectors cooperate with YopM to inhibit caspase-1 activation in LPS-activated macrophages has not been investigated. Toward this aim, epistasis analysis was carried out in which the phenotype of a Y. pseudotuberculosis yopM mutant was compared to that of a yopJ yopM, yopE yopM, yopH yopM, yopT yopM, or ypkA yopM mutant. Activation of caspase-1 was measured by cleavage of the enzyme, release of interleukin-1β (IL-1β), and pyroptosis in LPS-activated macrophages infected with wild-type or mutant Y. pseudotuberculosis strains. Results show enhanced activation of caspase-1 after infection with the yopJ yopM mutant relative to infection by any other single or double mutant. Similar results were obtained with the yopJ, yopM, and yopJ yopM mutants of Yersinia pestis. Following intravenous infection of mice, the Y. pseudotuberculosis yopJ mutant was as virulent as the wild type, while the yopJ yopM mutant was significantly more attenuated than the yopM mutant. In summary, through epistasis analysis this work uncovered an important role for YopJ in inhibiting caspase-1 in activated macrophages and in promoting Yersinia virulence.     

Molecular analysis of a novel Toll/interleukin-1 receptor (TIR)-domain containing virulence protein of Y. pseudotuberculosis among Far East scarlet-like fever serotype I strains

Pathogenicity of Yersinia pseudotuberculosis is determined by an arsenal of virulence factors. Particularly, the Yersinia outer proteins (Yops) and the Type III secretion system (T3SS) encoded on the pYV virulence plasmid are required for Yersinia pathogenicity. A specific group of Y. pseudotuberculosis, responsible for the clinical syndrome described as Far East scarlet-like fever (FESLF), is known to have an altered virulence gene cluster. Far East strains cause unique clinical symptoms for which the pYV virulence plasmid plays apparently a rather secondary role. Here, we characterize a previously unknown protein of Y. pseudotuberculosis serotype I strains (TcpYI) which can be found particularly among the FESLF strain group. The TcpYI protein shares considerable sequence homology to members of the Toll/IL-1 receptor family. Bacterial TIR domain containing proteins (Tcps) interact with the innate immune system by TIR-TIR interactions and subvert host defenses via individual, multifaceted mechanisms. In terms of virulence, it appears that the TcpYI protein of Y. pseudotuberculosis displays its own virulence phenotype compared to the previously characterized bacterial Tcps. Our results clearly demonstrate that TcpYI increases the intracellular survival of the respective strains in vitro. Furthermore, we show here that the intracellular survival benefit of the wild-type strain correlates with an increase in tcpYI gene expression inside murine macrophages. In support of this, we found that TcpYI enhances the survival inside the spleens of mice in a mouse model of peritonitis. Our results may point toward involvement of the TcpYI protein in inhibition of phagocytosis, particularly in distinct Y. pseudotuberculosis strains of the FESLF strain group where the pYV virulence plasmid is absent.     

Identification of the psaA Gene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other than Streptococcus pneumoniae

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.     

The Yersinia pseudotuberculosis YplA phospholipase differs in its activity,regulation and secretion from that of the Yersinia enterocolitica YplA

Analysis of the Yersinia pseudotuberculosis and Yersinia pestis genomes indicates that both species carry an identical copy of a gene that is predicted to encode a protein which shares 80% similarity to the Yersinia enterocolitica YplA, a secreted phospholipase that has been shown to contribute to virulence. In contrast to well tolerated production of the Y. enterocolitica YplA in Escherichia coli, Y. pseudotuberculosis YplA expression was found to be toxic; however, cell viability could be restored if the Y. pseudotuberculosis YplA was expressed in the presence of its accessory protein YplB. In vitro, Y. pseudotuberculosis YplB was shown to reduce the activity of its cognate phospholipase in a dose-dependent manner. To determine whether the Y. pseudotuberculosis and Y. enterocolitica YplAs were secreted and regulated in a similar manner, secretion and promoter activity assays were performed. Unlike the situation apparent in Y. enterocolitica, expression of the Y. pseudotuberculosis yplA gene did not appear to be controlled by the flagellar regulon, nor did the phospholipase appear to be efficiently exported through the flagellar apparatus. These results indicate that the Yersinia YplAs vary in many of their attributes despite their high degree of amino acid homology.     

Characterization of chromosomal regions conserved in Yersinia pseudotuberculosis and lost by Yersinia pestis

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37°C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37°C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.     

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.     

Bacterial conjugation in the cytoplasm of mouse cells

Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.     

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of P_tetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.     

Structural organization of intact and damaged by <Emphasis Type="Italic">IS</Emphasis>100 element porin genes adjacent to the PGM region in <Emphasis Type="Italic">Yersinia pestis</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> strains

The porin gene, which is adjacent to the pigmentation region (pgm), is usually damaged by IS100 element in highly virulent Yersinia pestis strains. In addition, the pgm region, which carries the genes responsible for virulence (high pathogenicity island) and biofilm generation (hms-operon), is flanked by direct IS100 copies (causing its destabilization). The study of distribution of intact and truncated porin genes was conducted among 240 Y. pestis strains from 39 natural foci of Russia and countries of the near abroad and 68 Yersinia pseudotuberculosis strains from different geographical regions. Most highly virulent Y. pestis strains and some phylogenetic Y. pseudotuberculosis lines of O:1 serotype contain truncated porin genes. At the same time, deletion of the pgm region by flanked IS100 in Y. pseudotuberculosis is impossible, since IS100 is integrated in the porin gene in an orientation opposite to that of Y. pestis. The intact porin gene is carried by all Y. pestis strains with low epidemic significance and certain phylogenetic lines of highly virulent Y. pestis strains from desert foci and Caspian sandy focus, as well as most Y. pseudotuberculosis strains of O:1 serotype. A continuous deletion, which includes the porin gene and a part of the astE gene, was detected in less virulent Y. pseudotuberculosis strains of O:3 serotype. The nucleotide sequence of porin genes is identical in Y. pestis and Y. pseudotuberculosis strains from different geographical regions. Three porin gene allele only differ by IS100 integration site and orientation or absence of its integration. The nucleotide sequence of IS100 introduced in the porin gene of Yersinia has small differences only for two Y. pestis strains isolated in America. The correlation of low frequency of Hms-mutants with the intact porin gene state in Y. pestis and the absence of such a correlation in Y. pseudotuberculosis were established.     

Vaccine Potential of Attenuated Mutants of Corynebacterium pseudotuberculosis in Sheep

Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-γ)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-γ-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.     

In situ transduction of stromal cells and thymocytes upon intrathymic injection of lentiviral vectors

Background

Congenic strains of mice are assumed to differ only at a single gene or region of the genome. These mice have great importance in evaluating the function of genes. However, their utility depends on the maintenance of this true congenic nature. Although, accumulating evidence suggests that congenic strains suffer genetic divergence that could compromise interpretation of experimental results, this problem is usually ignored. During coinfection studies with Salmonella typhimurium and Theiler's murine encephalomyelitis virus (TMEV) in major histocompatibility complex (MHC)-congenic mice, we conducted the proper F₂ controls and discovered significant differences between these F₂ animals and MHC-genotype-matched P₀ and F₁ animals in weight gain and pathogen load. To systematically evaluate the apparent non-MHC differences in these mice, we infected all three generations (P₀, F₁ and F₂) for 5 MHC genotypes (b/b, b/q and q/q as well as d/d, d/q, and q/q) with Salmonella and TMEV.

Results

Infected P₀ MHC q/q congenic homozygotes lost significantly more weight (p = 0.02) and had significantly higher Salmonella (p < 0.01) and TMEV (p = 0.02) titers than the infected F₂ q/q homozygotes. Neither weight nor pathogen load differences were present in sham-infected controls.

Conclusions

These data suggest that these strains differ for genes other than those in the MHC congenic region. The most likely explanation is that deleterious recessive mutations affecting response to infection have accumulated in the more than 40 years that this B10.Q-H-2 ^q MHC-congenic strain has been separated from its B10-H-2 ^b parental strain. During typical experiments with congenic strains, the phenotypes of these accumulated mutations will be falsely ascribed to the congenic gene(s). This problem likely affects any strains separated for appreciable time and while usually ignored, can be avoided with the use of F₂ segregants.     

Evaluation of the role of constitutive isocitrate lyase activity in Yersinia pestis infection of the flea vector and mammalian host

Yersinia pestis, unlike the closely related Yersinia pseudotuberculosis, constitutively produces isocitrate lyase (ICL). Here we show that the Y. pestis aceA homologue encodes ICL and is required for growth on acetate but not for flea infection or virulence in mice. Thus, deregulation of the glyoxylate pathway does not underlie the recent adaptation of Y. pestis to arthropod-borne transmission.     

Influence of the Salmonella typhimurium Pathogenicity Island 2 Type III Secretion System on Bacterial Growth in the Mouse

We have investigated the in vivo growth kinetics of a Salmonella typhimurium strain (P11D10) carrying a mutation in ssaJ, a Salmonella pathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice. Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route. Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared. Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not. Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo. At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo. The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in the Salmonella virulence plasmid spv genes. To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 and spv mutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation. These experiments failed to provide any evidence showing that the SPI2 and spv gene products interact together as part of the same virulence mechanism.     

Salmonella enterica serovar Typhi plasmid pRST98-mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts

pR_ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR_ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST₈ carrying pR_ST98, Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST₁₀ without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST₈ containing pR_ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR_ST98-mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR_ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR_ST98-mediated virulence in S. typhi.     

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.     

The luxS gene functions in the pathogenesis of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis, the most significant infectious bacterial disease of poultry worldwide. LuxS, the product of the luxS gene, mediates the quorum sensing (QS) mechanism. This involves the production of autoinducer-2 (AI-2), which regulates important physiological traits and a variety of adaptive processes in different bacteria. In this study, a luxS gene deleted APEC mutant strain, ΔDE17, was constructed using strain DE17. Analysis of bioluminescence indicated that deletion of the luxS gene abolished the production of the QS signal AI-2 in the bacteria. Further studies showed that deletion of the luxS gene in DE17 reduced the bacterial virulence by 31.5-fold in ducklings, based on the measurement of the 50% lethal dose. The mutant strain reduced significantly the abilities of adherence and invasion, by 50.0% and 40.7% respectively, compared with the wild strain DE17. The mutant strain also showed reduced survival in vivo: the bacterial loads of the mutant strain in infected liver, spleen and blood were 46.4-fold, 5.2-fold, and 3.7-fold reduced, respectively, compared with the wild-type strain DE17. Real-time polymerase chain reaction (PCR) demonstrated further that the mRNA levels of the virulence-related genes iucD, fyuA, vat, ompA, iss, fimC and tsh were significantly decreased in the mutant strain ΔDE17, when compared with DE17 (p < 0.05). In addition, the deletion of the luxS gene reduced the motility of the bacterium. This study suggests that the luxS gene functions in the pathogenesis of diseases caused by avian pathogenic E. coli.