Plasmacytoid Dendritic Cells Are Crucial in Bifidobacterium adolescentis-Mediated Inhibition of Yersinia enterocolitica Infection

In industrialized countries bacterial intestinal infections are commonly caused by enteropathogenic Enterobacteriaceae. The interaction of the microbiota with the host immune system determines the adequacy of an appropriate response against pathogens. In this study we addressed whether the probiotic Bifidobacterium adolescentis is protective during intestinal Yersinia enterocolitica infection. Female C57BL/6 mice were fed with B. adolescentis, infected with Yersinia enterocolitica, or B. adolescentis fed and subsequently infected with Yersinia enterocolitica. B. adolescentis fed and Yersinia infected mice were protected from Yersinia infection as indicated by a significantly reduced weight loss and splenic Yersinia load when compared to Yersinia infected mice. Moreover, protection from infection was associated with increased intestinal plasmacytoid dendritic cell and regulatory T-cell frequencies. Plasmacytoid dendritic cell function was investigated using depletion experiments by injecting B. adolescentis fed, Yersinia infected C57BL/6 mice with anti-mouse PDCA-1 antibody, to deplete plasmacytoid dendritic cells, or respective isotype control. The B. adolescentis-mediated protection from Yersinia dissemination to the spleen was abrogated after plasmacytoid dendritic cell depletion indicating a crucial function for pDC in control of intestinal Yersinia infection. We suggest that feeding of B. adolescentis modulates the intestinal immune system in terms of increased plasmacytoid dendritic cell and regulatory T-cell frequencies, which might account for the B. adolescentis-mediated protection from Yersinia enterocolitica infection.     

Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients

Background

Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF.

Methods

Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: ''Residual'': carrying at least one partial function CFTR mutation (class IV or V) and ''Minimal'' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories.

Results

Subjects with ''Minimal'' CFTR function had a higher hazard than patients with ''Residual'' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile).

Conclusions

Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.     

Mechanisms of protection against virulentYersinia pestis infection without participation of humoral antibody: H-2 restriction in athymic mouse model

Athymic mice, deficient in mature T-lymphocytes, were killed when subcutaneously challenged with 100 organisms of a highly virulent strain ofYersinia pestis. None of the methods for activating the macrophages protected the animals from death; however, the survival time of the mice primed withYersinia pseudotuberculosis containing VW antigen 14 days before the challenge was significantly prolonged. Neither IgM nor IgG specific forY. pestis was detectable in these mice 14 days after priming. The primed athymic mice given the spleen cells of the twice-primed thymus-bearing syngeneic or semiallogeneic mice resisted the challenge with virulentY. pestis. On the basis of these results, the protective activity of the immune T-cells againstY. pestis infection assayed in an adoptive transfer system resides in cell-mediated immunity with H-2 restriction.     

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 10⁷-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD₅₀ of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.     

Pasteurella Septica Infections in Humans

Five instances of infection by Pasteurella septica, an organism which closely resembles H. influenzae on methods of presumptive bacteriology, are reported from an acute treatment general hospital practice over a period of three months. This infection decidedly outnumbers those caused by other Pasteurella species in North America. The organism is apt to be identified as H. influenzae, Friedlander''s bacillus or Mima polymorpha. Unlike these, it shows extreme sensitivity to penicillin, sometimes combined with resistance to antibiotics that inhibit or kill these other organisms.While in the past Pasteurella septica infection was considered to be characterized by persistent local infection following domestic animal scratches or bites, it is clear that it is more often found in cases of respiratory tract infection and peripheral septic disease. In three of the five cases reported, exposure to animals was the source of infection.Because mixed infection with confusingly similar organisms may occur, lack of awareness of extreme penicillin sensitivity of Pasteurella septica may account for persistent failure of antibiotic therapy.     

The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages

Yersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y. pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.     

Characterization of the Na⁺/H⁺ antiporter from Yersinia pestis

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na/H antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein''s substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein''s structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na/H antiporter''s structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na/H antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.     

Caenorhabditis elegans Bacterial Pathogen Resistant bus-4 Mutants Produce Altered Mucins

Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and Yersinia pseudotuberculosis and have altered susceptibility to two Leucobacter species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the bus-4 N- and O-glycan pools. We observed dramatic changes in O-glycans and moderate ones in N-glycan pools compared to the parent strain. Ce core-I glycans, the nematode''s mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl O-glycans, Ce core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl N-glycans. Quantitative expression analysis revealed that two mucins, let-653 and osm-8, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.     

Remembering Malcolm J. Casadaban

Malcolm J. Casadaban died on 13 September 2009 from an infection and was found to have a weakened strain of the bacterium Yersinia pestis in his blood. This tragic event took the life of one of the most creative and influential geneticists of our time. In the late 1970s and ''80s, Malcolm invented novel approaches which changed the way many of us did science. Jon Beckwith, Tom Silhavy, and Olaf Schneewind have chronicled his scientific life from graduate school to his death and give us insight into Malcolm''s genius.Philip Matsumura Editor in Chief, Journal of Bacteriology     

The role of virulence antigens (VW) in the protection of mice againstYersinia pestis infection

Subcutaneous infection withYersinia enterocolitica harboring plasmid responsible for Ca²⁺ dependence at 37°C induced cell-mediated protective immunity against a lethal challenge withYersinia pestis; the isogenic derivative strain cured from this plasmid subverted the immunity in mice. This is the first identification of the antigen(s) responsible for the induction of cell-mediated protective immunity against the facultatively intracellular bacteria.     

Caspase-3 Mediates the Pathogenic Effect of Yersinia pestis YopM in Liver of C57BL/6 Mice and Contributes to YopM's Function in Spleen

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G⁺, F4/80⁺, and iNOS⁺ cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM''s action and supports the hypothesis that in liver YopM''s main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.     

Occurrence of Aphelenchoides besseyi in Louisiana Rice Seed and Its Interaction with Sclerotium oryzae in Selected Cultivars

Aphelenchoides besseyi, the nematode causal agent of white-tip disease of rice, was recovered from 5.5% of 474 seed samples obtained from rice seed warehouses in Louisiana. Laboratory tests in which A. besseyi-infested rice seed was treated with Phostoxin®, a compound used for control of insects in stored grain, indicate that it also has nematicidal properties. In 18-week-duration greenhouse tests, populations of A. besseyi increased 4-5-fold on the cultivars Saturn and Melrose and 3-fold on Nova ''76. Green weights of Nova ''76 plants inoculated with A. besseyi and Sclerotium oryzae, the causal agent of rice stem rot, were significantly reduced below those of plants inoculated with either organism alone or with distilled water. Weights of Melrose plants were reduced significantly by treatments with A. besseyi alone and A. besseyi plus S. oryzae, but not by S. oryzae alone. Saturn plant weights were not reduced significantly by either organism alone or by the two in combination.     

Expression of iron binding proteins and hemin binding activity in the dental pathogen Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans was found to express a polypeptide immunologically related to the Neisseria gonorrhoeae FbpA iron binding protein. In addition, the expression of hitB and hitC homologs was detected by Northern blot analysis. This periodontal pathogen also expresses a polypeptide homologous to the 31-kDa Haemophilus influenzae protein, which shows amino acid sequence homology with the FimA and YfeA proteins from Streptococcus parasanguis and Yersinia pestis, respectively. Both A. actinomycetemcomitans protein homologs were located within the periplasmic space, and their synthesis was regulated by the iron and hemin concentration of the culture medium. Southern and Western blot analysis together with molecular cloning revealed the presence of a Fur-like repressor, which may control the iron regulation of gene expression in this bacterium. Cultivation in the presence of hemin or Congo red revealed the ability of this organism to bind hemin. This binding activity was further confirmed by isolating Escherichia coli DH5α clones that produced red and brown colonies on agar plates containing Congo red and hemin, respectively, after transformation with an A. actinomycetemcomitans gene library.     

Yersiniosis in children

Sixty-five strains of Yersinia enterocolitica were isolated from stool specimens obtained from 35 patients over a 12-month period. The microbiologic characteristics and drug sensitivities are reported and the clinical patterns of disease associated with the organism are described. Gastroenteritis affecting infants and young children is the most frequent manifestation. The data for 1972 show the same epidemiological trend as in preceding years.     

Genotyping of Human and Porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri Strains from Switzerland by Amplified Fragment Length Polymorphism Analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin''s accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.     

Hemolymph responses in Heliothis zea to inoculation with Bacillus thuringiensis or Micrococcus lysodeikticus

Direct injection into the hemolymph of Heliothis zea of either an entomopathogen (Bacillus thuringiensis subsp. kurstaki) or a nonpathogen (Micrococcus lysodeikticus) is followed by a rapid phagocytosis and extensive removal of the organisms within 2 hr. The bacteria that survive this initial clearance initiate a new round of growth that is clearly evident 6–8 hr after injection. When the infecting organism is M. lysodeikticus, a second period of clearance occurs 8–12 hr after injection and nearly complete removal (many by lysis) is evident by the 12th hr. Larvae usually survive infection with this organism. When B. thuringiensis is the infecting organism, 60–80% of the phagocytized bacteria are lysed, however, the second wave of clearance seen with M. lysodeikticus does not occur; instead, the bacteria multiply extensively and death of the larvae results 12–16 hr after injection. This death does not appear to be caused either by crystalline protein or by the β-exotoxin. Analysis of hemolymph proteins using one-dimensional polyacrylamide gel electrophoresis indicated that although some quantitative changes were observed in some experiments, in the faster moving proteins when the infecting agent was B. thuringiensis, they were not consistent enough to support the idea that hemolymph proteins were either synthesized or used up during the time larvae were responding to the infectious agent. Dramatic changes were evident when the larvae were near death. No changes were ever observed when M. lysodeikticus was used as the infecting organism. A rapid response to infection using free spores of B. thuringiensis (sickness within 2–4 hr followed by death at 6–8 hr) may indicate that the spore germinating process is accompanied by release of a highly toxic material.