Proteomics of bacteroides fragilis and enterobacter cancerogenus

Bacteroides fragilis NCTC 9343 is a Gram-negative anaerobic bacterium with genomic DNA of 5205 Kb and a GC ratio of 43%. It is a commensal organism that can act as an opportunistic pathogen and is commonly present on the mucous membranes. It causes a variety of infections including intra abdominal infections, perirectal abscesses and decubitus ulcers. Enterotoxigenic forms are capable of causing diarrhoea in children and animals. Enterobacter cancerogenus ATCC 35316 is also a Gram-negative facultatively anaerobic bacterium with genomic DNA of 4602 Kb and a GC ratio of 55%. It is a naturally occurring human gut symbiont known to exhibit resistance to antibiotics like aminopenicillins. It has also been reported in cases of severe osteomyelitis and infections of bones and joints. This study aims to analyse the differential expression of proteins in the presence of mucin since it serves as the first site of adherence for the bacteria. The E. cancerogenus and B. fra gilis proteins were extracted and separated by two dimensional electrophoresis from logarithmic phase cultures grown in semi-defined media enriched with or without porcine gastric mucin Types II and III. The gel images were analysed using Bio-Rad PDQuest, Ludesi Redfin and Nonlinear Dynamics SameSpots softwares. It was observed that the presence of mucin in the media affected the expression of a number of proteins in E. cancerogenus and B. fragilis cells. The protein spots of interest were excised, hydrolysed using trypsin and subjected to electrospray ionisation based LC-MS analysis in order to determine the identity of the digested proteins and obtain a better understanding of the interactions of B. fra gilis and E. cancero genus with mucin. The outer membrane protein surface antigen X was found to be up-regulated in both mucin Type II and III enriched media in E. cancerogenus. Some of the other proteins that were differentially regulated in both E. cancerogenus and B. fra gilis included the elongation factor Ts, malate dehydrogenase, triose phosphate isomerase and thiol peroxidase proteins indicating that these proteins may be associated with the ability of bacteria to grow in mucin and may be potential virulence factors. Genes encoding the proteins CAH06598 and CAH09443 from the glycoside hydrolase families 95 and 97 in B. fra gilis strain NCTC9343 were cloned, overexpressed and purified using nickel affinity and gel filtration chromatography. The enzymes were found to be active by performing fluorimetric assays using methyl-umbelliferyl sugar substrates. Diffracting crystals of CAH09443 were obtained from the PACT ANION screens containing polyethylene glycol and sodium malonate as a precipitant. Structure determination was achieved via molecular replacement using the glycoside hydrolase Family 97 α-galactosidase, BtGH97b, from Bacteroides thetaiotaomicron as a starting model. The structure of CAH09443 was shown to be composed of a N-terminal β-super-sandwich domain and a canonical (β/α)₈ barrel, similar to the two other glycoside hydrolase family 97 enzyme structures reported

Proteomics of bacteroides fragilis and enterobacter cancerogenus

Bacteroides fragilis NCTC 9343 is a Gram-negative anaerobic bacterium with genomic DNA of 5205 Kb and a GC ratio of 43%. It is a commensal organism that can act as an opportunistic pathogen and is commonly present on the mucous membranes. It causes a variety of infections including intra abdominal infections, perirectal abscesses and decubitus ulcers. Enterotoxigenic forms are capable of causing diarrhoea in children and animals. Enterobacter cancerogenus ATCC 35316 is also a Gram-negative facultatively anaerobic bacterium with genomic DNA of 4602 Kb and a GC ratio of 55%. It is a naturally occurring human gut symbiont known to exhibit resistance to antibiotics like aminopenicillins. It has also been reported in cases of severe osteomyelitis and infections of bones and joints. This study aims to analyse the differential expression of proteins in the presence of mucin since it serves as the first site of adherence for the bacteria. The E. cancerogenus and B. fra gilis proteins were extracted and separated by two dimensional electrophoresis from logarithmic phase cultures grown in semi-defined media enriched with or without porcine gastric mucin Types II and III. The gel images were analysed using Bio-Rad PDQuest, Ludesi Redfin and Nonlinear Dynamics SameSpots softwares. It was observed that the presence of mucin in the media affected the expression of a number of proteins in E. cancerogenus and B. fragilis cells. The protein spots of interest were excised, hydrolysed using trypsin and subjected to electrospray ionisation based LC-MS analysis in order to determine the identity of the digested proteins and obtain a better understanding of the interactions of B. fra gilis and E. cancero genus with mucin. The outer membrane protein surface antigen X was found to be up-regulated in both mucin Type II and III enriched media in E. cancerogenus. Some of the other proteins that were differentially regulated in both E. cancerogenus and B. fra gilis included the elongation factor Ts, malate dehydrogenase, triose phosphate isomerase and thiol peroxidase proteins indicating that these proteins may be associated with the ability of bacteria to grow in mucin and may be potential virulence factors. Genes encoding the proteins CAH06598 and CAH09443 from the glycoside hydrolase families 95 and 97 in B. fra gilis strain NCTC9343 were cloned, overexpressed and purified using nickel affinity and gel filtration chromatography. The enzymes were found to be active by performing fluorimetric assays using methyl-umbelliferyl sugar substrates. Diffracting crystals of CAH09443 were obtained from the PACT ANION screens containing polyethylene glycol and sodium malonate as a precipitant. Structure determination was achieved via molecular replacement using the glycoside hydrolase Family 97 α-galactosidase, BtGH97b, from Bacteroides thetaiotaomicron as a starting model. The structure of CAH09443 was shown to be composed of a N-terminal β-super-sandwich domain and a canonical (β/α)₈ barrel, similar to the two other glycoside hydrolase family 97 enzyme structures reported.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Intranasal DNA vaccine for protection against respiratory infectious diseases: the delivery perspectives

published_or_final_versio

Infected Cell Protein (ICP)47 Enhances Herpes Simplex Virus Neurovirulence by Blocking the CD8+ T Cell Response

The herpes simplex virus (HSV) infected cell protein (ICP)47 blocks CD8+ T cell recognition of infected cells by inhibiting the transporter associated with antigen presentation (TAP). In vivo, HSV-1 replicates in two distinct tissues: in epithelial mucosa or epidermis, where the virus enters sensory neurons; and in the peripheral and central nervous system, where acute and subsequently latent infections occur. Here, we show that an HSV-1 ICP47− mutant is less neurovirulent than wild-type HSV-1 in mice, but replicates normally in epithelial tissues. The reduced neurovirulence of the ICP47− mutant was due to a protective CD8+ T cell response. When compared with wild-type virus, the ICP47− mutant expressed reduced neurovirulence in immunologically normal mice, and T cell–deficient nude mice after reconstitution with CD8+ T cells. However, the ICP47− mutant exhibited normal neurovirulence in mice that were acutely depleted of CD8+ T cells, and in nude mice that were not reconstituted, or were reconstituted with CD4+ T cells. In contrast, CD8+ T cell depletion did not increase the neurovirulence of an unrelated, attenuated HSV-1 glycoprotein (g)E− mutant. ICP47 is the first viral protein shown to influence neurovirulence by inhibiting CD8+ T cell protection

Antigen-specific immune responses to influenza vaccine in utero

Initial immune responses to allergens may occur before birth, thereby modulating the subsequent development of atopy. This paradigm remains controversial, however, due to the inability to identify antigen-specific T cells in cord blood. The advent of MHC tetramers has revolutionized the detection of antigen-specific T cells. Tetramer staining of cord blood after CMV infection has demonstrated that effective CD8+ antigen-specific immune responses can follow intrauterine viral infections. We hypothesized that sensitization to antigens occurs in utero in humans. We studied cord blood B and T cell immune responses following vaccination against influenza during pregnancy. Anti-Fluzone and anti-matrix protein IgM antibodies were detected in 38.5% (27 of 70) and 40.0% (28 of 70), respectively, of cord blood specimens. Using MHC tetramers, HA-specific CD4+ T cells were detected among 25.0% (3 of 12) and 42.9% (6 of 14) of cord blood specimens possessing DRB1*0101 and DRB1*0401 HLA types, respectively, and were detected even when the DRB1 HLA type was inherited from the father. Matrix protein–specific CD8+ T cells were detected among 10.0% (2 of 20) of HLA-A*0201+ newborns. These results suggest that B and T cell immune responses occur in the fetus following vaccination against influenza and have important implications for determining when immune responses to environmental exposures begin

Vaccination with hemagglutinin or neuraminidase DNA protects BALB/c mice against influenza virus infection in presence of maternal antibody

<p>Abstract</p> <p>Background</p> <p>Maternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.</p> <p>Results</p> <p>The results showed that: i) when the offspring were immunized with the same vaccine as their mothers, whether inactivated or DNA vaccine, the presence of maternal antibody inhibited offspring immune response and the offspring could not be protected from a lethal influenza virus infection; ii) when the offspring, born to mothers immunized with inactivated vaccine, were immunized with NA DNA vaccine, the interference of maternal antibody were overcome and the offspring could survive a lethal virus challenge; iii) when the offspring were immunized with different DNA vaccine from that for their mothers, the interference of maternal antibody were also overcome. In addition, high-dose inactivated vaccine in maternal immunization caused partial inhibition in offspring when the offspring were immunized with HA DNA vaccine, while lower dose caused no significant immunosuppression.</p> <p>Conclusion</p> <p>To avoid the interference of maternal antibody in influenza vaccination of offspring, mothers and their offspring shall not be immunized with the same vaccine. If mothers are immunized with inactivated vaccine, NA DNA vaccine for the offspring shall be effective; and if mothers are immunized with HA (NA) DNA, NA (HA) DNA for the offspring shall be effective.</p

Cd4+ T Cell Subsets during Virus Infection: Protective Capacity Depends on Effector Cytokine Secretion and on Migratory Capability

To analyze the antiviral protective capacities of CD4+ T helper (Th) cell subsets, we used transgenic T cells expressing an I-Ab–restricted T cell receptor specific for an epitope of vesicular stomatitis virus glycoprotein (VSV-G). After polarization into Th1 or Th2 effectors and adoptive transfer into T cell–deficient recipients, protective capacities were assessed after infection with different types of viruses expressing the VSV-G. Both Th1 and Th2 CD4+ T cells could transfer protection against systemic VSV infection, by stimulating the production of neutralizing immunoglobulin G antibodies. However, only Th1 CD4+ T cells were able to mediate protection against infection with recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G). Similarly, only Th1 CD4+ T cells were able to rapidly eradicate Vacc-IND-G from peripheral organs, to mediate delayed-type hypersensitivity responses against VSV-G and to protect against lethal intranasal infection with VSV. Protective capacity correlated with the ability of Th1 CD4+ T cells to rapidly migrate to peripheral inflammatory sites in vivo and to respond to inflammatory chemokines that were induced after virus infection of peripheral tissues. Therefore, the antiviral protective capacity of a given CD4+ T cell is governed by the effector cytokines it produces and by its migratory capability

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+T cell recognition

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo

Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage

DNA vaccination elicits humoral and cellular immune responses and has been shown to confer protection against several viral, bacterial, and parasitic pathogens. Here we report that optimized codon usage of an injected DNA sequence considerably increases both humoral and cellular immune responses. We recently generated a synthetic human immunodeficiency virus type 1 gp120 sequence in which most wild-type codons were replaced with codons from highly expressed human genes (syngp120). In vitro expression of syngp120 is considerably increased in comparison to that of the respective wild-type sequence. In BALB/c mice, DNA immunization with syngp120 resulted in significantly increased antibody titers and cytotoxic T-lymphocyte reactivity, suggesting a direct correlation between expression levels and the immune response. Moreover, syngp120 is characterized by rev-independent expression and a low risk of recombination with viral sequences. Thus, synthetic genes with optimized codon usage represent a novel strategy to increase the efficacy and safety of DNA vaccination