Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

BACKGROUND: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.

Characterization of two putative Dichelobacter nodosus footrot vaccine antigens identifies the first lysozyme inhibitor in the genus.

The Gram-negative anaerobic bacterium Dichelobacter nodosus (Dn) causes footrot in ruminants, a debilitating and highly contagious disease that results in necrotic hooves and significant economic losses in agriculture. Vaccination with crude whole-cell vaccine mixed with multiple recombinant fimbrial proteins can provide protection during species-specific outbreaks, but subunit vaccines containing broadly cross-protective antigens are desirable. We have investigated two D. nodosus candidate vaccine antigens. Macrophage Infectivity Potentiator Dn-MIP (DNO_0012, DNO_RS00050) and Adhesin Complex Protein Dn-ACP (DNO_0725, DNO_RS06795) are highly conserved amongst ~170 D. nodosus isolates in the https://pubmlst.org/dnodosus/ database. We describe the presence of two homologous ACP domains in Dn-ACP with potent C-type lysozyme inhibitor function, and homology of Dn-MIP to other putative cell-surface and membrane-anchored MIP virulence factors. Immunization of mice with recombinant proteins with a variety of adjuvants induced antibodies that recognised both proteins in D. nodosus. Notably, immunization with fimbrial-whole-cell Footvax vaccine induced anti-Dn-ACP and anti-Dn-MIP antibodies. Although all adjuvants induced high titre antibody responses, only antisera to rDn-ACP-QuilA and rDn-ACP-Al(OH)3 significantly prevented rDn-ACP protein from inhibiting lysozyme activity in vitro. Therefore, a vaccine incorporating rDn-ACP in particular could contribute to protection by enabling normal innate immune lysozyme function to aid bacterial clearance.

Streptococcus pneumoniae Cell Wall-Localized Trigger Factor Elicits a Protective Immune Response and Contributes to Bacterial Adhesion to the Host.

Trigger factor (TF) has a known cytoplasmic function as a chaperone. In a previous study we showed that pneumococcal TF is also cell-wall localized and this finding combined with the immunogenic characteristic of TF, has led us to determine the vaccine potential of TF and decipher its involvement in pneumococcal pathogenesis. Bioinformatic analysis revealed that TF is conserved among pneumococci and has no human homologue. Immunization of mice with recombinant (r)TF elicited a protective immune response against a pneumococcal challenge, suggesting that TF contributes to pneumococcal pathogenesis. Indeed, rTF and an anti-rTF antiserum inhibited bacterial adhesion to human lung derived epithelial cells, indicating that TF contributes to the bacterial adhesion to the host. Moreover, bacteria lacking TF demonstrated reduced adhesion, in vitro, to lung-derived epithelial cells, neural cells and glial cells. The reduced adhesion could be restored by chromosomal complementation. Furthermore, bacteria lacking TF demonstrated significantly reduced virulence in a mouse model. Taken together, the ability of rTF to elicit a protective immune response, involvement of TF in bacterial adhesion, conservation of the protein among pneumococcal strains and the lack of human homologue, all suggest that rTF can be considered as a future candidate vaccine with a much broader coverage as compared to the currently available pneumococcal vaccines.

Vaccination with MIP or Pgp3 induces cross-serovar protection against chlamydial genital tract infection in mice.

Previously we reported that recombinant Chlamydia muridarum macrophage infectivity potentiator (MIP) provided partial protection against C. muridarum genital tract infection in mice. On the other hand, Chlamydia trachomatis plasmid encoded Pgp3could induce the protection against C. muridarum air way infection. This study aimed to evaluate the immunogenicity of MIP and Pgp3 from C. trachomatis serovar D and further investigate whether MIP and Pgp3 provide cross-serovar protection against C. muridarum genital tract infection in mice. Our results showed that vaccination by any regimen, including MIP alone, Pgp3 alone or MIP plus Pgp3, induced specific serum antibody production and Th1-dominant cellular responses in mice. Live chlamydial shedding from the vaginal and inflammatory pathologies in the oviduct markedly reduced. However, MIP + Pgp3 vaccination did not provide better protection than the single immunization. In conclusion, this study demonstrated that both MIP and Pgp3 can induce cross-serovar protective against chlamydial genital tract infection, and provided the guide for the development of optimal multisubunit vaccines against C. trachomatis infection.

Peptidylprolyl Isomerases as In Vivo Carriers for Drugs That Target Various Intracellular Entities.

Analyses of sequences and structures of the cyclosporine A (CsA)-binding proteins (cyclophilins) and the immunosuppressive macrolide FK506-binding proteins (FKBPs) have revealed that they exhibit peculiar spatial distributions of charges, their overall hydrophobicity indexes vary within a considerable level whereas their points isoelectric (pIs) are contained from 4 to 11. These two families of peptidylprolyl cis/trans isomerases (PPIases) have several distinct functional attributes such as: (1) high affinity binding to some pharmacologically-useful hydrophobic macrocyclic drugs; (2) diversified binding epitopes to proteins that may induce transient manifolds with altered flexibility and functional fitness; and (3) electrostatic interactions between positively charged segments of PPIases and negatively charged intracellular entities that support their spatial integration. These three attributes enhance binding of PPIase/pharmacophore complexes to diverse intracellular entities, some of which perturb signalization pathways causing immunosuppression and other system-altering phenomena in humans.

Allergens involved in the cross-reactivity of Aedes aegypti with other arthropods.

BACKGROUND: Cross-reactivity between Aedes aegypti and mites, cockroaches, and shrimp has been previously suggested, but the involved molecular components have not been fully described. OBJECTIVE: To evaluate the cross-reactivity between A aegypti and other arthropods. METHODS: Thirty-four serum samples from patients with asthma and/or allergic rhinitis were selected, and specific IgE to A aegypti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Periplaneta americana. and Litopenaeus vannamei was measured by enzyme-linked immunosorbent assay. Cross-reactivity was investigated using pooled serum samples from allergic patients, allergenic extracts, and the recombinant tropomyosins (Aed a 10.0201, Der p 10, Blo t 10, Lit v 1, and Per a 7). Four IgE reactive bands were further characterized by matrix-assisted laser desorption/ionization tandem time of flight. RESULTS: Frequency of positive IgE reactivity was 82.35% to at least one mite species, 64.7% to A aegypti, 29.4% to P americana, and 23.5% to L vannamei. The highest IgE cross-reactivity was seen between A aegypti and D pteronyssinus (96.6%) followed by L vannamei (95.4%), B tropicalis (84.4%), and P americana (75.4%). Recombinant tropomyosins from mites, cockroach, or shrimp inhibited the IgE reactivity to the mosquito at a lower extent than the extracts from these arthropods. Several bands of A aegypti cross-reacted with arthropod extracts, and 4 of them were identified as odorant binding protein, mitochondrial cytochrome C, peptidyl-prolyl cis-trans isomerase, and protein with hypothetical magnesium ion binding function. CONCLUSION: We identified 4 novel cross-reactive allergens in A aegypti allergenic extract. These molecules could influence the manifestation of allergy to environmental allergens in the tropics.

Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins.

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.

A new ELISA method for serological diagnosis of Legionella pneumophila: use of five purified proteins, FLA, MOMP, MIP, IP, and PILE, as diagnostic antigen.

BACKGROUND: Legionella pneumophila plays an important role in human infection. Commercial ELISA kits commonly used, which take Legionella pneumophila whole-cell protein as the coating antigen, often have cross-reactivity among serogroups or species. In this study, five Legionella pneumophila proteins FLA, MOMP, MIP, IP, and PILE were purified and further applied in serological diagnosis of Legionella pneumophila infections compared with R & D Legionella ELISA kits. METHODS: The five recombinant plasmids pET-fla, pET-momp, pET-mip, pET-ip, and pET-pile were transformed into E. coli BL21 and then induced them with IPTG. The expression products were analyzed by SDS-PAGE and purified by affinity chromatography. Indirect enzyme-linked immunosorbent assays (ELISAs) were established with the five purified proteins FLA, MOMP, MIP, IP, and PILE altogether as the coating antigen and tested for the presence of IgG, IgM, and IgA antibody independently from 50 positive sera and 40 negative sera, compared with R & D IgG, IgM, and IgA Legionella ELISA kits. RESULTS: The FLA protein about 42 kDa in size, the MOMP protein about 45 kDa, the MIP protein about 40 kDa, the IP protein about 46 kDa, and the PILE protein about 35.7 kDa were separately expressed and purified. Compared with R & D IgG, IgM, and IgA Legionella ELISA kit, the outcome of indirect ELISAs set up with the five purified proteins showed that for IgG the sensitivity was 90.4%, the specificity was 97.4%, the area under ROC curve was 0.939, the kappa value was 0.865, the 95% confidence interval was 0.883 - 0.995. For IgM the sensitivity was 91.8%, the specificity was 95.1%, the area under ROC curve was 0.935, the kappa value was 0.866, the 95% confidence interval was 0.876 - 0.994. For IgA the sensitivity was 93.6%, the specificity was 95.3%, the area under ROC curve was 0.945, the kappa value was 0.889, the 95% confidence interval was 0.890 - 0.999. CONCLUSIONS: The proteins FLA, MOMP, MIP, IP, and PILE were successfully expressed and purified, and they seemed to be suitable coating antigens for the serological diagnosis of Legionella pneumophila.

Pin1 cysteine-113 oxidation inhibits its catalytic activity and cellular function in Alzheimer's disease.

The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.

Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.

[Expression and purification of Legionella pneumophila MIP protein and its application in serological diagnosis].

OBJECTIVE: To express and purify macrophage infectivity potentiator (MIP) protein of Legionella pneumophila(Lp), and explore its value in the serological diagnosis of Lp. METHODS: The recombinant plasmid pET-mip was transformed into E.coli BL21 competent cells. The expression of MIP protein was induced, and then analyzed by SDS-PAGE electrophoresis, purified by affinity chromatography. We screened out 40 positive blood serum and 30 negative blood serum using the DRG (Germany, IgG/IgM/IgA) Lp kit. The blood serum samples were detected for IgG, IgM, IgA antibody levels by indirect ELISA that we had established with the purified MIP protein as the coating antigen, as well as by R&D (USA, IgG/IgM/IgA) Lp kit. The two methods were compared in the sensitivity, specificity and consistency of the test results. RESULTS: The recombinant MIP protein was successfully expressed and purified with Mr; being 40 000 in E.coli BL21. In comparison of the indirect ELISA we developed with the R&D Lp kit for detecting Lp antibody IgG, IgM and IgA in blood serum, the specificity of IgG was 88.5% and the sensitivity was 95.5%, the Kappa value was 0.846 (P<0.05), the area under the ROC curve was 0.927; the specificity of IgM was 89.3% and the sensitivity was 97.6%, the Kappa value was 0.88 (P<0.05), the area under the ROC curve was 0.947; the specificity of IgA was 90% and the sensitivity was 95.2%, the Kappa value was 0.856 (P<0.05), the area under the ROC curve was 0.931. CONCLUSION: MIP proteins of L.pneumophila was expressed and purified successfully, and MIP protein can be used as a coating antigen in serological diagnosis of L.pneumophila.

Pathogen-free screening of bacteria-specific hybridomas for selecting high-quality monoclonal antibodies against pathogen bacteria as illustrated for Legionella pneumophila.

Antibodies are potent biological tools increasingly used as detection, diagnostic and therapeutic reagents. Many technological advances have optimized and facilitated production and screening of monoclonal antibodies. We report here an original method to screen for antibodies targeting biosafety level 2 or 3 pathogens without the fastidious handling inherent to pathogen use. A double ELISA screening was performed using as coated antigen transformed Escherichia coli expressing at its surface a protein specific to the pathogenic bacteria versus control untransformed E. coli. This method was applied to Legionella, using the surface-exposed Mip protein (macrophage infectivity potentiator). This screening proved to be an excellent means of selecting mAbs that bind Legionella pneumophila 1 surface-exposed Mip protein. This method also appears more biologically relevant than screening using the recombinant Mip protein alone and less tedious than a test performed directly on Legionella bacteria. We obtained 21 mAbs that bind strongly to L. pneumophila serogroups 1 to 13, and we validated their use in a rapid ELISA (performed in 4.5 h) and an immunochromatographic test (20 min).

Exocytosis of neutrophil granule subsets and activation of prolyl isomerase 1 are required for respiratory burst priming.

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs.

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPT(M)) and intratracheally (PAPT(Cp)), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPT(Cp). Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPT(Cp) yielded a serum IgA response.

Conserved anchorless surface proteins as group A streptococcal vaccine candidates.

Streptococcus pyogenes (group A Streptococcus (GAS)) causes ∼700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies cross-reactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting ≥99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease.

Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Novel role of Pin1 induction in type II collagen-mediated rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-alpha, and IL-1beta). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-kappaB, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-kappaB, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-kappaB, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.

Peptide microarrays for determination of cross-reactivity.

Polyclonal antibodies raised against full-length antigens are often used for localization experiments. Exact knowledge of epitopes in the antigen recognized by the antiserum is important if the target antigen belongs to a large family of proteins which are highly conserved. We have shown that epitope mapping using peptide microarrays represents a powerful tool for determination of immunodominat regions in a proteome-wide manner. As examples we show results of epitope mapping using peptide microarrays displaying overlapping peptide scans through either all human cyclophilins or all human FK506-binding proteins.

The peptidyl-prolyl isomerase Pin1 facilitates cytokine-induced survival of eosinophils by suppressing Bax activation.

The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.