Identification of the Most Immunoreactive Antigens of Candida auris to IgGs from Systemic Infections in Mice.

The delay in making a correct diagnosis of Candida auris causes concern in the healthcare system setting, and immunoproteomics studies are important to identify immunoreactive proteins for new diagnostic strategies. In this study, immunocompetent murine systemic infections caused by non-aggregative and aggregative phenotypes of C. auris and by Candida albicans and Candida haemulonii were carried out, and the obtained sera were used to study their immunoreactivity against C. auris proteins. The results showed higher virulence, in terms of infection signs, weight loss, and histopathological damage, of the non-aggregative isolate. Moreover, C. auris was less virulent than C. albicans but more than C. haemulonii. Regarding the immunoproteomics study, 13 spots recognized by sera from mice infected with both C. auris phenotypes and analyzed by mass spectrometry corresponded to enolase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase. These four proteins were also recognized by sera obtained from human patients with disseminated C. auris infection but not by sera obtained from mice infected with C. albicans or Aspergillus fumigatus. Spot identification data are available via ProteomeXchange with the identifier PXD049077. In conclusion, this study showed that the identified proteins could be potential candidates to be studied as new diagnostic or even therapeutic targets for C. auris.

Immunome and immune complex-forming components of Brugia malayi identified by microfilaremic human sera.

Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents.

Protective efficacy of liver fluke DNA vaccines: A systematic review and meta-analysis: Guiding novel vaccine development.

The immunogenicity and efficacy of Fasciola DNA vaccines have not yet been comprehensively summarised in the form of a systematic review and meta-analysis. Though multiple vaccine studies with respect to Fasciola vaccines exist, the variance in the experimental parameters has made comparison difficult. We conducted a bibliographic database search in Scopus, PubMed, Science Direct, Cochrane Library, EMBASE and Web of Science databases, limited to publications from 1998 to 2017. The key words: Liver fluke, Fasciola hepatica, Fasciola gigantica, DNA vaccination, and immunogenicity were used in combination to form search strings. A total of 4760 studies were identified after initial screening, of which 14 qualified for systematic review and 7 for meta-analysis. The mean Odds Ratio (OR) for all studies was 0.565 (95% confidence interval (CI) of 0.293 to 1.087), which means the percentage of protection in terms of decreased fluke burden in animals vaccinated with DNA vaccines was 43.5%. A moderate protective efficacy was observed for cysteine protease and phosphoglycerate kinase vaccine antigen candidates (pooled OR and 95% CI, [0.542; 0.179-1.721] and [0.616; 0.219-1.735], respectively). Vaccine effectiveness was observed in individual studies and cohorts; however, the overall pooled efficacy for all vaccine candidates was found to be non-significant. Despite multiple individual studies showing promising results for various DNA vaccine candidates against fascioliasis, the pooled studies showed the non-significant effect of the vaccine formulations against fluke burden, and displayed minimal protective efficacy against Fasciola infection. Though promising results are observed in isolated studies, further animal trials with standardised experimental parameters are required to develop new vaccine candidates effective against Fasciola.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II.

Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1ß (IL-1ß) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-ß. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.

Recombinant Toxoplasma gondii phosphoglycerate mutase 2 confers protective immunity against toxoplasmosis in BALB/c mice.

Toxoplasmosis is one of the most widespread zoonoses worldwide. It has a high incidence and can result in severe disease in humans and livestock. Effective vaccines are needed to limit and prevent infection with Toxoplasma gondii. In this study, we evaluated the immuno-protective efficacy of a recombinant Toxoplasma gondii phosphoglycerate mutase 2 (rTgPGAM 2) against T. gondii infection in BALB/c mice. We report that the mice nasally immunised with rTgPGAM 2 displayed significantly higher levels of special IgG antibodies against rTgPGAM 2 (including IgG1, IgG2a and IgAs) and cytokines (including IFN-γ, IL-2 and IL-4) in their blood sera and supernatant of cultured spleen cells compared to those of control animals. In addition, an increased number of spleen lymphocytes and enhanced lymphocyte proliferative responses were observed in the rTgPGAM 2-immunised mice. After chronic infection and lethal challenge with the highly virulent T. gondii RH strain by oral gavage, the survival time of the rTgPGAM 2-immunised mice was longer (P < 0.01) and the survival rate (70%) was higher compared with the control mice (P < 0.01). The reduction rate of brain and liver tachyzoites in rTgPGAM 2-vaccinated mice reached approximately 57% and 69% compared with those of the control mice (P < 0.01). These results suggest that rTgPGAM 2 can generate protective immunity against T. gondii infection in BALB/c mice and may be a promising antigen in the further development of an effective vaccine against T. gondii infection.

Screening diagnostic candidates for schistosomiasis from tegument proteins of adult Schistosoma japonicum using an immunoproteomic approach.

BACKGROUND: Schistosomiasis is one of the world's most prevalent zoonotic diseases and a serious worldwide public health problem. Since the tegument (TG) of Schistosoma japonicum is in direct contact with the host and induces a host immune response against infection, the identification of immune response target molecules in the schistosome TG is crucial for screening diagnostic antigens for this disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an immunoproteomics approach used TG proteins as screening antigens to identify potential diagnostic molecules of S. japonicum. Ten spots corresponding to six proteins were identified that immunoreacted with sera from S. japonicum-infected rabbits but not sera from uninfected rabbits and their specific IgG antibody levels declined quickly after praziquantel treatment. Recombinant phosphoglycerate mutase (PGM) and UV excision repair protein RAD23 homolog B (RAD23) proteins were expressed and their diagnostic potential for schistosomiasis was evaluated and compared with schistosome soluble egg antigen (SEA) using ELISA. The results showed high sensitivity and specificity and low crossreactivity when rSjPGM-ELISA and rSjRAD23-ELISA were used to detect water buffalo schistosomiasis. Moreover, antibodies to rSjPGM and rSjRAD23 might be short-lived since they declined quickly after chemotherapy. CONCLUSION/SIGNIFICANCE: Therefore, the two schistosome TG proteins SjPGM and SjRAD23 were identified as potential diagnostic markers for the disease. The two recombinant proteins might have the potential to evaluate the effectiveness of drug treatments and for distinguishing between current and past infection.

Cofactor independent phosphoglycerate mutase of Brugia malayi induces a mixed Th1/Th2 type immune response and inhibits larval development in the host.

Lymphatic filariasis is a major debilitating disease, endemic in 72 countries putting more than 1.39 billion people at risk and 120 million are already infected. Despite the significant progress in chemotherapeutic advancements, there is still need for other measures like development of an effective vaccine or discovery of novel drug targets. In this study, structural and immunological characterization of independent phosphoglycerate mutase of filarial parasite Brugia malayi was carried out. Protein was found to be expressed in all major parasite life stages and as an excretory secretory product of adult parasites. Bm-iPGM also reacted to all the categories of human bancroftian patient's sera including endemic normals. In vivo immunological behaviour of protein was determined in immunized BALB/c mice followed by prophylactic analysis in BALB/c mice and Mastomys coucha. Immunization with Bm-iPGM led to generation of a mixed Th1/Th2 type immune response offering 58.2% protection against larval challenge in BALB/c and 65-68% protection in M. coucha. In vitro studies confirmed participation of anti-Bm-iPGM antibodies in killing of B. malayi infective larvae and microfilariae through ADCC mechanism. The present findings reveal potential immunoprotective nature of Bm-iPGM advocating its worth as an antifilarial vaccine candidate.

[Cloning, expression and antigenicity analysis of phosphoglycerate mutase 2 gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein. METHODS: Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5alpha first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its antigenicity. RESULTS: The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum. CONCLUSION: The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.

Identification of antibodies as biological markers in serum from multiple sclerosis patients by immunoproteomic approach.

We identified the antibody against mitochondrial heat shock protein 70 (mtHSP70) in serum from multiple sclerosis (MS) patients by proteomics-based analysis. The prevalence of the anti-mtHSP70 antibody is significantly higher in serum from MS patients than in serum from Parkinson disease patients, multiple cerebral infarction patients, infectious meningoencephalitis patients, and healthy controls (HCs) (68% sensitivity; 74% specificity). We studied the clinical features and magnetic resonance imaging findings of MS patients with the anti-mtHSP70 antibody. As a result, there were no significant differences between the anti-mtHSP70-antibody-positive and -negative MS patients. Additionally, in our comprehensive analysis of the prevalence of both the anti-mtHSP70 antibody and the anti-phosphoglycerate mutase 1 (PGAM1) antibody, which was previously reported by us to also show a higher prevalence in serum from MS patients, the positivity rates of both these antibodies were significantly higher in serum from MS patients than in serum from patients with other neurological diseases and from HCs; moreover, the specificity of this combination assay was higher than that of the assay of only one antibody (57% sensitivity; 93% specificity). Results of our study suggest that not only the anti-PGAM1 antibody but also the anti-mtHSP70 antibody is good diagnostic markers of MS and the combination of both these antibodies is useful for a more specific diagnosis of MS.

The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress.

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H(2)O(2)-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

High prevalence of autoantibodies against phosphoglycerate mutase 1 in patients with autoimmune central nervous system diseases.

We identified the autoantibody against phosphoglycerate mutase 1 (PGAM1), which is a glycolytic enzyme, in sera from multiple sclerosis (MS) patients by proteomics-based analysis. We further searched this autoantibody in sera from patients with other neurological diseases. The prevalence of the anti-PGAM1 antibody is much higher in patients with MS and neuromyelitis optica (NMO) than in those with other neurological diseases and in healthy controls. It was reported that the anti-PGAM1 antibody is frequently detected in patients with autoimmune hepatitis (AIH). Results of our study suggest that the anti-PGAM1 antibody is not only a marker of AIH but also a nonspecific marker of central nervous system autoimmune diseases.

Immunohistochemical localization of Phosphoglycerate mutase in capillary endothelium of the brain and periphery.

To elucidate the cellular localization of Phosphoglycerate mutase (PGAM1) type B in the brain and periphery, immunocytochemical studies were performed. The purified antigen used to generate the antiserum to PGAM1 was run on an SDS-PAGE gel, stained with coomassie blue, which yielded one sharp band at 29 kDa. Immunocytochemistry of formalin perfused rats revealed distinct localization of PGAM1 in the endothelium of the capillaries and arteries of the brain, liver and kidneys. Since enhanced glycogenesis is a well-known characteristic of cancer cells, it is of interest that sustained angiogenesis is a hallmark that distinguishes cancer cells from their normal counterparts. In view of the fact that PGAM1 increases in a variety of tumors, we suggest that PGAM1 may have a pathological role of vascular invasion into cancerous tissue.

[Cloning, expressing and characterizing of a phosphoglycerate mutase gene of Schistosoma japonncum].

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.

Diversified serum IgG response involving non-myelin CNS proteins during experimental autoimmune encephalomyelitis.

We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis in SJL/J mice injected with CFA, Bordetella pertussis toxin (BPT) and proteolipid protein 139-151 peptide, compared with mice that received CFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenic bands (kappa=0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglycerate mutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associated with multiple sclerosis in our previous work.

Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.

We have previously reported the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001-1010). The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies. In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected. We report here the cross-species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins. Using this approach, we unambiguously identified the four C. albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase. Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C. albicans.

Recombinant M-, B- and MB-type isozymes of human phosphoglyceric acid mutase: their large-scale production and preparation of polyclonal antibodies specific to M- and B-type isozymes.

Human phosphoglyceric acid mutase is a dimer comprising M-, B- and MB-type isozymes composed from the combination of the muscle-specific (M) and non-muscle-specific (B) subunits. Human DNAs coding M and B subunits were, respectively, reconstructed at their 5' regions without changing amino acid sequences, and expressed directly in Escherichia coli under the control of the trp promoter. M- and B-type isozymes were over-produced in the bacterial cytoplasm as soluble, active forms, which have been purified and characterized. MB-type was synthesized in vitro by recombining M- and B-type. All three recombinant isozymes thus obtained showed the same properties as the naturally-occurring ones with respect to the properties tested. Polyclonal IgGs specific to the M-type, B-type and MB-type were prepared from rabbits immunized with M- and B-type, using columns bound with M- and B-type. A method for the immunoassay of MB-type which is specifically present in cardiac muscle, is now under development.

Higher-plant cofactor-independent phosphoglyceromutase: purification, molecular characterization and expression.

Cofactor-independent phosphoglyceromutase (PGM) was purified to homogeneity from developing castor seed endosperm. Immunological characterization using monospecific antisera raised against this protein indicates that the enzyme is located in the cytosol and that there is no immunologically related polypeptide in the leucoplast from this tissue. Isolation and sequence determination of full-length cDNA clones for castor and tobacco PGM demonstrate that the protein is highly conserved in these plants and is closely related to the maize enzyme. A comparison of the amino acid sequence of peptides derived from Neurospora crassa PGM with the cofactor-independent enzyme from higher plants demonstrated that they are related and may have diverged from a common ancestral gene. The previously proposed relationship between higher-plant PGM and alkaline phosphatases is not supported by sequence analysis of the castor and tobacco enzymes. Expression of the single castor cytosolic PGM gene correlates well with other cytosolic glycolytic genes in developing and germinating castor seeds, and with the appearance of enzyme activity and PGM polypeptides in these tissues.

Rabbit M type phosphoglyceromutase: comparative effects of two thiol reagents antibody reaction and hybridization studies.

1. Treatment of purified rabbit phosphoglyceromutase (M type) with N-ethylmaleimide or with iodoacetamide produces the concurrent loss of phosphoglyceromutase activity with its collateral glycerate-2,3-P2 phosphatase activity. 2. Differences are observed in the protective effect of glycerate-2,3-P2 and of glycolate-2-P against N-ethylmaleimide and iodoacetamide treatments. 3. Specific chicken antibodies obtained by injection of the purified rabbit M type phosphoglyceromutase do not cross-react with the B type but neutralize both rabbit and human M type phosphoglyceromutase. 4. Purified rabbit M type phosphoglyceromutase can hybridize in vitro with the purified human B type or with purified human glycerate-2,3-P2 synthase. 5. Its ability to hybridize with glycerate-2,3-P2 synthase is unchanged after iodoacetamide treatment.