Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse.

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Development of an artificial antibody specific for HLA/peptide complex derived from cancer stem-like cell/cancer-initiating cell antigen DNAJB8.

BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

A Biological and Immunological Characterization of Schistosoma Japonicum Heat Shock Proteins 40 and 90α.

We characterized Schistosoma japonicum HSP40 (Sjp40) and HSP90α (Sjp90α) in this study. Western blot analysis revealed both are present in soluble egg antigens and egg secretory proteins, implicating them in triggering the host immune response after secretion from eggs into host tissues. These observations were confirmed by immunolocalization showing both HSPs are located in the Reynolds' layer within mature eggs, suggesting they are secreted by miracidia and accumulate between the envelope and the eggshell. Both HSPs are present in the musculature and parenchyma of adult males and in the vitelline cells of females; only Sjp90α is present on the tegument of adults. Sjp40 was able to enhance the expression of macrophages, dendritic cells, and eosinophilic cells in mouse liver non-parenchymal cells, whereas rSjp90α only stimulated the expression of dendritic cells. T helper 1 (Th1), Th2, and Th17 responses were increased upon rSjp40 stimulation in vitro, but rSjp90 only stimulated an increased Th17 response. Sjp40 has an important role in reducing the expression of fibrogenic gene markers in hepatic stellate cells in vitro. Overall, these findings provide new information on HSPs in S. japonicum, improving our understanding of the pathological roles they play in their interaction with host immune cells.

[Study on immunological functions of heat shock protein 40 kDa of Schistosoma japonicum].

OBJECTIVE: To investigate the immunological functions of heat shock protein 40 kDa of Schistosoma japonicum (SjHSP40). METHODS: The homology of the SjHSP40 protein sequence was analyzed and the B and T cell epitopes of SjHSP40 were predicted using bioinformatics tools. The full-length SjHSP40 gene was amplified using a PCR assay, and cloned into the prokaryotic expression vector pGEX-6P-1, which was transformed into Escherichia coli BL-21. The protein expression was induced with isopropyl ß-D-thiogalactoside (IPDG), and then, the recombinant protein was purified with glutathione-sepharose 4B resin to yield the fusion protein GST-SjHSP40, which was checked with SDS-PAGE and Western blotting. Following immunization with GST-SjHSP40, the serum levels of anti-SjHSP40 IgG antibody and IgG1 and IgG2a subtypes were detected in BALB/c mice using ELISA. In addition, the effect of SjHSP40 on CD4+ T-cell subset differentiation was examined using flow cytometry. RESULTS: SjHSP40 contained 7 potential B cell epitopes and multiple T cell epitopes (CTL epitopes and Th epitopes). The prokaryotic expression plasmid pGEX-6p-1-SjSHP40 was successfully constructed, and the fusion protein GST-SjHSP40 was obtained following IPDG induction and protein purification. Significantly higher serum levels of anti-SjHSP40 IgG, IgG1 and IgG2a antibodies were detected in mice immunized with GST-SjHSP40 than in other groups; however, SjHSP40 showed no remarkable effects on CD4+ T-cell subset differentiation. CONCLUSIONS: SjHSP40 may induce specific humoral immune responses in mice; however, it does not affect the balance of Th immune responses. It is suggested that SjHSP40 may be a potential vaccine candidate.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Immunization with plasmid DNA expressing Heat Shock Protein 40 confers prophylactic protection against chronic Toxoplasma gondii infection in Kunming mice.

Toxoplasma gondii causes one of the most common protozoal diseases of humans and animals worldwide. With the aim of designing an effective vaccine against T. gondii infection, we examined the immunogenicity of a DNA vaccine expressing heat shock protein 40 (HSP40) against challenge with T. gondii (type I RH and type II Pru) strains in Kunming mice. The plasmid pVAX1-HSP40 was constructed and used to immunize mice by intramuscular injection for three sequential immunizations with two-week intervals. This immunization regimen significantly reduced parasite cyst burden in pVAX1-HSP40-immunized mice (1871.9 ± 142.3) compared with control mouse groups immunized with pVAX1 (3479.2 ± 204.4), phosphate buffered saline (3024.4 ± 212.8), or left untreated (3275.0 ± 179.8) as healthy controls (p < 0.01). However, immunization failed to protect mice against challenge with the virulent RH strain. There was a significant increase in T lymphocyte subclasses (CD3e+CD4+ T and CD3e+CD8a+ T lymphocytes) in splenic tissues in immunized mice compared with controls (p < 0.05). However, the level of antibodies, lymphocyte proliferation and concentration of cytokines (IFN-γ, IL-2, IL-4, IL-10 and IL-12p70) were not significantly different between immunized and control mouse groups (p < 0.05). These data indicate that pVAX1-HSP40 induced specific immune responses and achieved a significant reduction in the number of brain cysts in Pru-infected mice, and thus can be tested in future immunization studies along with plasmids containing other immunogenic proteins as a cocktail vaccine to fully abolish chronic toxoplasmosis.

Adaptive response of pearl oyster Pinctada fucata martensii to low water temperature stress.

The pearl oyster Pinctada fucata martensii is a warm-water shellfish that is sensitive to cold environments. To investigate its potential adaptation to low-temperature stress, the selected line (SL) and based population (BP) were sampled to undergo transcriptome sequence. Results of transcriptome analysis showed 572 significant differentially expressed genes. The typical HSP70 and HSP40 exhibited the polar expression model in the two groups. Meanwhile, the related genes that involved in energy release mediated by oxidative phosphorylation and the biosynthesis of unsaturated fatty acid were increased in the SL. The apparent enrichment of different expressed genes in amino acid metabolism indicated that the small molecule system with amino acids was one of the main regulator for low-temperature stress. The different expressions of immune-related and lysosome protein encoding genes also reflected the variation of immunity in the two groups and indicated that it could affect the adaptation ability in different temperature. In addition, the similar trends of different expression of typical genes between two groups were obtained by using RNA-seq and qRT-PCR. These results suggested that multi-system adjustments are involved in the processes of low water temperature stress in pearl oyster, providing insights into the response systems of shellfish to acclimatise with ambient environment change.

Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae.

The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2-/- mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2-/- mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4+ T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2-/- DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN.

Fibrillary GN is a rare form of GN of uncertain pathogenesis that is characterized by the glomerular accumulation of randomly arranged, nonbranching fibrils (12-24 nm) composed of Ig and complement proteins. In this study, we used mass spectrometry to comprehensively define the glomerular proteome in fibrillary GN compared with that in controls and nonfibrillary GN renal diseases. We isolated glomeruli from formalin-fixed and paraffin-embedded biopsy specimens using laser capture microdissection and analyzed them with liquid chromatography and data-dependent tandem mass spectrometry. These studies identified DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sampled protein detected only in fibrillary GN cases. The glomerular proteome of fibrillary GN cases also contained IgG1 as the dominant Ig and proteins of the classic complement pathway. In fibrillary GN specimens only, immunofluorescence and immunohistochemistry with an anti-DNAJB9 antibody showed strong and specific staining of the glomerular tufts in a distribution that mimicked that of the immune deposits. Our results identify DNAJB9 as a putative autoantigen in fibrillary GN and suggest IgG1 and classic complement effector pathways as likely mediators of the destructive glomerular injury in this disease.

Heterologous prime-boost immunization with live SPY1 and DnaJ protein of Streptococcus pneumoniae induces strong Th1 and Th17 cellular immune responses in mice.

Streptococcus pneumoniae is a leading cause of infectious diseases in children under 5-year-old. Vaccine has been used as an indispensable strategy to prevent S. pneumoniae infection for more than 30 years. Our previous studies confirmed that mucosal immunization with live attenuated strain SPY1 can protect mice against nasopharyngeal colonization of S. pneumoniae and lethal pneumococcal infection, and the protective effects are comparable with those induced by commercially available 23-valent polysaccharide vaccine. However, live attenuated vaccine SPY1 needs four inoculations to get satisfactory protective effect, which may increase the risk of virulence recovery. It is reported that heterologous primeboost approach is more effective than homologous primeboost approach. In the present study, to decrease the doses of live SPY1 and improve the safety of SPY1 vaccine, we immunized mice with SPY1 and DnaJ protein alternately. Our results showed that heterologous prime-boost immunization with SPY1 and DnaJ protein could significantly reduce the colonization of S. pneumoniae in the respiratory tract of mice, and induce stronger Th1 and Th17 cellular immune responses than SPY1 alone. These results indicate heterologous prime-boost immunization method not only elicits better protective effect than SPY1 alone, but also reduces the doses of live SPY1 and decreases the risk of SPY1 vaccine. This work is the first time to study the protective efficiency with two different forms of S. pneumoniae candidate vaccine, and provides a new strategy for the development of S. pneumoniae vaccine.

DnaJ (hsp40) of Streptococcus pneumoniae is involved in bacterial virulence and elicits a strong natural immune reaction via PI3K/JNK.

As a heat shock protein, DnaJ plays an important role in the pathogenesis of pneumococcal infection. However, how the virulence factor-DnaJ elicits host natural immunity still remains unclear. In this study, we investigated the effects of dnaJ deficiency in Streptococcus pneumoniae (S. pneumoniae) on bacterial virulence, and further explored the related molecular mechanisms in vivo and in vitro. By generating dnaJ deficient mutant (ΔdnaJ), the virulence and colonization were detected in murine pneumonia and sepsis models in vivo. Compared with wild-type parent strain, the abilities of rapid colonization and induction of inflammatory responses of ΔdnaJ in mouse lungs were significantly impaired. Simultaneously, recombinant DnaJ purified from E. coli expression system (rDnaJ) induced macrophage strain RAW264.7 to secrete IL-6 by activation of PI3K and JNK signal pathways, which were confirmed by the specific signaling inhibitors. In conclusion, DnaJ, a novel virulence protein, was essential for the virulence and colonization of S. pneumoniae and induced pro-inflammatory cytokine production in macrophages through PI3K/JNK.

Pneumococcal DnaJ modulates dendritic cell-mediated Th1 and Th17 immune responses through Toll-like receptor 4 signaling pathway.

Pneumococcal DnaJ was recently shown to be a potential protein vaccine antigen that induces strong Th1 and Th17 immune response against streptococcus pneumoniae infection in mice. However, how DnaJ mediates T cell immune response against S. pneumoniae infection has not been addressed. Here, we investigate whether DnaJ contributes to the development of T cell immunity through the activation of bone marrow-derived dendritic cells (BMDCs). We found that endotoxin-free recombinant DnaJ (rDnaJ) induced activation and maturation of BMDCs via recognition of Toll-like receptor 4 (TLR4) and activation of MAPKs, NF-κB and PI3K-Akt pathways. rDnaJ-treated BMDCs effectively stimulated naïve CD4+ T cells to secrete IFN-γ and IL-17A. Splenocytes from mice that were adoptively transferred with rDnaJ-pulsed BMDCs secreted higher levels of IFN-γ and IL-17A compared with those that received PBS-activated BMDCs. Splenocytes from TLR4-/- mice immunized with rDnaJ produced lower levels of IFN-γ and IL-17A compared with those from wild type mice. Our findings indicate that DnaJ can induce Th1 and Th17 immune responses against S. pneumoniae through activation of BMDCs in a TLR4-dependent manner.

Co-expression of heat shock protein (HSP) 40 and HSP70 in Pinctada martensii response to thermal, low salinity and bacterial challenges.

Heat shock protein (HSP) 40 proteins are a family of molecular chaperones that bind to HSP70 through their J-domain and regulate the function of HSP70 by stimulating its adenosine triphosphatase activity. In the present study, a HSP40 homolog named PmHSP40 was cloned from the hemocytes of pearl oyster Pinctada martensii using EST and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of PmHSP40 was 1251 bp in length, which included a 5' untranslated region (UTR) of 75 bp, an open reading frame (ORF) of a 663 bp, and a 3' UTR of 513 bp. The deduced amino acid sequence of PmHSP40 contains a J domain in the N-terminus. In response to thermal and low salinity stress challenges, the expression of PmHSP40 in hemocytes and the gill were inducible in a time-dependent manner. After bacterial challenge, PmHSP40 transcripts in hemocytes increased and peaked at 6 h post injection. In the gill, PmHSP40 expression increased, similar to expression in hemocytes; however, transcript expression of PmHSP40 was significantly up-regulated at 12 h post injection. Furthermore, the transcripts of PmHSP70 showed similar kinetics as that of PmHSP40, with highest induction during thermal, low salinity stress and bacterial challenges. Altogether these results demonstrate that PmHSP40 is an inducible protein under thermal, low salinity and bacterial challenges, suggesting its involvement in both environmental and biological stresses, and in the innate immunity of the pearl oyster.

[Pneumococcal HSP40 induces the immune response in mouse macrophages via p38MAPK and JNK signaling pathways].

OBJECTIVE: To investigate the mechanism of immune response in mouse macrophage induced by Pneumococcal heat shock protein 40 (HSP40). METHODS: After recombinant HSP40 (rHSP40) underwent expression detection and purification, lipopolysaccharide (LPS) was removed from it. Then rHSP40 was used to stimulate bone marrow derived macrophages (BMDMs) derived from C57BL/6 wild-type mice. The mRNA levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-1ß, IL-23p19, IL-12p40, IL-12p35 and IL-10 in BMDMs were determined by reverse transcription PCR; the expressions of TNF-α, IL-6 and IL-12p40 were measured by ELISA. After stimulated by rHSP40, the levels of TNF-α and IL-6 expressed by wide-type, TLR2-/- and TLR4-/- BMDMs were detected by ELISA. The effects of the pretreatment of mitogen-activated protein kinases (MAPK) inhibitors on the secretion of TNF-α and IL-6 induced by rHSP40 were also evaluated by ELISA in BMDMs. The phosphorylation levels of p38MAPK and c-Jun N-terminal kinase (JNK) were determined by Western blotting. RESULTS: The rHSP40 protein reached a purity of more than 90%. It significantly enhanced the phosphorylation levels of p38MAPK and JNK as well as the expressions of TNF-α and IL-6. The p38MAPK and JNK inhibitors significantly suppressed the expressions of TNF-α and IL-6. The expressions of TNF-α and IL-6 in TLR4-/- BMDMs significantly decreased compared with wide-type BMDMs. CONCLUSION: HSP40-induced immune response of mouse macrophages is regulated by p38MAPK and JNK signaling pathways, and this induction process depends on TLR4.

Increased levels of antibodies against heat shock proteins in stroke patients.

Ischemic stroke is the second leading cause of death worldwide. One of the main risk factors of the ischemic stroke is atherosclerosis which is a chronic inflammatory and immune-mediated disease. Bacterial infections generate specific human antibodies against various antigens, including Hsps. It has been demonstrated that Hsps are selectively overexpressed in the atherosclerotic lesions. The amino acid sequence homology between human and bacterial Hsps may lead to an autoimmune response by immunological cross-reaction. Such immune response against Hsps overexpressed in the blood vessels under stressful conditions may contribute to inflammatory processes and subsequent development of atherosclerosis. In this study we determined the antibody levels against bacterial and human Hsp by ELISA in blood plasma obtained from stroke patients. Using ANOVA we analyzed levels of Hsp-antibodies in control and patient groups and correlate them with several stroke risk factors. The group of stroke patients had elevated levels of anti-Hsp antibodies compared to the control group. We also discovered an antibody level increase in patients that previously underwent another stroke. Our data provide evidence that autoimmunity could underlie formation of atherosclerosis plaque leading to stroke.

Protection against pneumococcal infection elicited by immunization with multiple pneumococcal heat shock proteins.

Heat shock proteins (HSPs) play important roles in the pathogenesis of pneumococcal infection, and they are considered as potential protein vaccine antigens. In this study, we investigated the efficacy of immunization with pneumococcal HSPs, including ClpP (hsp100/Clp peptidase subunit), DnaJ (hsp40) and GroEL (hsp60), to protect against pneumococcal carriage, lung colonization and sepsis in mouse models using different serotypes of Streptococcus pneumoniae. In a nasopharyngeal colonization model by serotype 6B or 14 and in a lung colonization model by serotype 19F, immunization with pneumococcal HSPs could elicit effective protection. Likewise, vaccination with ClpP, DnaJ or GroEL allowed significantly longer mouse survival times after lethal intranasal challenge with serotype pneumococcal 2, 3 or 4. Interestingly, combinations of these HSPs could consistently enhance the protection against nasopharynx carriage, lung colonization as well as invasive infection caused by different pneumococcal serotypes. In an in vitro killing assay, anti-sera against ClpP, DnaJ or GroEL could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way, and combinations of multiple anti-sera against these HSPs could increase the killing ability compared with single anti-sera. Finally, passive immunization studies with anti-sera against pneumococcal HSPs also demonstrated that an additive effect could be achieved by using multiple anti-sera when compared with single anti-sera. Thus, inclusion of multiple pneumococcal HSPs is important for the development of protein-based pneumococcal vaccines.

The ERdj5-Sel1L complex facilitates cholera toxin retrotranslocation.

Cholera toxin (CT) traffics from the host cell surface to the endoplasmic reticulum (ER), where the toxin's catalytic CTA1 subunit retrotranslocates to the cytosol to induce toxicity. In the ER, CT is captured by the E3 ubiquitin ligase Hrd1 via an undefined mechanism to prepare for retrotranslocation. Using loss-of-function and gain-of-function approaches, we demonstrate that the ER-resident factor ERdj5 promotes CTA1 retrotranslocation, in part, via its J domain. This Hsp70 cochaperone regulates binding between CTA and the ER Hsp70 BiP, a chaperone previously implicated in toxin retrotranslocation. Importantly, ERdj5 interacts with the Hrd1 adaptor Sel1L directly through Sel1L's N-terminal lumenal domain, thereby linking ERdj5 to the Hrd1 complex. Sel1L itself also binds CTA and facilitates toxin retrotranslocation. By contrast, EDEM1 and OS-9, two established Sel1L binding partners, do not play significant roles in CTA1 retrotranslocation. Our results thus identify two ER factors that promote ER-to-cytosol transport of CTA1. They also indicate that ERdj5, by binding to Sel1L, triggers BiP-toxin interaction proximal to the Hrd1 complex. We postulate this scenario enables the Hrd1-associated retrotranslocation machinery to capture the toxin efficiently once the toxin is released from BiP.

Human Hsp40 proteins, DNAJA1 and DNAJA2, as potential targets of the immune response triggered by bacterial DnaJ in rheumatoid arthritis.

Hsp40 proteins of bacterial and human origin are suspected to be involved in the pathogenesis of rheumatoid arthritis (RA). It has been shown that sera of RA patients contain increased levels of antibodies directed to bacterial and human Hsp40s. The aim of this work was to explore immunological similarities between the bacterial (DnaJ) and human (DNAJA1 and DNAJA2) Hsp40 proteins in relation to their possible involvement in the RA. Using polyclonal antibodies directed against a full-length DnaJ or its domains, against DNAJA1 and DNAJA2, as well as monoclonal anti-DnaJ antibodies, we found immunological similarities between the bacterial and human Hsp40s. Both ELISA and Western blotting showed that these similarities were not restricted to the conserved J domains but were also present in the C-terminal variable regions. We also found a positive correlation between the levels of the anti-DnaJ and anti-DNAJA1 antibodies in the sera of RA patients. This finding supports the molecular mimicry hypothesis that human Hsp40 could be the targets of antibodies originally directed against bacterial DnaJ in RA.

Multiplex immune-related genes expression analysis response to bacterial challenge in mud crab, Scylla paramamosain.

Crabs lack an acquired adaptive immune system and host defense is believed to depend entirely on innate, non-adaptive mechanisms to resist invasion by pathogens. Discovery of immune-related factors are helpful for understanding the molecular response of crabs to pathogens. The mud crab Scylla paramamosain is an important marine species for aquaculture in China because of its high nutritional value for humans. In recent years, the crab is prone to being infected by microbes with the enlargement of breeding scale. In this study, eight immune-related genes were analyzed by multiplex genes expression analysis using the GenomeLab GeXP analysis system (Beckman Coulter). The expression levels of all the detected genes rose after challenged by the live bacteria, but the levels of only four genes (C-type lectin, alpha 2-macroglobulin, HSP70 and thioredoxin 1) increased after challenge in heat-killed bacteria group. So the live bacteria were more effective in motivating expressions of immune factors than heat-killed bacteria. However, the transcript of C-type lectin firstly increased at 1 h after challenge in both heat-killed and live bacteria group. This indicated that C-type lectin was a quite susceptive immune factor responding to external pathogen. In group challenged by live bacteria, the genes of alpha 2-macroglobulin, HSP40, thioredoxin 1 and prophenoloxidase activating factor (PPAF) showed response earlier than the other genes. The rise of PPAF expression preceded prophenoloxidase (proPO), which suggested that PPAF might trigger production of proPO transcripts in the early stage of phenoloxidase reaction system. C-type lectin, proPO, thioredoxin 1, HSP40, and alpha 2-macroglobulin are very important immunity factors in response to bacterial infection. According to the result of heat-killed group, HSP70 is a sensitively inductive factor to foreign stimulus compared with the other genes. The multi-gene analysis presented an alternative approach for screening of immune-related genes, and provided a more global overview of genes transcript alteration in response to bacterial challenge.