GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

Heat Shock Protein 60 Restricts Release of Mitochondrial dsRNA to Suppress Hepatic Inflammation and Ameliorate Non-Alcoholic Fatty Liver Disease in Mice

Non-alcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease, consists of fat deposited (steatosis) in the liver due to causes besides excessive alcohol use. The folding activity of heat shock protein 60 (HSP60) has been shown to protect mitochondria from proteotoxicity under various types of stress. In this study, we investigated whether HSP60 could ameliorate experimental high-fat diet (HFD)-induced obesity and hepatitis and explored the potential mechanism in mice. The results uncovered that HSP60 gain not only alleviated HFD-induced body weight gain, fat accumulation, and hepatocellular steatosis, but also glucose tolerance and insulin resistance according to intraperitoneal glucose tolerance testing and insulin tolerance testing in HSP60 transgenic (HSP60Tg) compared to wild-type (WT) mice by HFD. Furthermore, overexpression of HSP60 in the HFD group resulted in inhibited release of mitochondrial dsRNA (mt-dsRNA) compared to WT mice. In addition, overexpression of HSP60 also inhibited the activation of toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and phosphorylated-interferon regulatory factor 3 (p-IRF3), as well as inflammatory biomarkers such as mRNA of il-1β and il-6 expression in the liver in response to HFD. The in vitro study also confirmed that the addition of HSP-60 mimics in HepG2 cells led to upregulated expression level of HSP60 and restricted release of mt-dsRNA, as well as downregulated expression levels of TLR3, MDA5, and pIRF3. This study provides novel insight into a hepatoprotective effect, whereby HSP60 inhibits the release of dsRNA to repress the TLR3/MDA5/pIRF3 pathway in the context of NAFLD or hepatic inflammation. Therefore, HSP60 may serve as a possible therapeutic target for improving NAFLD.

Proteomic Analysis of Retinal Tissue in an S100B Autoimmune Glaucoma Model

Glaucoma is a neurodegenerative disease that leads to damage of retinal ganglion cells and the optic nerve. Patients display altered antibody profiles and increased antibody titer, e.g., against S100B. To identify the meaning of these antibodies, animals were immunized with S100B. Retinal ganglion cell loss, optic nerve degeneration, and increased glial cell activity were noted. Here, we aimed to gain more insights into the pathophysiology from a proteomic point of view. Hence, rats were immunized with S100B, while controls received sodium chloride. After 7 and 14 days, retinae were analyzed through mass spectrometry and immunohistology. Using data-independent acquisition-based mass spectrometry, we identified more than 1700 proteins on a high confidence level for both study groups, respectively. Of these 1700, 43 proteins were significantly altered in retinae after 7 days and 67 proteins revealed significant alterations at 14 days. For example, α2-macroglobulin was found significantly increased not only by mass spectrometry analysis, but also with immunohistological staining in S100B retinae at 7 and 14 days. All in all, the identified proteins are often associated with the immune system, such as heat shock protein 60. Once more, these data underline the important role of immunological factors in glaucoma pathogenesis.

Zhen-Wu-Tang Induced Mitophagy to Protect Mitochondrial Function in Chronic Glomerulonephritis via PI3K/AKT/mTOR and AMPK Pathways

Chronic glomerulonephritis (CGN) is one of the major causes of end-stage kidney disease. Zhen-wu-tang (ZWT), as a famous Chinese herbal prescription, is widely used in China for CGN therapy in clinic. However, the mechanism of ZWT in CGN has not been fully understood. The present study explored the therapeutic effect and the underlying mechanism of ZWT on mitochondrial function in cationic bovine serum albumin (C-BSA)-induced CGN model rats and tumor necrosis factor (TNF-α)-damaged mouse podocytes. The renal functions were measured by serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathological changes and ultrastructure of kidney tissues were evaluated by periodic acid-Schiff (PAS) staining and transmission electron microscopy. The levels of antioxidases, including mitochondrial catalase (CAT), superoxide dismutase 2 (SOD2), and peroxiredoxin 3 (PRDX3), in CGN rats were examined by real-time PCR. The mitochondrial functions of podocytes were measured by ATP concentration, mitochondrial membrane potential (MMP), and mitochondrial ROS (mtROS). For mitophagy level detection, the expressions of mitophagy-related proteins, including LC3, p62, heat shock protein 60 (HSP60), and translocase of outer mitochondrial membrane 20 (TOMM20), were measured by Western blot, as the colocation of LC3 and mitochondrial marker COX IV were evaluated by immunofluorescence. Our results manifested that ZWT ameliorated CGN model rats by a remarkable decrease in Scr and BUN, inhibition of mesangial matrix proliferation, protection against foot processes fusion, and basement membrane thickening. More importantly, ZWT protected against mitochondrial dysfunction by increasing the expressions of CAT, SOD2, and PRDX3 in CGN model rats, increased ATP content and MMP in podocytes, and decreased excessive mtROS. Furthermore, ZWT induced mitophagy in CGN through increasing the expression of LC3, and decreasing p62, HSP60, TOMM20, and ZWT also enhanced the colocation of LC3 to the mitochondria. We found that ZWT inhibited the PI3K/AKT/mTOR pathway, which could be disturbed by PI3K inhibitor LY294002 and agonist insulin-like growth factor 1. Moreover, ZWT reversed the inhibition of the AMPK pathway in CGN. Overall, ZWT ameliorated renal mitochondrial dysfunction probably by inducing mitophagy via the PI3K/AKT/mTOR and AMPK pathways.

Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

The Heat Shock Protein 60 and Pap1 Participate in the Sporothrix schenckii-Host Interaction

Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 μg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen–host interaction.

Identification, Characterization, and Antioxidant Potential of Bifidobacterium longum subsp. longum Strains Isolated From Feces of Healthy Infants

Increasing evidence has indicated that oxidative stress is associated with the health of infants. Bifidobacterium, especially B. longum subsp. longum strains, are abundant in the gut microbiota of infants, which may have the potential to ameliorate oxidative damage. Thus, this study aimed to isolate and screen B. longum subsp. longum strains with probiotic characters and antioxidant properties as infants’ dietary supplements. In this study, 24 B. longum subsp. longum strains were isolated from 15 healthy infants identified via 16S rRNA and heat shock protein 60 (hsp60) sequences. B. longum subsp. longum B13, F2, K4, K5, K10, K13, and K15 strains were selected based on high values obtained from autoaggregation, hydrophobicity, and adhesion assays to HT-29 cells. Among these seven strains, B. longum subsp. longum F2, K5, K10, and K15 were selected according to the high tolerance of gastrointestinal tract conditions compared to Bifidobacterium animalis subsp. lactis BB-12. Among these four strains, B. longum subsp. longum K5 was susceptible to common antibiotics and showed the highest intestinal epithelial cell proliferation of CCD 841 CoN. Additionally, B. longum subsp. longum K5 showed a strong antioxidant capacity, and its supernatant exhibited better activity of reducing power, hydroxyl radical scavenging, and DPPH radical scavenging than that of the intact cells with cell-free extracts. The findings indicated that B. longum subsp. longum K5 could be used as a probiotic candidate in infant nutrition.

First Report of Sclerotinia Blight on Peanut Caused by Sclerotinia sclerotiorum in Qinghai Province, China

Historically, peanut has not been produced in Qinghai province located in Northwest China because of the high elevation and cold climates. However, since 2020 field studies have been conducted to evaluate peanut cultivars for suitability to field production. In 2020, peanut cultivation was successful for the first time in Haidong city, Qinghai province, China. In August 2020, brown, irregular-shaped lesions were observed on peanut stems from Qinghai province in China. In the early stage, the watersoaked spots were formed on the stems, then lesions expanded rapidly and became brown. In advanced stages of the disease, stems became bleached and eventually died. The inside of the stems was rotten and hollow, and the diseased stem wilted and died. White hyphae and black irregular shaped sclerotia were observed on the infected stems. Finally, local or whole plant rotted and died at the end. Approximately 10% of the plants in a field were infected. Symptomatic stems were cut into small pieces, disinfected with 75% ethanol for 1 minute, 0.5% NaClO for two minutes, and sterile water for three times. Pieces then were plated on potato dextrose agar (PDA) media and incubated at 25°C in darkness. Fungal colonies were initially white, becoming gray, then black sclerotia (2.4 to 6.0 mm in diameter) were appeared at the edge of colonies. Genomic DNA of the pure cultures of an isolate (ZHX7) was extracted and PCR was carried out using glyceraldehydes-3-phosphate dehydrogenase gene (G3PDH) region primers G3PDH-F/G3PDH-R, heat-shock protein 60 gene (HSP60) region primers HSP60-F/HSP60-R, and DNA-dependent RNA polymerase subunit gene (RPB2) region primers RPB2-F/RPB2-R (Staats et al., 2005), respectively. G3PDH region (Accession No. MZ388475) showed 99.44% sequence identity (887 bp out of 909 bp) to Sclerotinia sclerotiorum (Accession No. AJ705044, 887 bp out of 887 bp). HSP60 region (Accession No. MZ388476) showed 99.90% sequence identity (972 bp out of 984bp) to S. sclerotiorum (Accession No. AJ716048, 972 bp out of 980 bp). RPB2 region (Accession No. MZ388477) showed 100.00% sequence identity (1096 bp out of 1129 bp) to S. sclerotiorum (Accession No. AJ745716, 1096 bp out of 1096 bp). Phylogenetic analysis was done using Neighbor-Joining (NJ) analysis based on those gene sequences. The isolate was identified as S. sclerotiorum based on molecular analysis and morphological characteristics. For pathogenicity assay, ten-days-old potted peanut (Luhua No.12) seedlings were inoculated with one mycelial plug (8 mm in diameter ) by placing the inoculum on the base of the stem in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). All inoculated seedlings exhibited typical basal stem rot, and root showed different degrees of damage, and wilted 5 days after inoculation. No symptoms were observed on control plants treated with sterile distilled mycelial plugs, and S. sclerotiorum was consistently re-isolated from symptomatic tissue. S. sclerotiorum has been reported on peanut in Northeastern China (Yan et al., 2005). To our knowledge, this is the first report of S. sclerotiorum causing Sclerotinia Blight on peanut in Qinghai province, China. The peanut planting area in Qinghai has been further expanded this year, and S. sclerotiorum has a broad host range (Boland and Hall, 1994), so Sclerotinia Blight is a potential threat to peanut production, and as a result, it is critical for commercial producers to monitor plants for S. sclerotiorum.

First Report of Post-Harvest Gray Mold on Hyacinth bean (Lablab purpureus) Caused by Botrytis cinerea in Korea

Hyacinth bean (Lablab purpureus L.) is a highly proteineous legume under the family Fabaceae. It is native to Africa, cultivated throughout the world, and recently introduced vegetable in Korea. In April 2020, approximately 10 to 15% of the total harvested pods showed gray mold rot symptoms after 3–5 days of storage at 4 °C in Jeonju, Jeonbuk province, Korea. The symptoms observed were irregular, water-soaked spots become brown or gray with white hyphae were appeared on the infected pods. Diseased tissue was excised, and surface sterilized by immersing in 1% sodium hypochlorite (NaOCl) for 1 min, rinsed three times with sterilized distilled water, placed on potato dextrose agar (PDA) plates, and incubated at 20 ± 2°C for 7 days. A total of five morphologically similar fungal isolates (HBGM001 to HBGM005) were obtained from diseased samples; isolate HBGM002 and HBGM005 were selected for identification. The fungus produced initially white colonies, after 7 days it changes to gray to dark colonies with dark mycelium that sporulated abundantly on PDA at 20ºC. The conidia (n = 50) were single-celled, ellipsoid or ovoid in shape, and 6.11 to 13.9 × 4.8 to 9.4 μm in size for HBGM001 isolate and 5.81 to 14.1× 4.5 to 9.6 μm in size for HBGM005. Conidiophores (n = 15) arose solitary or in groups, straight or flexuous, septate, with an inflated basal cell brown to light brown, and measured 103 to 420× 7 to 25 μm for HBGM001 isolate and 101 to 415 × 5 to 23 μm for HBGM005 isolate. After two weeks, the fungus formed several black sclerotia (n = 20) ranging from 0.5 to 4.2 × 0.5 to 3.4 mm for HBGM001 isolate and 0.4 to 4.4 × 0.3 to 3.3 mm for HBGM005 isolate near the edge of the Petri dish. Morphological characters were consistent with those of Botrytis cinerea Pers.: Fr. (Ellis 1971). As for molecular identification, the internal transcribed spacer (ITS) and three nuclear protein-coding genes (glyceraldehydes-3-phosphate dehydrogenase gene [G3PDH], heat-shock protein 60 gene [HSP60], and DNA-dependent RNA polymerase subunit gene [RPB2]) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), G3PDH-F/G3PDH-R, HSP60-F/HSP60-R, and RPB2-F/RPB2-R (Staats et al. 2005), respectively. The ITS, G3PDH, HSP60, and RPB2 sequences of HBGM002 and HBGM005 isolates (GenBank accession number MT439648 and MT968495 for ITS; MT439649 and MT968496 for G3PDH; MT439650 and MT968497 for HSP60; MT439651 and MT968498 for RPB2 respectively) were 99% to 100% identical to those of B. cinerea (KY364366, KF015583, KJ018758, and KJ018756, respectively). To determine pathogenicity, five disinfected pods were pinpricked (3 sites per pod) with sterile needles and 50 µl of conidial suspension (1 × 105 conidia/ml) was inoculated by pipetting into the wounds. An analogous five pods, serving as controls, were inoculated with sterile distilled water. All the pods were placed in a growth chamber and maintained a temperature of 20±2ºC and a relative humidity >80%. After 5 days, gray mold symptoms developed on the inoculated pods, whereas no symptoms appeared on control pods. The pathogen was re-isolated from the inoculated pods, fulfilling Koch’s postulates. B. cinerea has been reported causing gray mold in Hyacinth bean in China, Taiwan and India (Farr and Rossman 2021). To our knowledge, this is the first report of B. cinerea causing post-harvest gray mold on hyacinth bean in Korea. The disease could represent a threat for hyacinth bean post-harvest and storage and management strategies should be investigated and applied.

Characterization of Heat Shock Protein 60 as an Interacting Partner of Superoxide Dismutase 2 in the Silkworm, Bombyx mori, and Its Response to the Molting Hormone, 20-Hydroxyecdysone

Oxidative stress promotes pupation in some holometabolous insects. The levels of superoxide, a reactive oxygen species (ROS), are increased and superoxide dismutase 1 (BmSod1) and superoxide dismutase 2 (BmSod2) are decreased during metamorphic events in silkworm (Bombyx mori). These observations strongly suggest that pupation is initiated by oxidative stress via the down-regulation of BmSod1 and BmSod2. However, the molecular mechanisms underlying ROS production during metamorphic events in silkworm remain unknown. To investigate these molecular mechanisms, the peripheral proteins of BmSod1 and BmSod2 were identified and characterized using dry and wet approaches in this study. Based on the results, silkworm heat shock protein 60 (BmHsp60) was identified as an interacting partner of BmSod2, which belongs to the Fe/MnSOD family. Furthermore, the present study results showed that BmHsp60 mRNA expression levels were increased in response to oxidative stress caused by ultraviolet radiation and that BmHsp60 protein levels (but not mRNA levels) were decreased during metamorphic events, which are regulated by the molting hormone 20-hydroxyecdysone. These findings improve our understanding of the mechanisms by which holometabolous insects control ROS during metamorphosis.