Evaluation of Immunodiagnostic Performances of Neospora caninum Peroxiredoxin 2 (NcPrx2), Microneme 4 (NcMIC4), and Surface Antigen 1 (NcSAG1) Recombinant Proteins for Bovine Neosporosis.

Bovine neosporosis is among the main causes of abortion in cattle worldwide, causing serious economic losses in the beef and dairy industries. A highly sensitive and specific diagnostic method for the assessment of the epidemiology of the disease, as well as it surveillance and management, is imperative, due to the absence of an effective treatment or vaccine against neosporosis. In the present study, the immunodiagnostic performance of Neospora caninum peroxiredoxin 2 (NcPrx2), microneme 4 (NcMIC4), and surface antigen 1 (NcSAG1) to detect IgG antibodies against N. caninum in cattle were evaluated and compared with that of the indirect fluorescent antibody test (IFAT). The results revealed that NcSAG1 had the highest sensitivity and specificity, with values of 88.4% and 80.7%, respectively, followed by NcPrx2, with a high sensitivity of 87.0% but a low specificity of 67.0%, whereas NcMIC4 showed sensitivity and specificity of 84.1% and 78.9%, respectively, when compared with IFAT. A high degree of agreement was observed for NcSAG1 (k = 0.713) recombinant protein, showing the highest diagnostic capability, followed by NcMIC4 (k = 0.64) and NcPrx2 (k = 0.558). The present study demonstrates that NcSAG1 is helpful as an antigen marker and also demonstrates the potential immunodiagnostic capabilities of NcPrx2 and NcMIC4, which could serve as alternative diagnostic markers for detecting N. caninum infection in cattle. These markers may find utility in future treatment management, surveillance, and risk assessment of neosporosis in livestock or other animal host species. Further research should be directed toward understanding the in vivo immune response differences resulting from immunization with both recombinant proteins.

Metabolite profiling of Trichinella spiralis adult worms and muscle larvae identifies their excretory and secretory products.

Human trichinellosis is a parasitic infection caused by roundworms belonging to the genus Trichinella, especially Trichinella spiralis. Early and accurate clinical diagnoses of trichinellosis are required for efficacious prognosis and treatment. Current drug therapies are limited by antiparasitic resistance, poor absorption, and an inability to kill the encapsulating muscle-stage larvae. Therefore, reliable biomarkers and drug targets for novel diagnostic approaches and anthelmintic drugs are required. In this study, metabolite profiles of T. spiralis adult worms and muscle larvae were obtained using mass spectrometry-based metabolomics. In addition, metabolite-based biomarkers of T. spiralis excretory-secretory products and their related metabolic pathways were characterized. The metabolic profiling identified major, related metabolic pathways involving adenosine monophosphate (AMP)-dependent synthetase/ligase and glycolysis/gluconeogenesis in T. spiralis adult worms and muscle larvae, respectively. These pathways are potential drug targets for the treatment of the intestinal and muscular phases of infection. The metabolome of larva excretory-secretory products was characterized, with amino acid permease and carbohydrate kinase being identified as key metabolic pathways. Among six metabolites, decanoyl-l-carnitine and 2,3-dinor-6-keto prostaglandin F1α-d9 were identified as potential metabolite-based biomarkers that might be related to the host inflammatory processes. In summary, this study compared the relationships between the metabolic profiles of two T. spiralis growth stages. Importantly, the main metabolites and metabolic pathways identified may aid the development of novel clinical diagnostics and therapeutics for human trichinellosis and other related helminthic infections.

Sericin coated thin polymeric films reduce keratinocyte proliferation via the mTOR pathway and epidermal inflammation through IL17 signaling in psoriasis rat model.

Therapeutic treatment forms can play significant roles in resolving psoriatic plaques or promoting wound repair in psoriatic skin. Considering the biocompatibility, mechanical strength, flexibility, and adhesive properties of silk fibroin sheets/films, it is useful to combine them with anti-psoriatic agents and healing stimulants, notably silk sericin. Here, we evaluate the curative properties of sericin-coated thin polymeric films (ScF) fabricated from silk fibroin, using an imiquimod-induced psoriasis rat model. The film biocompatibility and psoriatic wound improvement capacity was assessed. A proteomics study was performed to understand the disease resolving mechanisms. Skin-implantation study exhibited the non-irritation property of ScF films, which alleviate eczema histopathology. Immunohistochemical and gene expression revealed the depletion of ß-defensin, caspase-3 and -9, TNF-α, CCL-20, IL-1ß, IL-17, TGF-ß, and Wnt expressions and S100a14 mRNA level. The proteomics study suggested that ScF diminish keratinocyte proliferation via the mTOR pathway by downregulating mTOR protein, corresponding to the modulation of TNF-α, Wnt, and IL-1ß levels, leading to the enhancement of anti-inflammatory environment by IL-17 downregulation. Hematology data demonstrated the safety of using these biomaterials, which provide a potential therapeutic-option for psoriasis treatment due to desirable effects, especially anti-proliferation and anti-inflammation, functioning via the mTOR pathway and control of IL-17 signaling.

Mass Spectrometry-Based Metabolomics Revealed Effects of Metronidazole on Giardia duodenalis.

Giardia duodenalis is a significant protozoan that affects humans and animals. An estimated 280 million G. duodenalis diarrheal cases are recorded annually. Pharmacological therapy is crucial for controlling giardiasis. Metronidazole is the first-line therapy for treating giardiasis. Several metronidazole targets have been proposed. However, the downstream signaling pathways of these targets with respect to their antigiardial action are unclear. In addition, several giardiasis cases have demonstrated treatment failures and drug resistance. Therefore, the development of novel drugs is an urgent need. In this study, we performed a mass spectrometry-based metabolomics study to understand the systemic effects of metronidazole in G. duodenalis. A thorough analysis of metronidazole processes helps identify potential molecular pathways essential for parasite survival. The results demonstrated 350 altered metabolites after exposure to metronidazole. Squamosinin A and N-(2-hydroxyethyl)hexacosanamide were the most up-regulated and down-regulated metabolites, respectively. Proteasome and glycerophospholipid metabolisms demonstrated significant differential pathways. Comparing glycerophospholipid metabolisms of G. duodenalis and humans, the parasite glycerophosphodiester phosphodiesterase was distinct from humans. This protein is considered a potential drug target for treating giardiasis. This study improved our understanding of the effects of metronidazole and identified new potential therapeutic targets for future drug development.

Untargeted serum metabolomics analysis of Trichinella spiralis-infected mouse.

BACKGROUND: Trichinellosis, caused by a parasitic nematode of the genus Trichinella, is a zoonosis that affects people worldwide. After ingesting raw meat containing Trichinella spp. larvae, patients show signs of myalgia, headaches, and facial and periorbital edema, and severe cases may die from myocarditis and heart failure. The molecular mechanisms of trichinellosis are unclear, and the sensitivity of the diagnostic methods used for this disease are unsatisfactory. Metabolomics is an excellent tool for studying disease progression and biomarkers; however, it has never been applied to trichinellosis. We aimed to elucidate the impacts of Trichinella infection on the host body and identify potential biomarkers using metabolomics. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with T. spiralis larvae, and sera were collected before and 2, 4, and 8 weeks after infection. Metabolites in the sera were extracted and identified using untargeted mass spectrometry. Metabolomic data were annotated via the XCMS online platform and analyzed with Metaboanalyst version 5.0. A total of 10,221 metabolomic features were identified, and the levels of 566, 330, and 418 features were significantly changed at 2-, 4-, and 8-weeks post-infection, respectively. The altered metabolites were used for further pathway analysis and biomarker selection. A major pathway affected by Trichinella infection was glycerophospholipid metabolism, and glycerophospholipids comprised the main metabolite class identified. Receiver operating characteristic revealed 244 molecules with diagnostic power for trichinellosis, with phosphatidylserines (PS) being the primary lipid class. Some lipid molecules, e.g., PS (18:0/19:0)[U] and PA (O-16:0/21:0), were not present in metabolome databases of humans and mice, thus they may have been secreted by the parasites. CONCLUSIONS/SIGNIFICANCE: Our study highlighted glycerophospholipid metabolism as the major pathway affected by trichinellosis, hence glycerophospholipid species are potential markers of trichinellosis. The findings of this study represent the initial steps in biomarker discovery that may benefit future trichinellosis diagnosis.

Discovery of Schistosoma mekongi circulating proteins and antigens in infected mouse sera.

Schistosomiasis is a neglected tropical disease caused by an infection of the parasitic flatworms schistosomes. Schistosoma mekongi is a restricted Schistosoma species found near the Mekong River, mainly in southern Laos and northern Cambodia. Because there is no vaccine or effective early diagnosis available for S. mekongi, additional biomarkers are required. In this study, serum biomarkers associated with S. mekongi-infected mice were identified at 14-, 28-, 42-, and 56-days post-infection. Circulating proteins and antigens of S. mekongi in mouse sera were analyzed using mass spectrometry-based proteomics. Serine protease inhibitors and macrophage erythroblast attacher were down-regulated in mouse sera at all infection timepoints. In addition, 54 circulating proteins and 55 antigens of S. mekongi were identified. Notable circulating proteins included kyphoscoliosis peptidase and putative tuberin, and antigens were detected at all four infection timepoints, particularly in the early stages (12 days). The putative tuberin sequence of S. mekongi was highly similar to homologs found in other members of the genus Schistosoma and less similar to human and murine sequences. Our study provided the identity of promising diagnostic biomarkers that could be applicable in early schistosomiasis diagnosis and vaccine development.

Proteomic Analysis Reveals Distinct Protein Corona Compositions of Citrate- and Riboflavin-Coated SPIONs.

Superparamagnetic iron oxide nanoparticles (SPIONs) are recognized as one of the most beneficial tools for biomedicine, especially in theranostic applications. Even though SPIONs have excellent properties regarding their biocompatibility and unique magnetic properties, they lack stability in biological fluids. To stabilize and increase the specificity of the SPIONs to target desirable cells or tissues, several surface coatings have been introduced. These surface coatings can lead to different preferences of serum protein bindings, which ultimately determine their behaviors in vitro and in vivo. Thus, understanding the interaction of SPIONs with biological systems is important for their biocompatible design and clinical applications. In this study, using proteomic analyses, we analyzed the protein corona fingerprints on SPIONs with two different coatings, including citrate and riboflavin, that have been widely used as surface coatings and ligands for enhancing cellular uptake in breast cancer cells. Though both citrate-coated SPIONs (C-SPIONs) and riboflavin-coated SPIONs (Rf-SPIONs) showed similar sizes and zeta potentials, we found that Rf-SPIONs adsorbed more serum proteins than bare SPIONs (B-SPIONs) or C-SPIONs, which was likely due to the higher hydrophobicity of the riboflavin. The enriched proteins consisted mainly of immune-responsive and blood coagulation proteins with different fingerprint profiles. Cellular uptake studies in MCF-7 breast cancer cells comparing the activities of preformed and in situ coronas showed different uptake behaviors, suggesting the role of protein corona formation in promoting the interaction between the SPIONs and the cells. The results obtained here provide the essential information for further development of the potential strategy to reduce or stimulate immune response in vivo to increase therapeutic applications of both C-SPIONs and Rf-SPIONs.

Protein and antigen profiles of third-stage larvae of Gnathostoma spinigerum assessed with next-generation sequencing transcriptomic information.

Gnathostomiasis is a food-borne zoonotic disease that can affect humans who eat improperly cooked meat containg infective third-stage larvae. Definitive diagnosis is through larval recovery. However, this is an invasive technique and is impractical if the larvae have encysted in inaccessible areas of the body. Antigen or antibody detection might be more interesting techniques for diagnosis. Proteomic could elucidate diagnostic markers and improve our understanding of parasite biology. However, proteomic studies on Gnathostoma spinigerum are hampered by the lack of a comprehensive database for protein identification. This study aimed to explore the protein and antigen profiles of advanced third-stage G. spinigerum larvae (aL3Gs) using interrogation of mass spectrometry data and an in-house transcriptomic database for protein identification. Immunoproteomic analysis found 74 proteins in 24-kDa SDS-PAGE bands, which is size-specific for the immunodiagnosis of gnathostomiasis. Moreover, 13 proteins were found in 2-DE 24-kDa bands. The data suggest that collagenase 3, cathepsin B, glutathione S-transferase 1, cuticle collagen 14, major antigen, zinc metalloproteinase nas-4, major egg antigen, peroxiredoxin, and superoxide dismutase [Cu-Zn] may be good candidates for novel human gnathostomiasis diagnostic assays. These findings improve our understanding of the parasite's biology and provide additional potential targets for novel therapeutics, diagnostics, and vaccines.

Identification and profiling of Trichinella spiralis circulating antigens and proteins in sera of mice with trichinellosis.

Trichinellosis is a zoonotic disease caused by the ingestion of the Trichinella nematode. With a worldwide incidence of approximately 10,000 cases per year, Trichinella spiralis is responsible for most human infections. There are no specific signs or symptoms of this parasitic infection. Muscle biopsy is the gold diagnostic standard for trichinellosis, but the technique is invasive and unable to detect the early stage of infection. Although immunodiagnostics are also available, antibody detection usually occurs after 3 weeks and prolonged up to 19 years after the acute phase. Therefore, additional diagnostic biomarkers must be identified to improve trichinellosis diagnosis. This study aimed to measure concentration changes in mouse serum proteins prior to T. spiralis infection and 2, 4 and 8 weeks after infection, and to identify T. spiralis circulating proteins and antigens using mass spectrometry-based proteomics. Mouse muscle-related proteins including inter-alpha-trypsin inhibitor heavy chain H2, a protein involved in the response to muscle tissue damage, were up-regulated in mouse sera during the T. spiralis larvae invasion. Additionally, 33 circulatory parasite proteins were identified in infected mouse sera. Notably, T. spiralis long-chain fatty acid transport protein 1 could be detected in the early stage of infection and peroxidasin-like protein was identified 2, 4 and 8 weeks after infection. Seventeen T. spiralis circulating antigens were detected in mouse immune complexes, with PX domain protein being found 2, 4 and 8 weeks after infection. Because peroxidasin-like protein and PX domain protein were detected at all post-infection time points, sequence alignments of these proteins were performed, which showed they are conserved among Trichinella spp. and have less similarity to the human and murine sequences. Integrative analysis of T. spiralis biomarkers throughout the course of infection may reveal additional diagnostic targets to improve early diagnosis of trichinellosis.

Cytoskeletal Alteration Is an Early Cellular Response in Pulmonary Epithelium Infected with Aspergillus fumigatus Rather than Scedosporium apiospermum.

Invasive aspergillosis and scedosporiosis are life-threatening fungal infections with similar clinical manifestations in immunocompromised patients. Contrarily, Scedosporium apiospermum is susceptible to some azole derivative but often resistant to amphotericin B. Histopathological examination alone cannot diagnose these two fungal species. Pathogenesis studies could contribute to explore candidate protein markers for new diagnosis and treatment methods leading to a decrease in mortality. In the present study, proteomics was conducted to identify significantly altered proteins in A549 cells infected with or without Aspergillus fumigatus and S. apiospermum as measured at initial invasion. Protein validation was performed with immunogold labelling alongside immunohistochemical techniques in infected A549 cells and lungs from murine models. Further, cytokine production was measured, using the Bio-Plex-Multiplex immunoassay. The cytoskeletal proteins HSPA9, PA2G4, VAT1, PSMA2, PEX1, PTGES3, KRT1, KRT9, CLIP1 and CLEC20A were mainly changed during A. fumigatus infection, while the immunologically activated proteins WNT7A, GAPDH and ANXA2 were principally altered during S. apiospermum infection. These proteins are involved in fungal internalisation and structural destruction leading to pulmonary disorders. Interleukin (IL)-21, IL-1α, IL-22, IL-2, IL-8, IL-12, IL-17A, interferon-γ and tumour necrosis factor-α were upregulated in both aspergillosis and scedosporiosis, although more predominately in the latter, in accordance with chitin synthase-1 and matrix metalloproteinase levels. Our results demonstrated that during invasion, A. fumigatus primarily altered host cellular integrity, whereas S. apiospermum chiefly induced and extensively modulated host immune responses.

Curcuminoid supplementation in canine diabetic mellitus and its complications using proteomic analysis.

Introduction: Inflammation and oxidative stress contribute to diabetes pathogenesis and consequences. Therapeutic approaches for canine diabetes remain a challenge. Curcumin has anti-inflammatory and anti-oxidative effects and is beneficial for humans with diabetes mellitus (DM); however, data on its impact on canine diabetes is limited. This study aimed to evaluate the potential for causing adverse effects, anti-inflammatory effects, anti-oxidative effects and proteomic patterns of curcuminoid supplementation on canine DM. Methods: Altogether, 18 dogs were divided into two groups: DM (n = 6) and healthy (n = 12). Curcuminoid 250 mg was given to the DM group orally daily for 180 days. Blood and urine sample collection for hematological parameters, blood biochemistry, urinalysis, oxidative stress parameters, inflammatory markers and proteomics were performed every 6 weeks. Results and discussion: Curcuminoid supplementation with standard therapy significantly decreased oxidative stress with the increased glutathione/oxidized glutathione ratio, but cytokine levels were unaffected. According to the proteomic analysis, curcuminoid altered the expression of alpha-2-HS-glycoprotein, transthyretin, apolipoprotein A-I and apolipoprotein A-IV, suggesting that curcuminoid improves insulin sensitivity and reduces cardiovascular complications. No negative impact on clinical symptoms, kidneys or liver markers was identified. This study proposed that curcuminoids might be used as a targeted antioxidant strategy as an adjunctive treatment to minimize diabetes complications in dogs.

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

Proteomics of Gnathostomiasis: A Way Forward for Diagnosis and Treatment Development.

Gnathostoma spinigerum is the most common cause of gnathostomiasis in humans. It has a complex life cycle, which requires two intermediate hosts and a definitive host, and poses a high risk for zoonosis. Definitive prognosis of gnathostomiasis relies mainly on the isolation of advanced-stage larvae (aL3), which is very challenging especially if the aL3 is sequestered in difficult-to-reach organs. There is also a lack of a confirmatory diagnostic test for gnathostomiasis. With the ongoing advancement of proteomics, a potential diagnostic approach is underway using immunoproteomics and immunodiagnostics. In addition to this, the employment of mass spectrometry could further elucidate not only understanding the biology of the parasite but also determining potential targets of prospective drugs and vaccines. This article reports the past, present, and future application of proteomics in the study of gnathostomiasis.

Identification of Low Molecular Weight Proteins and Peptides from Schistosoma mekongi Worm, Egg and Infected Mouse Sera.

Schistosoma mekongi is found in the lower Mekong river region and causes schistosomiasis. Low sensitivity of diagnosis and development of drug resistance are problems to eliminate this disease. To develop novel therapies and diagnostics for S. mekongi, the basic molecular biology of this pathogen needs to be explored. Bioactive peptides have been reported in several worms and play important roles in biological functions. Limited information is available on the S. mekongi peptidome. Therefore, this study aimed to identify S. mekongi peptides using in silico transcriptome mining and mass spectrometry approaches. Schistosoma peptide components were identified in adult worms, eggs, and infected mouse sera. Thirteen neuropeptide families were identified using in silico predictions from in-house transcriptomic databases of adult S. mekongi worms. Using mass spectrometry approaches, 118 peptides (from 54 precursor proteins) and 194 peptides (from 86 precursor proteins) were identified from adult worms and eggs, respectively. Importantly, eight unique peptides of the S. mekongi ubiquitin thioesterase, trabid, were identified in infected mouse sera 14, 28, and 56 days after infection. This protein may be a potential target for diagnosis of schistosomiasis. The S. mekongi peptide profiles determined in this study could be used for further drug and diagnostic development.

Potential benefits of L. acidophilus in dyslipidemic rats.

Several studies have shown that probiotics and synbiotics ameliorate dyslipidemia. However, the molecular mechanisms mediating their effects remain to be determined. Therefore, we aimed to compare the effects of a probiotic, a prebiotic, and a synbiotic in dyslipidemic Sprague-Dawley rats, and explore the mechanisms involved using a proteomic approach. The rats were allocated to five groups: a control group that was fed normal chow, and four high-fat diet-fed groups, three of which were administered a probiotic (Lactobacillus acidophilus), a prebiotic (inulin), or a combination of the two (a synbiotic) for 30 days. We showed that the administration of inulin, and especially L. acidophilus, improved the lipid profile and reduced the serum concentrations of inflammatory markers in high-fat diet-fed rats. Proteomic analysis showed changes in lipid elongation, glycerolipid metabolism, activation of antioxidants, and a reduction in the activation of the mitogen-activated protein kinase signaling pathway in the livers of rats administered L. acidophilus, which likely mediate its beneficial effects on inflammation and dyslipidemia by reduced the levels of 18.56% CRP, 35.71% TNF-α 25.6% LDL-C and 28.57% LDL-C/HDL-C ratio when compared to HF group. L. acidophilus and inulin may represent effective natural means of maintaining inflammation and dyslipidemia.

Effect of Praziquantel on Schistosoma mekongi Proteome and Phosphoproteome.

Schistosoma mekongi causes schistosomiasis in southeast Asia, against which praziquantel (PZQ) is the only treatment option. PZQ resistance has been reported, thus increasing the requirement to understand mechanism of PZQ. Herein, this study aimed to assess differences in proteome and phosphoproteome of S. mekongi after PZQ treatment for elucidating its action. Furthermore, key kinases related to PZQ effects were predicted to identify alternative targets for novel drug development. Proteomes of S. mekongi were profiled after PZQ treatment at half maximal inhibitory concentration and compared with untreated worms. A total of 144 proteins were differentially expressed after treatment. In parallel, immunohistochemistry indicated a reduction of phosphorylation, with 43 phosphoproteins showing reduced phosphorylation, as identified by phosphoproteomic approach. Pathway analysis of mass spectrometric data showed that calcium homeostasis, worm antigen, and oxidative stress pathways were influenced by PZQ treatment. Interestingly, two novel mechanisms related to protein folding and proteolysis through endoplasmic reticulum-associated degradation pathways were indicated as a parasiticidal mechanism of PZQ. According to kinase-substrate predictions with bioinformatic tools, Src kinase was highlighted as the major kinase related to the alteration of phosphorylation by PZQ. Interfering with these pathways or applying Src kinase inhibitors could be alternative approaches for further antischistosomal drug development.

Altered proteome of a Burkholderia pseudomallei mutant defective in short-chain dehydrogenase affects cell adhesion, biofilm formation and heat stress tolerance.

Burkholderia pseudomallei is a Gram-negative bacillus that causes melioidosis and is recognized as an important public health problem in southeast Asia and northeast Australia. The treatment of B. pseudomallei infection is hampered by resistance to a wide range of antimicrobial agents and no vaccine is currently available. At present, the underlying mechanisms of B. pseudomallei pathogenesis are poorly understood. In our previous study, we reported that a B. pseudomallei short-chain dehydrogenase (SDO; BPSS2242) mutant constructed by deletion mutagenesis showed reduced B. pseudomallei invasion and initial intracellular survival. This indicated that SDO is associated with the pathogenesis of melioidosis. In the present study, the role of B. pseudomallei SDO was further investigated using the SDO deletion mutant by a proteomic approach. The protein profiles of the SDO mutant and wild-type K96243 were investigated through gel-based proteomic analysis. Quantitative intensity analysis of three individual cultures of the B. pseudomallei SDO mutant revealed significant down-regulation of five protein spots compared with the wild-type. Q-TOF MS/MS identified the protein spots as a glutamate/aspartate ABC transporter, prolyl-tRNA synthetase, Hsp70 family protein, quinone oxidoreductase and a putative carboxypeptidase. Functional assays were performed to investigate the role of these differentially expressed proteins in adhesion to host cells, biofilm induction and survival under heat stress conditions. The SDO deletion mutant showed a decreased ability to adhere to host cells. Moreover, biofilm formation and the survival rate of bacteria under heat stress conditions were also reduced in the mutant strain. Our findings provide insight into the role of SDO in the survival and pathogenesis of B. pseudomallei at the molecular level, which may be applied to the prevention and control of B. pseudomallei infection.

Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni).

The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry-based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims.

Proteomic analysis of adult Schistosoma mekongi somatic and excretory-secretory proteins.

Schistosoma mekongi is a causative agent of human schistosomiasis. There is limited knowledge of the molecular biology of S. mekongi and very few studies have examined drug targets, vaccine candidates and diagnostic biomarkers for S. mekongi. To explore the biology of S. mekongi, computational as well as experimental approaches were performed on S. mekongi males and females to identify excretory-secretory (ES) proteins and proteins that are differentially expressed between genders. According to bioinformatic prediction, the S. mekongi ES product was approximately 4.7% of total annotated transcriptome sequences. The classical secretory pathway was the main process to secrete proteins. Mass spectrometry-based quantification of male and female adult S. mekongi proteins was performed. We identified 174 and 156 differential expression of proteins in male and female worms, respectively. The dominant male-biased proteins were involved in actin filament-based processes, microtubule-based processes, biosynthetic processes and homeostatic processes. The major female-biased proteins were related to biosynthetic processes, organelle organization and signal transduction. An experimental approach identified 88 proteins in the S. mekongi secretome. The S. mekongi ES proteins mainly contributed to nutrient uptake, essential substance supply and host immune evasion. This research identifies proteins in the S. mekongi secretome and provides information on ES proteins that are differentially expressed between S. mekongi genders. These findings will contribute to S. mekongi drug and vaccine development. In addition, the study enhances our understanding of basic S. mekongi biology.

Phosphoproteomics analysis of male and female Schistosoma mekongi adult worms.

Schistosoma mekongi is one of the major causative agents of human schistosomiasis in Southeast Asia. Praziquantel is now the only drug available for treatment and there are serious concerns about parasite resistance to it. Therefore, a dataset of schistosome targets is necessary for drug development. Phosphorylation regulates signalling pathways to control cellular processes that are important for the parasite's growth and reproduction. Inhibition of key phosphoproteins may reduce the severity of schistosomiasis. In this research, we studied the phosphoproteomes of S. mekongi male and female adult worms by using computational and experimental approaches. Using a phosphoproteomics approach, we determined that 88 and 44 phosphoproteins were male- and female-biased, respectively. Immunohistochemistry using anti-phosphoserine antibodies demonstrated phosphorylation on the tegument and muscle of male S. mekongi worms and on the vitelline gland and gastrointestinal tract of female worms. This research revealed S. mekongi sex-dependent phosphoproteins. Our findings provide a better understanding of the role of phosphorylation in S. mekongi and could be integrated with information from other Schistosoma species to facilitate drug and vaccine development.