Project description:Background: Streptococcus dysgalactiae subsp. equisimilis (SDSE), similar to Lancefield group A Streptococcus pyogenes (GAS), causes invasive diseases such as life-threatening streptococcal toxic shock syndrome (STSS). Despite their similar genome sequences, SDSE lacks several important virulence factors of GAS, suggesting that SDSE has specific disease-causing systems. Using microarray analysis, we analyzed SDSE for specific transcriptional regulatory systems involved in stress responses, such as the LytSR/LrgAB system, and their transcriptional profiles under stress conditions. Methods: Transcriptional profiling was performed using microarrays to test the effects of eight antibiotics and five growth conditions. These findings were compared with those obtained during intraperitoneal infection in mice. Results: Genes encoding LrgAB, which modulates the murein hydrolase activity of CidAB and inhibits autolysis in Staphylococcus aureus, were upregulated by exposure to antibacterial agents, phosphate buffered saline (PBS) and stationary phase conditions and during intraperitoneal infection in mice. Starvation and anaerobic conditions stimulated the expression of the streptolysin S operon and polysaccharide lyases in SDSE. Catabolite-responsive elements (cre) were present in the promoter regions of these genes, suggesting that carbon catabolite repression (CCR) is involved in regulating SDSE virulence factors. Comparative genome analysis showed the presence in SDSE of the LytSR/LrgAB system and an additional sigma factor (SDEG_0623), both of which were absent from the GAS genome. Conclusions: These results suggest that the LytSR/LrgAB and CCR play important roles in bacterial resistance to stress microenvironments. Microarray data also indicated that starvation and low oxygen tension partly mimic the microenvironment present during invasive diseases.
Project description:Streptococcus dysgalactiae subsp. equeisimilis (SDSE) has Lancefield group G or C antigens. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe and US. Although SDSE possesses similar virulence factors to S. pyogenes including streptolysin S (SLS) and streptolysin O (SLO), some important S. pyogenes virulence factors including active superantigens, SpeB and a hyarulonic acids capsule are missing in SDSE genome. The mechanisms and the key virulence factors for causing invasive diseases by SDSE are poorly understood. Here, we analyzed the transcriptome of SDSE in vivo using the murine sepsis model, revealing the strategy of SDSE to destruct host tissues with the virulence factors and to scavenge depleted nutrients. The expression of SLO operon increased at relatively early stage of infection while the SLS and hyaluronidases upregulated after 4h post infection. Microarray data suggested that SDSE degraded host tissue polysaccharides by streptococcal-secreting poly/oligosaccharide lyases and simultaneously used the Entner-Doudoroff pathway to metabolize acquired carbohydrates. A global negative virulence gene regulator CsrRS of SDSE modulated the expressions of genes encoding SLS and the carbohydrate metabolism enzymes. Moreover, csrS deficient mutant induced sever systemic hemolysis in mice. The most frequently isolated stG6792 strains from invasive disease secreted abundant SLS and SLO rather than other SDSE emm types, indicating the relationship between the SLS and SLO productions and poor outcome by the stG6792 strain infection. Our findings suggest that the concomitant regulation of virulence factors destructing the host tissues and metabolic enzymes play an important role to produce invasive diseases by SDSE.
Project description:Streptococcus dysgalactiae subsp. equeisimilis (SDSE) has Lancefield group G or C antigens. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe and US. Although SDSE possesses similar virulence factors to S. pyogenes including streptolysin S (SLS) and streptolysin O (SLO), some important S. pyogenes virulence factors including active superantigens, SpeB and a hyarulonic acids capsule are missing in SDSE genome. The mechanisms and the key virulence factors for causing invasive diseases by SDSE are poorly understood. Here, we analyzed the transcriptome of SDSE in vivo using the murine sepsis model, revealing the strategy of SDSE to destruct host tissues with the virulence factors and to scavenge depleted nutrients. The expression of SLO operon increased at relatively early stage of infection while the SLS and hyaluronidases upregulated after 4h post infection. Microarray data suggested that SDSE degraded host tissue polysaccharides by streptococcal-secreting poly/oligosaccharide lyases and simultaneously used the Entner-Doudoroff pathway to metabolize acquired carbohydrates. A global negative virulence gene regulator CsrRS of SDSE modulated the expressions of genes encoding SLS and the carbohydrate metabolism enzymes. Moreover, csrS deficient mutant induced sever systemic hemolysis in mice. The most frequently isolated stG6792 strains from invasive disease secreted abundant SLS and SLO rather than other SDSE emm types, indicating the relationship between the SLS and SLO productions and poor outcome by the stG6792 strain infection. Our findings suggest that the concomitant regulation of virulence factors destructing the host tissues and metabolic enzymes play an important role to produce invasive diseases by SDSE. To analyze gene expressions in group G streptococci with the murine infection model, we developed a custom microarray for Streptococcus dysgalactiae subsp. equisimilis (SDSE) based on the genome sequences of three SDSE strains; GGS_124, ATCC12923 and RE378. We intraperitoneally inoculated 10^8 CFU of GGS_124 stain and the csrS deficient mutant into ddY mice. Bacterial cells were collected from the abdominal cavity at 0, 2, 4 and 8 h post infection. GGS_124 cells were also collected from OD600=0.6 culture in brain heart infusion broth as a control.
Project description:A genome reduced E. coli strain MDS42ΔgalK::Ptet-gfp-kan were applied for the comparative transcriptome analysis. Genome-wide transcriptional changes under high osmotic prresure, high temperature condition and starvation were evaluated.
Project description:To validate the functions that iron might play in B cell proliferation and function we used deferoxamine (DFO, a widely used iron chelator) to create an iron-deficient environment for cell culture in vitro.
Project description:IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions.
Project description:BackgroundCampylobacter jejuni is a leading foodborne pathogen worldwide. Despite the fastidious nature of C. jejuni growth, increasing numbers of human campylobacteriosis suggest that C. jejuni may possess unique mechanisms to survive under various stress conditions. C. jejuni possesses only three sigma factors (FliA, RpoD, and RpoN) and lacks stress-defense sigma factors. Since FliA and RpoD are dedicated to flagella synthesis and housekeeping, respectively, in this study, we investigated the role of RpoN in C. jejuni's defense against various stresses.ResultsSurvivability of an rpoN mutant was compared with the wild-type C. jejuni under various stress conditions. While the growth of the rpoN mutant was as comparably as that of the wild type in shaking cultures, the rpoN mutant exhibited significant survival defects when cultured statically. The rpoN mutant was more sensitive to osmotic stress (0.8% NaCl) with abnormally-elongated cell morphology. Compared to the wile type, the rpoN mutant was more susceptible to acid stress (pH 5) and more resistant to hydrogen peroxide. However, the rpoN mutation had little effect on the resistance of C. jejuni to alkaline pH, heat, cold and antimicrobials.ConclusionsThe results demonstrate that RpoN plays an important role in C. jejuni's defense against various stresses which this bacterial pathogen may encounter during transmission to and infection of humans.
Project description:Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, β-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, β-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.