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Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram‐negative bacteria are well‐established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram‐positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha‐type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence‐associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus. [ABSTRACT FROM AUTHOR]

Abstract Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram‐negative bacteria are well‐established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram‐positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha‐type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence‐associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus.

The diversity and severity of infections caused and the rapid emergence of antibiotic resistance necessitates the development of a vaccine against Staphylococcus aureus. None of the antigens tried as vaccine candidates so far has been translated into a clinically viable vaccine. Recent research data suggest that antigens with the potential to activate cell mediated immunity along with humoral immunity would be the key to the development of a vaccine. Alkaline shock protein 23, a membrane-anchored protein involved in the stress response, has been identified as a CD4+ T cell antigen from S. aureus. In the present study, we report the evaluation of immunogenicity and protective efficacy of a recombinant alkaline shock protein 23 from S. aureus in mouse models. The gene coding for the protein was cloned and expressed in Escherichia coli, purified using immobilized metal iron affinity chromatography, sequence-confirmed using mass spectrometry and intraperitoneally administered to BALB/c mice. Serum titers of IgG, IgG1, and IgG2a in response to the protein were measured on post-immunization days 21, 35 and 42 using indirect ELISA and compared to control mice injected with PBS. Our results showed that the protein induced significantly higher (p < 0.01) antibody responses in immunized mice compared to the control mice. The mean serum antibody titers of IgG, IgG1 and IgG2a three weeks after the last immunization were found to be 25600, 25600 and 12800 respectively. Moreover, we found that immunization with Asp23 protected mice from a lethal dose of S. aureus strain USA300.

The diversity and severity of infections caused and the rapid emergence of antibiotic resistance necessitates the development of a vaccine against Staphylococcus aureus. None of the antigens tried as vaccine candidates so far has been translated into a clinically viable vaccine. Recent research data suggest that antigens with the potential to activate cell mediated immunity along with humoral immunity would be the key to the development of a vaccine. Alkaline shock protein 23, a membrane-anchored protein involved in the stress response, has been identified as a CD4+ T cell antigen from S. aureus. In the present study, we report the evaluation of immunogenicity and protective efficacy of a recombinant alkaline shock protein 23 from S. aureus in mouse models. The gene coding for the protein was cloned and expressed in Escherichia coli, purified using immobilized metal iron affinity chromatography, sequence- confirmed using mass spectrometry and intraperitoneally administered to BALB/c mice. Serum titers of IgG, IgG1, and IgG2a in response to the protein were measured on post-immunization days 21, 35 and 42 using indirect ELISA and compared to control mice injected with PBS. Our results showed that the protein induced significantly higher (p < 0.01) antibody responses in immunized mice compared to the control mice. The mean serum antibody titers of IgG, IgG1 and IgG2a three weeks after the last immunization were found to be 25600, 25600 and 12800 respectively. Moreover, we found that immunization with Asp23 protected mice from a lethal dose of S. aureus strain USA300. [ABSTRACT FROM AUTHOR]

Lactobacillus (L. casei) Zhang is a koumiss-originated probiotic strain, which was used as a model in a long-term antibiotics-driven evolution experiment to reveal bacterial evolutionary dynamics; and we isolated gentamycin-resistant L. casei Zhang descendents. To decipher the gentamycin resistance mechanism, here we cultivated the parental L. casei Zhang and its descendent cells in an antibiotics-containing environment to compare their global protein expression profiles using the iTRAQ-based proteomic approach. A total of 72 proteins were significantly up-regulated (>2.0-fold, P < 0.05), whilst 32 proteins were significantly down-regulated

Due to rising worldwide industrial competitiveness the machining sector is compelled to improve day by day. In this way, CO2 cryogenic machining is presented as alternative to conventional hard turning. So, in the tests carried out in this research, dry and CO2 cryogenic hard turning with two types of inserts were compared. The variables measured were tool life, superficial roughness and the piece microstructure. The results show an increase of tool life over 60%, a similar superficial roughness and, when positive insert is used, the absence of the white layer. [ABSTRACT FROM AUTHOR]

In this study, diamond-like carbon (DLC) films are coated onto oxynitriding-treated ASP23 high-speed steel by DC-pulsed PECVD. The main research parameters of the DC-pulsed PECVD process are the various duty cycles (5%, 10%, 15% and 20%) used to examine the coating characteristics. In order to investigate the DLC film properties, a Raman spectroscopy analysis, wear test, adhesion, and hardness tests are performed. The experimental results show that the duplex coating layers have optimal properties when DLC films are treated with a low-pulse voltage (− 1.5 kV), a coating time of 90 min and duty cycles maintained at 10%. As a result, the DLC/oxynitriding duplex-treated specimens with the highest surface hardness (Hv 0.01 1931) and the lowest wear volume loss (3 × 10 − 3 mm 3 ) can be acquired. In addition, compared with non-treated (polarization resistance R p = 5 × 10 3 Ω cm 2 ) and oxynitriding-treated (R p = 8 × 10 3 Ω cm 2 ) specimens, the optimal DLC/oxynitriding duplex-treated ASP23 high-speed steel possesses the lowest corrosion current (I corr = 8 × 10 −6 A cm − 2 ) and highest polarization resistance (R p = 1 × 10 4 Ω cm 2 ) in 3.5 wt.% NaCl solutions. This result confirms that the DLC/oxynitriding duplex treatment enhances optimal wear and corrosion resistance. [ABSTRACT FROM AUTHOR]

The alkaline shock protein Asp23 was identified as a σB-dependent protein in Staphylococcus aureus. In Bacillus subtilis, the asp23 promoter from S. aureus is regulated like other σB-dependent promoters, which are strongly induced by heat and ethanol stress. However, almost no induction of asp23 expression was found after heat or ethanol stress in S. aureus MA13 grown in a synthetic medium, where the basal expression level of asp23 is high. Under the same experimental conditions the σ B gene itself showed a similar expression pattern: it was highly expressed in synthetic medium but not induced by heat or ethanol stress. In contrast, σB activity was increased by heat stress when the cells were grown in a complex medium. The constitutive expression of sigB and σB-dependent stress genes in S. aureus MA13 grown in a synthetic medium is in a sharp contrast to the regulation of σB activity in B. subtilis, and needs further investigation. A deletion of 11 bp in the rsbU gene, which encodes the phosphatase that acts on RsbV (the anti-anti-sigma factor), in S. aureus NCTC 8325-4 might be responsible for the failure of heat stress to activate σB in complex medium, and thus reduce the initiation of transcription at σB-dependent promoters in this strain. [ABSTRACT FROM AUTHOR]

Lactobacillus ( L. casei ) Zhang is a koumiss-originated probiotic strain, which was used as a model in a long-term antibiotics-driven evolution experiment to reveal bacterial evolutionary dynamics; and we isolated gentamycin-resistant L. casei Zhang descendents. To decipher the gentamycin resistance mechanism, here we cultivated the parental L. casei Zhang and its descendent cells in an antibiotics-containing environment to compare their global protein expression profiles using the iTRAQ-based proteomic approach. A total of 72 proteins were significantly up-regulated (>2.0-fold, P < 0.05), whilst 32 proteins were significantly down-regulated <-2.0-fold, P < 0.05) in the descendent line. The gentamycin-resistant descendent line showed elevated expression in some carbohydrates, amino acids, and purine metabolic pathways. Several stress-related proteins were also differentially expressed. Among them, one alkaline shock protein, asp 23, was up-regulated most in the gentamycin-resistant strain (21.9-fold increase compared with the parental strain). The asp 23 gene disruption mutant was significantly more sensitive to gentamycin compared with the wild type, suggesting an important role of this gene in developing the gentamycin-resistant phenotype in L. casei . Our report has described the adaptation of a probiotic strain that has acquired antibiotics resistance through long-term antibiotics exposure at the proteome level, and we revealed a novel mechanism of gentamycin resistance.