Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Characterization of the lytic phage MSP1 for the inhibition of multidrug-resistant Salmonella enterica serovars Thompson and its biofilm.

The increasing prevalence of multidrug-resistant (MDR) Salmonella is a serious public health threat. Intervention strategies available to control Salmonella mostly target Salmonella enterica serovars Typhimurium and Enteritidis, and little has been investigated to control serovars in serogroup C, such as S. enterica serovar Thompson, despite their increasing prevalence. Here, we isolated phages targeting MDR S. Thompson and characterized the antimicrobial activities of MSP1 phage, a virulent phage with a broad host range. MSP1 phage strongly infected S. Thompson and S. Mbandaka isolates from retail chicken and also other serovars, including Dublin, Enteritidis, Heidelberg, Paratyphi, and Typhimurium. MSP1 phage was able to inhibit the biofilm formation on stainless steel and glass formation by around 42.7-47.9 %. MSP1 phage was robust to withstand wide ranges of pH (4-12) and temperature (30-60 °C), and no genes associated with antibiotic resistance and virulence were found in the phage genome, suggesting that this phage is suitable for food application. When MSP1 phage was tested on foods (chicken meat and milk), MSP1 phage significantly reduced the level of MDR S. Thompson below the detection limit. Our findings suggest that MSP1 phage is a promising antimicrobial agent for the control of food contamination by MDR S. Thompson.

Inhibition of Antimicrobial-Resistant Escherichia coli Using a Broad Host Range Phage Cocktail Targeting Various Bacterial Phylogenetic Groups.

Antimicrobial-resistant (AMR) commensal Escherichia coli is a major reservoir that disseminates antimicrobial resistance to humans through the consumption of contaminated foods, such as retail poultry products. This study aimed to control AMR E. coli on retail chicken using a broad host range phage cocktail. Five phages (JEP1, 4, 6, 7, and 8) were isolated and used to construct a phage cocktail after testing infectivity on 67 AMR E. coli strains isolated from retail chicken. Transmission electron microscopic analysis revealed that the five phages belong to the Myoviridae family. The phage genomes had various sizes ranging from 39 to 170 kb and did not possess any genes associated with antimicrobial resistance and virulence. Interestingly, each phage exhibited different levels of infection against AMR E. coli strains depending on the bacterial phylogenetic group. A phage cocktail consisting of the five phages was able to infect AMR E. coli in various phylogenetic groups and inhibited 91.0% (61/67) of AMR E. coli strains used in this study. Furthermore, the phage cocktail was effective in inhibiting E. coli on chicken at refrigeration temperatures. The treatment of artificially contaminated raw chicken skin with the phage cocktail rapidly reduced the viable counts of AMR E. coli by approximately 3 log units within 3 h, and the reduction was maintained throughout the experiment without developing resistance to phage infection. These results suggest that phages can be used as a biocontrol agent to inhibit AMR commensal E. coli on raw chicken.

Development of new strategy combining heat treatment and phage cocktail for post-contamination prevention.

Heat treatment is an effective method for ensuring food safety and quality by controlling microbial contamination. However, food poisoning outbreaks have continuously occurred in heat-treated products due to improper thermal treatment and/or post-contamination of foodborne pathogens. This study proposes a novel strategy combining thermostable bacteriophages with thermal processing of food production plants to control foodborne pathogens and even bacterial contamination. Typically, bacteriophages' susceptibility to heat is a major challenge to their application with thermal processing, we isolated thermostable bacteriophages by a modified isolation method of applying heat to samples and characterized the thermostable bacteriophages. Furthermore, we optimized the bacteriophage cocktail components to expand the controllable host range and reduce the risk of bacteriophage resistance development. Finally, we verified this antibacterial strategy by combining heat treatment with thermostable bacteriophages in model systems, including milk and chicken breast. After the phage cocktail and heat treatment, we artificially contaminated the food products to mimic the post-contamination event. Surprisingly, the remaining bacteriophages that withstood heat treatment significantly reduced the number of post-contaminated Salmonella. Altogether, thermostable phages could be applied as complementary tools to control post-contamination after thermal processing of food products.

A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals.

The rapid, accurate diagnosis of Plasmodium spp. is essential for the effective control of malaria, especially in asymptomatic infections. In this study, we developed a sensitive, genus-specific, real-time quantitative PCR assay. It was compared with the microscopic examination of Giemsa-stained blood smears and two different molecular diagnostic techniques: nested PCR and multiplex PCR. For the effective quantitative detection of malaria parasites, all reagents were designed with a lyophilized format in one tube. Plasmodium was detected successfully in all 112 clinically suspected malaria patients, including 32 individuals with low parasitemia (1-100 parasites/µl). The sensitivity threshold was 0.2 parasites/µl and no PCR-positive reaction occurred when malaria parasites were not present. This may be a useful method for detecting malaria parasites in endemic areas.

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Identification of cystatin B as a potential serum marker in hepatocellular carcinoma.

PURPOSE: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. EXPERIMENTAL DESIGN: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). RESULTS: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. CONCLUSION: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and alpha-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

Decreased expression of CD3zeta and nuclear transcription factor kappa B in patients with pulmonary tuberculosis: potential mechanisms and reversibility with treatment.

BACKGROUND: The protective immune response against Mycobacterium tuberculosis relies both on antigen-presenting cells and on T lymphocytes. In patients with different forms of tuberculosis, varying degrees of T cell function--ranging from positive delayed-type hypersensitivity, in asymptomatic infected healthy individuals, to the absence of the response, in patients with miliary or pulmonary tuberculosis (PTB)--have been reported. The decreased expression of CD3zeta reported in T cells from patients with either cancer or leprosy has provided possible explanations for the altered immune response observed in these diseases. METHODS: The present study aimed to compare the expression of CD3zeta , nuclear transcription factor- kappa B (NF- kappa B), arginase activity, and cytokine production in 20 patients with PTB, in 20 tuberculin-positive asymptomatic subjects, and in 14 tuberculin-negative control subjects. RESULTS: Compared with those in tuberculin (purified protein derivative)-negative control subjects, peripheral-blood T lymphocytes from patients with active PTB had significantly (P < .001) decreased expression of CD3zeta and absence of the p65/p50 heterodimer of NF- kappa B. These alterations were reversed only in patients who responded to treatment. Also reported here for the first time is that the presence of arginase activity in peripheral-blood mononuclear-cell lysates of patients with PTB parallels high production of interleukin-10. CONCLUSIONS: The presence of arginase could, in part, explain the decreased expression of CD3zeta . These findings provide a novel mechanism that may explain the T cell dysfunction observed in patients with PTB.

Structural requirements for assembly and homotypic interactions of the hepatitis C virus core protein.

The hepatitis C virus (HCV) core protein is involved in the assembly of nucleocapsid particles, as well as regulation of cellular and viral gene expression. To investigate the biological properties of the viral core protein and viral RNA assembly, two recombinant core proteins, the mature core protein (named C179) and a C-terminal truncated protein (named C124), were expressed and purified. To confirm their ability to generate viral particles, the production of nucleocapsid-like particles was monitored using transmission electron microscopy (EM). The EM analysis revealed that exposure of these proteins to the 5' untranslated region (5' UTR) of the viral RNA resulted in generation of spherical particles of 30-140nm in diameter. Interestingly, a cross-linking analysis revealed that C124 required an RNA component for homotypic interactions. In contrast, C179 successfully assembled in the absence of nucleic acids. Additionally, RNA-mediated conversion of the C124 structure into a more stable state was maintained even after RNase treatment. Therefore, our results indicate that the basic N-terminal domain of the viral core protein utilizes RNA components to induce conformational changes or efficient homotypic interactions, while the C-terminal domain may contain key peptide sequences for initiating spontaneous multimerization at the early stages of viral assembly.

Proteomic analysis of phosphotyrosyl proteins in morphine-dependent rat brains.

Morphine has been used as a potent analgesic, having a high propensity to induce tolerance and physical dependence following their repeated administration. Although the mechanisms that underlie the development of dependence on morphine remain unclear, previous studies suggested that phosphorylations of diverse types of cellular proteins are crucial determinants of the neuroadaptive mechanisms associated with morphine dependence. Thus, understanding global phosphorylation events induced by chronic morphine administration is essential for understanding the complex signaling mechanisms of morphine dependence. This study characterized the alteration of tyrosine phosphorylation of frontal cortical proteins in morphine-dependent rat brains using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microl/h) for 72 h via osmotic minipumps in rats. Phosphotyrosyl (p-Tyr) protein spots in brain frontal cortical regions were detected by two-dimensional electrophoresis (2-DE) and immunoblotting with anti-p-Tyr-specific antibodies. The protein spots showing significant changes in tyrosine phosphorylation were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Similar patterns of protein expression were detected by 2-DE gels in morphine-dependent and saline-treated control rat brains. However, phosphotyrosine 2-DE images of the frontal cortical proteins from saline-treated control and morphine-dependent rat brains were apparently different. The densities of most matched p-Tyr protein spots were increased in morphine-dependent rat brains compared with that of control samples. Additional p-Tyr protein spots were detected in 2-DE image of morphine-dependent rat brains. Fifty of p-Tyr protein spots, corresponding to 40 different proteins, were identified from 2-DE gels of morphine-dependent rat brains. The identified proteins include enzymes, cytoskeletal proteins, cell signaling molecules, and other proteins. In conclusion, the first available phosphotyrosine proteomic resources of morphine dependence were established using an animal model. The findings illustrate the potential of proteomics as an effective technique for studying phosphorylation events of morphine dependence in brains.

Proteomic analysis of phosphotyrosyl proteins in the rat brain: effect of butorphanol dependence.

Butorphanol (17-cyclobutylmethyl-3,14-dihydroxymorphinan) tartrate (Stadol) is a mixed agonist-antagonist opioid analgesic agent that is about five to seven times as potent as morphine in analgesic effects. The chronic use of butorphanol produces physical dependence in humans and animals. Phosphorylation plays a very important role in developing butorphanol dependence; however, global phosphorylation events induced by chronic butorphanol administration have not been reported. The aim of this study is to determine the alteration of tyrosine phosphorylation of brain frontal cortical proteins in butorphanol-dependent rats using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/microl/hr) for 72 hr via osmotic minipump in rats. Similar patterns of protein expression were detected by two-dimensional electrophoresis (2-DE) in brain frontal cortex of butorphanol-dependent and saline-treated control rats. All 65 phosphotyrosyl (p-Tyr) protein spots detected in pH 3-10 phosphotyrosine 2-DE of control rat brains were detected in butorphanol-dependent rat brains. The densities of most p-Tyr protein spots were increased in butorphanol-dependent rat brains compared to saline-treated control samples. Eighteen additional p-Tyr protein spots were detected in pH 3-10 2-DE images of butorphanol-dependent rat brains. Immobilized pH strips with three different narrow pH ranges were examined to improve the resolution of p-Tyr proteins in 2-DE gels. Fifty-three p-Tyr protein spots were identified as known proteins involved in cell cytoskeleton, cell metabolism, and cell signaling. This proteomic approach can provide useful information for understanding the complex mechanism of butorphanol dependence in vivo.

L-Arginine modulates CD3zeta expression and T cell function in activated human T lymphocytes.

Engagement of the T cell receptor (TCR) by antigen or anti-CD3 antibody results in a cycle of internalization and re-expression of the CD3zeta. Following internalization, CD3zeta is degraded and replaced by newly synthesized CD3zeta on the cell surface. Here, we provide evidence that availability of the amino acid L-arginine modulates the cycle of internalization and re-expression of CD3zeta and cause T cell dysfunction. T cells stimulated and cultured in presence of L-arginine, undergo the normal cycle of internalization and re-expression of CD3zeta. In contrast, T cells stimulated and cultured in absence of L-arginine, present a sustained down-regulation of CD3zeta preventing the normal expression of the TCR, exhibit a decreased proliferation, and a significantly diminished production of IFNgamma, IL5, and IL10, but not IL2. The replenishment of L-arginine recovers the expression of CD3zeta. The decreased expression of CD3zeta is not caused by a decreased CD3zeta mRNA, an increased CD3zeta degradation or T cell apoptosis.