ABSTRACT
Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.
Subject(s)
Amino Acid Substitution , Lysostaphin , Chemistry , Polyethylene Glycols , Chemistry , Protein Engineering , Recombinant Proteins , Chemistry , Staphylococcus aureusABSTRACT
Objective To investigate the phenotypic and genetic characteristics of the lysostaphin‐resistant Staphylococcus aureus variants induced by recombinant lysostaphin in vitro .Methods Three clinical isolates of S . aureus ,including two resistant to methicillin (MRSA ) and one susceptible to methicillin (MSSA ) were induced by treatment with sub‐MIC of recombinant lysostaphin via one‐step selection in vitro .Susceptibility of the variants to antibiotics were determined and compared with their parental strains .The full length of femABX genes was amplified by polymerase chain reaction and sequenced to identify the potential mutation sites in these genes .The growth‐curve in liquid medium and virulence in a mouse systemic infection model of both parental and variant strains were observed . Results The frequency of lysostaphin resistance in S . aureus was between 10-4 to 10-8 following induction by lysostaphin . Resistance to lysostaphin was associated with a significant decrease in growth rate in vitro and virulence in vivo ,as well as increased susceptibility toβ‐lactams evidenced by the M IC of β‐lactams against the variants as low as 1/4 000 to 1/2 of the M IC against their parental strains . Sequencing of f emA BX genes showed mutation in femA gene in both variants ,which resulted in a premature termination codon .Conclusions Resistance of S . aureus to lysostaphin may develop following induction by recombinant lysostaphin in vitro . The lysostaphin‐resistant S . aureus variants are characteristic of lower growth rate , decreased virulence ,and higher susceptibility to β‐lactams .
ABSTRACT
Seroprevalence of antibodies against influenza viruses from 1000 people between the ages of 0 to 90 years of age (100 samples for each decade of life) in the Pittsburgh, PA, USA was measured. One year removed from the outbreak of novel H1N1 influenza into the human population in the Northern Hemisphere and following the emergence of a new H3N2 influenza isolate, sera was collected to determine the hemagglutination-inhibition antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses in the vaccine used for the 2010-2011 influenza season. The seroprevalence of antibodies to influenza virus, A/California/7/2009 (H1N1), increased from the previously reported November 2009 samples and the samples collected at the end of the 2010 influenza season (June 2010) during the 2010-2011 season in all age groups, but people the under the age of 20 had the highest rise in the number of positive samples. The number of individuals positive for H1N1 stayed the same through the entire influenza season. In contrast, there were little to no positive serum samples against the H3N2 virus, A/Perth/16/2009, from samples collected during the 2009-2010 influenza season, however, titers against these viruses rose significantly during the early months of the 2010-2011 season with the highest number of positive samples detected in the very young and very old populations. However, these titers waned by May, 2011 in those over the age of 40. There was a rise in adults to the B/Brisbane/60/2008 influenza virus in adults in samples collected in October, 2010, but these titers quickly declined. The highest titers to B influenza were detected in people between the ages of 10-30 years of age. These findings may have implications for the development of vaccination strategies aiming at the protection against seasonal and/or pandemic influenza virus infection and pre-pandemic preparedness activities.
