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1.  Contributions of sigB and sarA to distinct multiple antimicrobial resistance mechanisms of Staphylococcus aureus 
Multiple antimicrobial resistance in Staphylococcus aureus can result from mutations leading to reduced susceptibility to Pine oil-based cleaners (PSRS) as well as following growth with the non-steroidal anti-inflammatory salicylate. We now define the contributions of alternative sigma factor (sigB) and staphylococcal accessory regulator (sarA) to these mechanisms. We conclude that sarA plays a more prominent role than sigB in overall intrinsic multiple antimicrobial resistance. Both genes have similar effects on intrinsic vancomycin resistance, and the salicylate-inducible mechanism is not sigB- or sarA-dependent. Furthermore, analyses determined that altered expression of sigB and sarA is not responsible for the salicylate-inducible mechanism, and sarA upregulation is associated with the PSRS phenotype.
PMCID: PMC3551609  PMID: 16777384
Staphylococcus aureus; Multiple antimicrobial resistance; Alternative sigma factor; Staphylococcal accessory regulator
2.  Mouse salivary glands and human β-defensin-2 as a study model for antimicrobial gene therapy: technical considerations☆ 
Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands that secrete these peptides into saliva to control bacterial/fungal infection in the oral cavity. However, an animal study model to test this potential has not been established. Therefore, we determined to test (i) whether the potent antimicrobial peptide human β-defensin-2 (hBD-2) can be overexpressed in saliva after transduction of salivary glands and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Lentiviral vector SIN18cPPTRhMLV bearing hBD-2 cDNA was introduced into SCID mouse submandibular glands via cannulation. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or enzyme-linked immunosorbent assay (ELISA) were performed to detect hBD-2 expression in glands or in saliva. Candida albicans 613p was inoculated orally into SCID mice to establish oral candidiasis. Whilst expression of hBD-2 was detected in mouse salivary glands by RT-PCR and immunohistochemistry 1 day or 1 week following delivery of lentivirus, hBD-2 was not detected in saliva. There was recoverable C. albicans from the oral cavity and gastrointestinal tract 4 days to 4 weeks after infection, but there was no establishment of observable oral candidiasis in SCID mice under a stereomicroscope. Our data indicate that lentiviral vectors transduce mouse salivary glands, but not at a sufficient level to allow hBD-2 detection in saliva. Other vectors for gene transduction and additional treatment of SCID mice to establish oral candidiasis are needed in order to utilise mouse salivary glands to test antimicrobial gene therapy.
PMCID: PMC3285981  PMID: 16963233
hBD-2; Lentiviral vectors; Mouse salivary glands; Candida albicans; SCID mice
3.  Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene 
Nucleoside hydrolase (NH) (EC. is an essential enzyme in the purine–pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH was amplified using polymerase chain reaction with L. major genomic DNA as the template, cloned into an expression vector and used to transform a yeast mutant unable to grow on uridine as the sole source of uracil. Expression of LmNH yielded an ca. 35.6 kDa protein, which was shown to be functional as the mutant strain was able to grow on medium containing uridine as the sole source of uracil. Importantly, this work has resulted in a strain that can be used to screen compounds as potential inhibitors of this essential Leishmania enzyme.
PMCID: PMC1847609  PMID: 17141482
Leishmania major; Nucleoside hydrolase; Drug screening; Leishmaniasis

Results 1-3 (3)