Serum folate receptor alpha, mesothelin and megakaryocyte potentiating factor in ovarian cancer: association to disease stage and grade and comparison to CA125 and HE4.

BACKGROUND: Evaluate and compare the utility of serum folate receptor alpha (FRA) and megakaryocyte potentiating factor (MPF) determinations relative to serum CA125, mesothelin (MSLN) and HE4 for the diagnosis of epithelial ovarian cancer (EOC). METHODS: Electrochemiluminescent assays were developed for FRA, MSLN and MPF and used to assess the levels of these biomarkers in 258 serum samples from ovarian cancer patients. Commercial assays for CA125 and HE4 were run on a subset of 176 of these samples representing the serous histology. Data was analyzed by histotype, stage and grade of disease. A comparison of the levels of the FRA, MSLN and MPF biomarkers in serum, plasma and urine was also performed in a subset of 57 patients. RESULTS: Serum and plasma levels of FRA, MSLN and MPF were shown to be highly correlated between the two matrices. Correlations between all pairs of markers in 318 serum samples were calculated and demonstrated the highest correlation between HE4 and MPF, and the lowest between FRA and MPF. Serum levels of all markers showed a dependence on both stage and grade of disease. A multi-marker logistic regression model was developed resulting in an AUC=0.91 for diagnosis of serous ovarian cancer, a significant improvement over the AUC for any of the individual markers, including CA125 (AUC=0.84). CONCLUSIONS: FRA has significant potential as a biomarker for ovarian cancer, both as a stand-alone marker and in combination with other known markers for EOC. The lack of correlation between the various markers analyzed in the present study suggests that a panel of markers can aid in the detection and/or monitoring of this disease.

Novel chromosomally encoded multidrug efflux transporter MdeA in Staphylococcus aureus.

Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae.

The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.