Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold. Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.

We have used a polyclonal antiserum t o cell wall proteins of Candida albicans to isolate several clones from a cDNA i l g t l l expression library. Affinitypurified antibody prepared to the fusion protein of one clone identified a 40 kDa moiety present in cell wall extracts from both morphologies of the organism. Indirect immunof luorescence demonstrated expression of this moiety at the C. albicans cell surface. Sequencing of a pBluescript II genomic clone identified with the cDNA clone revealed an open reading frame for a 417 amino acid protein. The nucleotide sequence showed significant homology with 3-phosphoglycerate kinase (PGK) genes, with 88%, 77% and 76% nucleotide homology with the PGK genes from Candida maltosa, Sacchammyces cere visiae and Kluyveromyces lactis, respectively. The deduced amino acid sequence was consistent with this identification of the sequence as PGKI of C. albicans. This finding was confirmed by a positive immunological response of a commercially available purified PGK from S. cerevisiae with the affinity-purified antibody against the fusion protein of the cDNA clone. The presence of PGK in the cell wall was confirmed by two additional methods.Cell wall proteins were biotinylated with a derivative that does not permeate the cell membrane to distinguish extracellular from cytosolic proteins. Biotinylated PGK was detected among the biotinylated proteins obtained following streptavidin affinity chromatography. lmmunoelectron microscopy revealed that the protein was present at the outer surface of the cell membrane and cell wall as well as expected in the cytoplasm. Northern blot analysis revealed that the gene transcript was present in C. albicans cells growing under different conditions, including different media, temperatures and morphologies. Most of the enzyme activity was found in the cytosol. Low enzymic activity was detected in intact cells but not in culture filtrates. These observations confirmed that PGK is a bona fide cell wall protein of C. albicans.

We have previously reported the isolation of several clones from a cDNA expression library from Candida albicans, one of which was associated with a constitutively expressed 70-kDa protein. The moiety was present in the ␤-mercaptoethanol extracts of cell walls from both blastoconidia and germ tubes. The surface expression of this moiety was revealed by an indirect immunofluorescence assay using affinity-purified antibody to the fusion protein produced by the clone. The 0.68-kb cDNA insert was sequenced. A database search revealed extensive homology with the 70-kDa family of stress or heat shock proteins (hsps). The 77% homology with another C. albicans HSP70 sequence suggested that this fragment represented a second member of the HSP70 family in this organism. Homology ranging from 65 to 76% was observed with members of four subfamilies (SSA, SSB, SSC, and SSD) of the Saccharomyces cerevisiae HSP70 gene family. The nucleic acid sequence and the deduced amino acid sequence of the open reading frame showed greatest homology with SSA1 and SSA2 sequences, and the gene corresponding to the cDNA clone was designated C. albicans SSA2. The relationship with the SSA family was supported by reactivity of the 70-kDa component with antibody recognizing the Ssa proteins of S. cerevisiae. The presence of an hsp70 in the cell wall was confirmed by two additional methods. Cell wall proteins were biotinylated with a non-membrane-permeable derivative to distinguish extracellular from cytosolic proteins. Biotinylated hsp70 was detected by Western blotting (immunoblotting) among the biotinylated components affinity purified by chromatography on streptavidin, thereby establishing its presence in the cell wall. Immunoelectron microscopy showed that the 70-kDa component was present at the cell surface as well as the outer surface of the plasma membrane and extended through the cell wall, occasionally appearing to reach the cell surface through channels. Northern (RNA) blot analysis showed that the gene was expressed in yeast cells growing in yeast extract-peptone medium at both 25 and 37؇C and in Lee medium at 25؇C and during formation of germ tubes in Lee medium 37؇C. No obvious increase in the expression level was detected after the temperature shift. Members of the hsp70 family have been reported to be immunoreactive. The fusion protein produced by the cDNA clone was recognized by serum from healthy individuals and patients with candidiasis. Since members of the hsp70 family of eucaryotic proteins are associated with chaperone and translocation functions, in addition to being immunogenic, this protein may play a role in the assembly and function of other cell wall proteins.

No abstract

BACKGROUND The nucleoside analog cytarabine (Ara-C [cytosine arabinoside]) is the key agent for treating acute myeloid leukemia (AML); however, up to 30% of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. This project aimed to construct and evaluate a self-bioluminescent reporter strain of Escherichia coli for use as an Ara-C biosensor and to design an in vitro assay to predict Ara-C response in clinical samples. METHODS We used transposition mutagenesis to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to Ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine Ara-C uptake and phosphorylation by leukemic cells. RESULTS Intracellular concentrations of 0.025 μmol/L phosphorylated Ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to Ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for Ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to Ara-C within 8 h. CONCLUSIONS The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to Ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced Ara-C dose levels. The 8-h assay monitors intracellular Ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings.

No abstract

A bioluminescent clinical isolate of Streptococcus pneumoniae was used to test the real-time effects of the oxazolidinone antibiotic, linezolid, on metabolism compared with effects on cell replication. Viable counts and bioluminescence measurements showed that linezolid has little bactericidal effect, which was similar at minimum (6 mg/L), intermediate (13 mg/L) and maximum (20 mg/L) serum concentrations. The post-antibiotic effect, however, was shorter when measured by light output than by viable counts. The results demonstrate that bioluminescence provides a rapid and sensitive means of measuring the effect of antimicrobials on bacterial metabolism, and that the latter recovers earlier than commencement of cell replication after linezolid exposure.

Background Enteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively. Methods Salmonella -free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm 2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 ( S. Enteritidis), Tϕ7 phage suspension ( S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonella s was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S . Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h. Results The median levels of Salmonella reduction following phage treatment were 1.38 log 10 MPN (Enteritidis) and 1.83 log 10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer ( p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls ( p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface. Conclusions The results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observ...