11 research outputs found

    Apd1 and Aim32 Are Prototypes of Bis-Histidinyl-Coordinated Non-Rieske [2Fe-2S] Proteins

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    Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = -164±5 mV, pKox1,2=7.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 ≈ -150 mV, pKox1,2=9.8 and 11.5) and cytochrome bc1/b6f Rieske clusters (Em,7 ≈ +300 mV, pKox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of [2Fe-2S] cluster containing proteins with bis-histidinyl coordination and proton-coupled electron transfer.<br /

    Secreted <i>Gaussia princeps</i> Luciferase as a Reporter of <i>Escherichia coli</i> Replication in a Mouse Tissue Cage Model of Infection

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    <div><p>Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for <i>in vivo</i> bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic <i>Escherichia coli</i> (<i>E. coli</i>) strain, ATCC25922, to secrete the marine copepod <i>Gaussia princeps</i> luciferase (Gluc), and assessed the use of Gluc as both an <i>in situ</i> and <i>ex situ</i> reporter for bacterial burden in mouse tissue cage infections. The <i>E. coli</i> expressing Gluc demonstrates <i>in vivo</i> imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an <i>ex situ</i> indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic <i>E. coli in vivo</i> to enable a facile tool for longitudinal evaluation of persistent bacterial infection.</p></div

    PelB secretion signal derived from pectate lyase of <i>Erwinia carotovora</i> (<i>E. carotovora</i>) promotes an efficient secretion of <i>Gaussia princeps</i> luciferase (Gluc) by <i>E. coli</i>.

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    <p>M: lane for protein marker, B: whole <i>E. coli</i> cell lysate before induction with IPTG, A: whole cell lysate after induction, T: total whole cell lysate after French press lysis, S: soluble fraction of lysate after French press, I: insoluble fraction of lysate after French press. Arrow: the predicted position of Gluc-HlyA on gel. Asterisk: the predicted position of Gluc, PelB-Gluc, and SS-Gluc fusion protein. For panel A and B, lysate corresponding to 10<sup>8</sup> cells was loaded in each lane. (A) The expression of Gluc without secretion signal (Gluc) or Gluc with C-terminal secretion signal derived from α-hemolysin (Gluc-HlyA) was induced from pCOLDI-based vectors in <i>E. coli.</i> Expressions were detected by Western blotting using a rabbit polyclonal antibody against Gluc. (B) The expression of Gluc with N-terminal secretion signal derived from pectate lyase (PelB-Gluc) or Gluc with its native secretion signal from <i>Gaussia princeps</i> (SS-Gluc) was induced from pCOLDI-based vectors in <i>E. coli</i> and detected by the above rabbit polyclonal antibody. (C) For panel C, the bacterial culture supernatant corresponding to secreted protein from 2×10<sup>8</sup> cells was loaded in each lane. −: Culture supernatant of <i>E. coli</i> expressing Gluc without secretion signal, H: supernatant of <i>E. coli</i> expressing Gluc-HlyA, P: supernatant of <i>E. coli</i> expressing pelB-Gluc, +: supernatant of <i>E. coli</i> expressing SS-Gluc. Note: The exposure time for panel A and B was shorter than for panel C. There is a gel crack in the lane of “+” which appears two bands at the position of SS-Gluc.</p

    Detection sensitivity of Gluc secreted by bacteria recovered from <i>in vivo</i> infection increases with CTZ concentration, but is less affected by integration time.

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    <p>All results have been normalized with OD<sub>600</sub>. (A) <i>E. coli</i> clone 6189 (ML6189) was grown in LB medium for 16 hr and secreted Gluc in culture supernatant was measured as the “before infection” sample. <i>E. coli</i> from the above culture was used for mice thigh infection. 24 hr later, <i>E. coli</i> was recovered from infected thigh tissues and grown as colonies on Agar plate. 7 colonies were randomly picked from the plate and inoculated into LB for 16 hr. Culture supernatant of the above 7 colonies was measured for secreted Gluc activity. CTZ: coelenterazine, substrate of Gluc. For panel A, integration time for Gluc assay is 1000 millisecond (ms). (B) For panel B, 39 µM CTZ was used for Gluc assay. Overnight culture supernatant of the 7 colonies recovered from mice infection was measured for secreted Gluc activity.</p

    Integration of the <i>pelB</i> tagged <i>gluc</i> gene into chromosomal <i>lacZ</i> locus is stable during <i>in vitro</i> passage.

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    <p>All results have been normalized against OD<sub>600</sub> of the culture analyzed. For all panels, data represent mean and standard error of triplicate samples. Error bars may be too small to see for some samples. CPS: photon counts per second (A) <i>E. coli</i> clones with the <i>pelB</i> tagged <i>gluc</i> gene were grown in LB medium without kanamycin for one passage and measured for the secretion of Gluc into culture supernatant. All clones in panel A harbor the kanamycin resistance gene accompanying the <i>pelB</i> tagged <i>gluc</i> gene. Clones ATCC25922, 618 and 619 are the same shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090382#pone-0090382-g003" target="_blank">Figure 3</a>. Clone R8 is an <i>E. coli</i> clone with the <i>pelB</i> tagged <i>gluc</i> gene integrated in the same orientation as <i>lacZ</i>, however, the <i>gluc</i> gene is inserted into <i>lacZ</i> ORF instead of replacing <i>lacZ</i> ORF (see Experimental Procedure Table1). (B) <i>E. coli</i> clones from panel A have undergone 5 more serial passages either in media supplied with kanamycin (black bar) or without kanamycin (grey bar). Culture supernatant corresponding to the 6<sup>th</sup> passage was measured for the secretion of Gluc. (C) Kanamycin resistance gene was eliminated from clone R8 to generate clone R85 and R86. Clones 6181 and 6189 are kanamycin sensitive (Kan<sup>S</sup>) derivatives from clone 618. Clones 6192 and 61911 are Kan<sup>S</sup> derivatives from clone 619. Clones were passaged in LB medium without kanamycin. Culture supernatant collected from the 1<sup>st</sup> passage (grey bar) and the 6<sup>th</sup> passage (black bar) were measured for secreted Gluc activity.</p

    CFU burden of the recombinant <i>E. coli</i> in tissue cage fluid (TCF) and secreted Gluc activity in TCF correlate to a high degree for the established tissue cage infection.

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    <p>Mouse tissue cages were inoculated with 10<sup>5</sup> CFU of the recombinant <i>E .coli</i> strain ML6189 expressing Gluc or the parental <i>E. coli</i> strain ATCC25922. TCF was collected at different time points and analyzed by both CFU plating and measurement of secreted Gluc activity (details seen Experimental Procedures). CPS: photon counts per second. For all panels, the mean value for each group of samples is represented by a black bar. Data from two independent experiments were pooled. (A) CFU burden of ML6189 in TCF at different time points during 18 days of tissue cage infection. (B) CFU burden of ATCC25922 in TCF at different time points along 18 days of tissue cage infection. (C) Secreted Gluc activity in TCF at different time points of ML6189 infection. The red bars represent mean value of the background Gluc signal calculated from data in panel D. Note: for ML6189 infection, 4 mice (day 11) and 5 mice (day 18) were removed from the group for imaging experiments and were excluded from quantification of Gluc activity in TCF (panel C). (D) Secreted Gluc activity in TCF at different time points of ATCC25922 infection.</p

    Secreted Gluc permeates from the established tissue cage infection loci to the blood stream and serves as an <i>ex situ</i> indicator for the localized tissue cage infection.

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    <p>Mouse plasma was collected at the denoted time points during tissue cage infection and measured for Gluc activity present in plasma (details seen Experimental Procedures). (A) Mouse tissue cages were inoculated with 10<sup>3</sup> CFU of ML6189 or ATCC25922 at the beginning of infection. (B) Mouse tissue cages were inoculated with 10<sup>5</sup> CFU of ML6189 or ATCC25922 at the beginning of infection. Unpaired and two tailed Student's <i>t</i>-test was used for all two comparisons with one variable (indicated by the connected lines in the figure), <i>P</i><0.01 was considered as statistically significant and marked by “*”. For all panels, the mean value for each group of samples is represented by a black bar. Data from two independent experiments were pooled.</p

    The <i>pelB</i> tagged <i>gluc</i> gene integrated in the same orientation as the original <i>lacZ</i> ORF yields a high level of secretion of Gluc to bacterial culture supernatant by <i>E. coli</i>.

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    <p>All <i>E. coli</i> clones were grown in LB medium without kanamycin supplement for overnight and 50 µl culture supernatant was measured for secreted Gluc activity. Note: all results have been normalized with OD<sub>600</sub>. CPS: photon counts per second. <i>E. coli</i> strain ATCC25922 is the negative control strain without integration of <i>gluc</i> gene on the chromosome. <i>E. coli</i> clones 618 and 619 harbor the <i>pelB</i> tagged <i>gluc</i> gene integrated in the same orientation as the original <i>LacZ</i> ORF on the chromosome. Clones 622 and 623 have the <i>pelB</i> tagged <i>gluc</i> gene integrated in the orientation opposite to <i>LacZ</i>. Clones 641 and 642 have the <i>SS</i> tagged <i>gluc</i> gene integrated in the orientation opposite to <i>LacZ</i>. Data represent mean and standard error of triplicate samples. Error bars may be too small to see for some samples.</p
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