Table 1.
Table 1. Members of the putative porin family of P. amoebophila are predicted to be localized to the outer membrane by in silico analysis.
Figure 1.
Toxic effect of the heterologous expression of PomS, PomT, PomU, and PomV in E. coli.
(A) Survival of E. coli BL21 (DE3) carrying different pet16b plasmid constructs after induction of protein expression (labeled with *) and without induction. As controls, the vector pet16b without insert and pet16b containing a gene fragment coding for the inclusion membrane protein IncA (pc0399) of P. amoebophila were used [32]. Induction of expression of proteins of the putative porin family lead to a rapid decrease in the number of colony forming units (cfus) compared to the non-toxic protein IncA. Ten minutes after induction, the numbers of cfus of E. coli expressing the putative porins were significantly lower than those of all controls (p<0.05, one-way ANOVA test and Dunnett’s post test); this difference was not significant anymore at sixty minutes after induction. The mean number of cfus for three independent replicates is shown +/− the standard error of the mean (SEM). (B) Visualization of the toxic effect of the putative porins on E. coli 10 min after induction of protein expression. Colonies formed by 10 µl droplets of the same dilutions for the non-toxic IncA (left) and the putative porin PomU (right) are shown after incubation overnight at 37°C. Similar results were obtained for all four putative porins tested. Dilutions range from 1∶10 to 1∶10,000. (C) Detection of protein expression by SDS-PAGE (left) and Western blot analysis (right) for PomT and IncA. Time in min after induction of protein expression by addition of IPTG is indicated above the lanes. Expression of IncA can be detected by SDS-PAGE and Western blot analysis whereas expression of PomT can be detected only by the more sensitive Western blot analysis.
Figure 2.
Detection of PomS and PomT after overexpresson in E. coli and in outer membrane fractions of P. amoebophila.
Upper panel: An additional band in SDS-PAGE gels (left) is present after induction of expression of leaderless PomS or PomT in E. coli (lanes labeled “+”) compared to uninduced samples (lanes labeled “−”). Western blot analysis (right) using polyclonal anti-PomS and anti-PomT antibodies demonstrates specificity for the heterologously expressed proteins. The anti-PomT antibodies additionally target one E. coli protein with a lower molecular mass. Bands at the correct molecular mass for the leaderless proteins (33.9 kDa for PomS and 36.9 kDa for PomT) are indicated by arrow heads. Molecular mass of marker bands (M) in kDa. Lower panel: Bands for leaderless PomS (33.9 kDa) and PomT (36.9 kDa) can be observed in the sarkosyl-insoluble outer membrane fraction (OM) and in the sarkosyl-soluble (S) fraction using specific polyclonal antibodies (bands at the correct molecular mass are indicated by arrow heads). The cytoplasmic heat-shock protein DnaK, which served as a control, is detected only in the sarkosyl-soluble fraction. For comparison, a SDS gel stained with colloidal coomassie is shown on the left. Molecular mass of marker bands (M) in kDa.
Figure 3.
Detection of PomS and PomT in P. amoebophila within its natural amoeba host.
Left panel: Localization of PomS and PomT by immunofluorescence in an asynchronous culture of A. castellanii containing P. amoebophila. Halo-shaped fluorescence signals were observed around intracellular P. amoebophila. In contrast, signals for the heat-shock protein DnaK were confined to the cytoplasm. No differences were observed for methanol- and PFA- fixed samples. Identical microscopic fields are shown. Bar, 5µm. Right panel: Magnification of intracellular P. amoebophila; overlay images of fluorescence signals for PomS and PomT (red), respectively, with DnaK (green) are shown. Bars, 2 µm.
Figure 4.
Localization of PomS in the outer membrane of P. amoebophila by immuno-transmission electron microscopy.
Immunogold labeling with pre-immune serum (left) and polyclonal anti-PomS antibodies (right). Gold particles indicating PomS were confined to the outer membrane of P. amoebophila. White arrow head, gold particle in the outer membrane; grey arrowhead, cytoplasmic membrane; black arrowhead, inclusion membrane. Bars, 200 nm.
Figure 5.
Expression of PomS during the developmental cycle of P. amoebophila in its amoeba host.
Upper panel: Relative levels of pomS transcripts measured by real-time quantitative PCR. pomS transcripts were normalized to the 16S rRNA to account for an increase in copy numbers due to multiplication of P. amoebophila. Data are shown as the mean of five replicates +/− SEM from a total of three independent infection experiments. Lower panel: Expression of PomS at the same time points as in the upper panel detected by anti-PomS antibody in methanol-fixed cells. Outlines of the amoebae are drawn in white. Bars 5 µm.
Figure 6.
Infection-inhibition assays using anti-Pam and anti-PomS antibodies.
Left panel: Incubation of host-free P. amoebophila EBs with anti-PomS and anti-Pam antibodies prior to fixation demonstrated that these antibodies can bind unfixed cells. Fluorescence signals derived from specific antibodies (left) and 4′, 6-Diamidino-2-phenylindol (DAPI; right) are shown for identical microscopic fields. Bars, 2 µm. The absence of DAPI signals for some cells indicates cells that lysed during the purification procedure. Right panel: Infection-inhibition assay using preincubations of EBs with anti-Pam and anti-PomS antibodies in different dilutions. The proportion of infected amoebae compared to all counted amoebae of three replicates at 48 h p.i. is shown +/− SEM. Heat-inactivated EBs, used as negative controls, were taken up by the amoebae but did not multiply.
Figure 7.
Purification of PomS from P. amoebophila EBs.
A gel stained with colloidal coomassie is shown; 1, outer membrane fraction after incubation of EBs with n-octyl-POE; 2, outer membrane fraction after precipitation with acetone; 3, column flow through; 4–7, fractions after elution with 0.1, 0.25, 0.3, 0.35 and 0.4 M NaCl. Molecular mass of marker bands (M) in kDa; the expected size of PomS (33.9 kDa) is indicated by an arrow head.
Figure 8.
Porin function of purified PomS.
Single channel experiments using a PC/n-decane membrane in the presence of purified PomS. The aqueous phase contained 1 M KCl and 10 ng ml−1 PomS dissolved in 1% Genapol. The applied membrane potential was 20 mV; T = 20°C. Left panel: Single-channel recordings show a uniform stepwise increase as expected for a highly enriched purified porin. Right panel: Frequency of observed conductance increments. P(G) was calculated by dividing the number of fluctuations with a given conductance increment by the total number of conductance fluctuations. Data from both panels suggest that the purified protein fraction contains mainly PomS (about 82% of the total number of pores) and that there is only a very minor contribution of other pores in the histogram (about 18% of the total number of pores) caused either by contaminant porins or by degradation of PomS. The average single-channel conductance was 3.25 nS for 230 single-channel events.
Table 2.
Average single-channel conductance of PomS in different salt solutions.
Table 3.
Zero-current membrane potentials of PC/n-decane membranes in the presence of PomS measured for a 2.5-fold gradient of different salts (300 mM versus 750 mM).
Figure 9.
PomS was added in a concentration of 500 ng ml−1 to the trans-side side of a PC/n-decane membrane in multi-channel experiments. The aqueous phase contained 1 M KCl, pH 6.0. After 30 min the conductance had increased considerably. At this point different potentials were applied to the membrane. The ratio of the conductance G at a given membrane potential (Vm) divided by the conductance Go at 10 mV was calculated as a function of the membrane potential Vm [79]. The membrane potential refers to the cis-side of the membrane. T = 20°C. Means (± SD) of three membranes are shown.