Construction of 23 mutant strains.
To construct strains missing two, three, or four of Yersinia surface factors (YadA, Ail, O-ag, and OC oligosaccharide), we first stemmed from the wild-type (wt) Y. enterocolitica O:3 strain 6471/76 six single-negative direct derivatives: (i) the pYV-cured strain YeO3-c; (ii) the yadA strain YeO3-028 (ΔyadA::Km-GenBlock); (iii) the ail strain YeO3-Ail (Δail::Km-GenBlock); (iv and v) the two types OC mutants, YeO3-OC [Δ(wzx-wbcQ), with the whole OC cluster deleted] and YeO3-trs11 [Δ(wzx-wbcL)::Km-GenBlock], and (vi) the rough strain YeO3-R2 (spontaneous O-ag-negative mutant). The first five served as starting points for further construction steps. Detailed description of the construction strategies of the strains is given in Materials and Methods.
The YadA, O-ag, OC, and Ail expression levels of all the strains were compared. Immunoblotting and dot blotting showed that there were small differences in YadA expression levels between the studied strains (Fig. and data not shown). Compared to the wild type, YeO3-Ail, YeO3-OC, YeO3-Ail-OC, and YeO3-OCR produced slightly more YadA, while YeO3-trs11-R and YeO3-Ail-OCR expressed significantly less protein. When O-ag expression was compared between the strains by immunoblotting, only YeO3-c-trs8 and YeO3-trs11 showed a little fainter O-ag smear than that of the wild type (Fig. and data not shown). A little more O-ag was expressed only by a few strains (for example, YeO3-c; Fig. and data not shown). Most strains expressed amounts of OC similar to those of YeO3, as demonstrated by immunoblotting and dot blotting. There seemed to be marginally more OC in the O-ag-negative strains (Fig. and data not shown). The comparison of Ail expression was performed by analysis of outer membrane proteins (Fig. ). These were run on SDS-PAGE and visualized by silver staining. There was, however, a protein comigrating with the Ail band that gave a faint background band in the Ail-negative strains, YeO3-Ail and E. coli carrying pBR322 (Fig. ). Based on the band intensities, YeO3-R2, YeO3-c-OC, YeO3-O28-R, YeO3-OCR, and YeO3-trs11 produced slightly more Ail, while YeO3-OC and YeO3-trs11-R produced slightly less Ail, than the wild-type strain.
FIG. 1. Detection and analysis of YadA, O-ag, OC, and Ail expression in different Y. enterocolitica O:3 strains. (A) Immunoblot analysis using monoclonal antibodies specific for YadA (top), O-ag (middle), and OC (bottom). (B) Silver-stained SDS-PAGE gel showing (more ...) CP/AP- and AP-mediated killing of bacteria grown to stationary phase.
In general, when grown at RT all the studied Y. enterocolitica strains were highly susceptible to AP- and CP/AP-mediated killing, being totally killed already after 30 min of incubation (data not shown). On the contrary, after growing at 37°C the strains expressed various degrees of serum resistance. Several serum resistance phenotypes could be distinguished based on the survival percentages and killing kinetics of the strains (Table , Fig. and ). Below we discuss these results, organized under the construction lines of strains, to demonstrate how stepwise loss of the individual factors affected the bacterial serum resistance.
Complement resistance phenotypes and killing kinetics of Y. enterocolitica O:3 wt and mutant strains (grown at 37°C) in 66.7% NHS (based on data presented in Fig. and )
FIG. 2. Survival percentages of YadA-positive Y. enterocolitica O:3 strains (grown to stationary phase at 37°C) in normal and EGTA-Mg-treated sera after 30 or 120 min of incubation. Survival in HIS was set to 100%. In each panel, the left-hand columns (more ...)
Survival percentages of YadA-negative Y. enterocolitica O:3 strains grown at 37°C in normal and in EGTA-Mg-treated sera after 30 or 120 min of incubation. See the legend to Fig. for further explanations.
The pYV-positive YadA-positive strains.
In the line YeO3→YeO3-Ail→YeO3-Ail-R→YeO3-Ail-OCR (Fig. , top row), all the strains expressed YadA as we deprived the wt strain YeO3 first of Ail and then stepwise of O-ag and OC. Resistance to both sera (resistance to EGTA-Mg serum was more prominent) displayed by the wt strain was significantly weakened by removal of Ail (YeO3-Ail) and then of O-ag (YeO3-Ail-R). Interestingly, final loss of OC (YeO3-Ail-OCR) decreased resistance to NHS even more, but it had no effect on resistance to EGTA-Mg serum.
In the two genetically different but phenotypically identical lines YeO3→YeO3-OC→YeO3-OCR and YeO3→YeO3-trs11→YeO3-trs11-R (Fig. and data not shown), we first deprived the wt strain of OC by complete deletion of the OC gene cluster (YeO3-OC) or by Km-GenBlock insertion (YeO3-trs11). YeO3-OC and YeO3-trs11 displayed comparable and higher resistance to both sera than the wt strain. In both mutant lines, further loss of O-ag (YeO3-OCR and YeO3-trs11-R) caused a decrease in resistance to NHS and EGTA-Mg serum after 2 h of incubation.
In line YeO3→YeO3-OC→YeO3-Ail-OC (Fig. ), YeO3-OC (see above) was further deprived of Ail, which resulted in decreased resistance to both types of sera.
The last pYV-positive YadA-positive line was YeO3-R2 (Fig. , bottom). Compared to the wt strain, YeO3-R2, missing only the O-ag, displayed a significant increase in resistance to NHS. Resistance to EGTA-Mg serum presented by both strains was at about the same level, but surprisingly, YeO3-R2 was more resistant to NHS than to EGTA-Mg serum.
The YadA-negative strains.
Two different YadA-negative strains, YeO3-028 (ΔyadA::Km-GenBlock) and YeO3-c (pYV negative), served as starting points for construction of strains missing Ail, O-ag, and OC. Both YeO3-028 and YeO3-c were very rapidly killed in NHS (Fig. ). They displayed high-level resistance at 0.5 h in EGTA-Mg serum, but at 2 h they were efficiently eliminated.
In both the pYV-positive, YadA-negative line YeO3→YeO3-028→YeO3-028-R→YeO3-028-OCR and the pYV-negative line YeO3→YeO3-c→YeO3-R1 (Fig. ), loss of O-ag did not alter the resistance of bacteria to EGTA-Mg serum, but it increased the resistance of bacteria to NHS (see YeO3-028-R and YeO3-R1). However, there were pronounced differences between these two Ail- and OC-positive strains, especially in NHS: YeO3-028-R was more sensitive than YeO3-R1. An explanation for this may be the presence of a fully functional type III secretion system (12
) in YeO3-028 and its derivatives but not in YeO3-R1. Type III secretion utilizes a syringe-like structure that extends through the bacterial cell wall. One could speculate that it could impair the OM integrity and make it more susceptible to complement components.
The OC-negative strain YeO3-028-OCR, derived from YeO3-028-R and expressing only Ail, displayed increased resistance to complement, especially at 2 h, in both sera (Fig. ).
In the pYV-positive YadA-negative line YeO3→YeO3-028→YeO3-028-OC (Fig. ) as well as the pYV-negative lines YeO3→YeO3-c→YeO3-c-OC→YeO3-c-OCR (Fig. ) and YeO3→YeO3-c→YeO3-c-trs8→YeO3-c-trs8-R (data not shown), three strains sharing the O-ag Ail-positive phenotype were constructed using different genetic approaches to inactivate or delete the yadA gene and the OC gene cluster (Table ). Irrespective of their genetic history, serum resistance of the strains was similar (compare YeO3-028-OC, YeO3-c-OC, and YeO3-c-trs8); loss of OC did not dramatically change bacterial resistance to the complement. Of note was the slight increase in resistance of YeO3-c-OC and YeO3-c-trs8 (but not that of YeO3-028-OC) after 0.5 h of incubation in both types of sera. The O-ag-negative derivatives YeO3-c-OCR and YeO3-c-trs8-R (Fig. and data not shown) showed significantly increased resistance, especially to NHS, compared to that of their O-ag-positive precursors. Again, as noted above, the effect of removal of O-ag and exposure of Ail resulted in increased serum resistance.
In the pYV-negative lines YeO3→YeO3-c→YeO3-c-Ail→YeO3-c-Ail-OC and YeO3→YeO3-c→YeO3-c-Ail→YeO3-c-Ail-R (Fig. ), strains expressing only O-ag and/or OC (YeO3-c-Ail, YeO3-c-Ail-OC, and YeO3-c-Ail-R) were highly susceptible to complement killing, as was YeO3-c-Ail-OCR, derived from YeO3-c-Ail-R and missing all four surface factors (data not shown). These results were identical to those obtained with all the strains grown at RT (see above).
Influence of growth phase on serum resistance.
The growth phase experiments were performed with the wt strain YeO3 and strains expressing only one of the four factors: Ail (YeO3-c-OCR), YadA (YeO3-Ail-OCR), OC (YeO3-c-Ail-R), and O-ag (YeO3-c-OC). The bacteria grown at RT and at 37°C exposed to NHS and EGTA-Mg serum were either in the logarithmic or stationary phase of growth. The results (Fig. ) clearly showed that wt strain YeO3, when growing exponentially at 37°C (expressing YadA, Ail, O-ag, and OC), was more resistant to NHS and EGTA-Mg serum than when it had reached stationary phase. Interestingly, YeO3 grown at RT (expressing only O-ag and OC) both in the exponential and stationary phase of growth was highly sensitive to NHS and EGTA-Mg serum (Fig. ). Thus, YadA- and Ail-negative bacteria were rapidly killed irrespective of the growth phase.
Effect of growth phase on the survival of the wt strain YeO3 in normal and in EGTA-Mg treated sera. Y. enterocolitica O:3 bacteria, grown at RT and at 37°C to stationary or to exponential phase, were exposed to sera for 0.5 and 2 h.
Independent of the phase of growth, strains expressing only OC (YeO3-c-Ail-R grown at 37°C) or O-ag (YeO3-c-OC grown at RT) were highly sensitive to NHS and to EGTA-Mg serum (data not shown). Ail-expressing bacteria (YeO3-c-OCR) at exponential phase were significantly more resistant to serum, especially after 2 h of incubation, than bacteria at the stationary phase of growth (Fig. ). The same also applied to the YadA-expressing strain YeO3-Ail-OCR (Fig. ) at 0.5 h of incubation in sera. After 2 h of incubation, differences between YadA-positive bacteria at stationary and exponential phase were very small, and the bacteria were highly sensitive to serum killing.
Effect of growth phase on the survival of strains YeO3-c-OCR (Ail positive) and YeO3-Ail-OCR (YadA positive) grown at 37°C and incubated in normal and in EGTA-Mg-treated sera.
Deposition and degradation of complement component C3 on Y. enterocolitica O:3 strains.
To check C3 deposition and cleavage on Yersinia
, we applied the monoclonal antibody, specific for the α-chain of C3 and binding to the C-terminal 40-kDa fragment of iC3b, called MAb755 (48
). It detects intact C3, C3b, and iC3b as 118-, 109-, and 40-kDa fragments, respectively (Fig. ). In addition, C3b molecules covalently bound to Yersinia
surfaces are detected as high-molecular-weight (HMW) bands with molecular masses of over 200 kDa. For the deposition experiments, we used Yersinia
mutants representing different phenotypes and allowed the bacteria to interact with NHS and EGTA-Mg serum for 3, 15, and 30 min before processing the samples for immunoblotting as described in Materials and Methods. In the interpretation of the C3 deposition data, one needs to remember that some strains were already completely killed at 30 min, thus, significant amounts of C3 originally bound to bacteria could have been washed away with bacterial debris.
FIG. 6. C3 deposition and degradation on different Y. enterocolitica O:3 mutant bacteria. Bacteria were incubated in EGTA-Mg-treated serum and NHS for 3, 15, and 30 min. After being washed, C3 and its derivatives deposited on bacteria were identified by immunoblotting (more ...) Deposition in EGTA-Mg-treated serum. (i) HMW C3.
Ten out of 11 O-ag-positive strains used in the experiment did not display HMW C3 binding at 3 min (Fig. ). Among those strains, there were two single-positive O-ag mutants (YeO3-Ail-OC [at 22°C] and YeO3-Ail-OC), but there were also strains additionally expressing YadA, Ail, or OC in different combinations (Fig. ). The only O-ag-positive strain that bound some HMW C3 already at 3 min was YeO3-trs11; interestingly, the expression level of O-ag for this strain was lower than that in others (Fig. ).
Five of the 10 strains still resisted HMW C3 deposition at 15 min or were very efficient in cleaving it (YeO3, YeO3-Ail, YeO3-Ail-OC, YeO3-Ail [at 22°C], and YeO3-c-Ail). Interestingly, three of those strains expressed YadA (YeO3, YeO3-Ail, and YeO3-Ail-OC). The only O-ag-positive Ail-positive strain that did not bind HMW C3 was the wild-type strain expressing all four factors. Ail-positive, O-ag-positive, and YadA- and/or OC-negative strains (YeO3-028, YeO3-trs11, YeO3-OC, and YeO3-c-trs8) were HMW C3 positive at 15 min, as were two single-positive O-ag strains (YeO3-Ail-OC [at 22°C] and YeO3-Ail-OC). The only O-ag-positive YadA-negative strains that did not bind HMW C3 at 15 min were those that expressed OC (YeO3-Ail and YeO3-c-Ail).
Finally, the only strain that did not display HMW C3 on the surface at 30 min was the wild-type strain YeO3.
In general, O-ag-negative strains already deposited C3b on the cell surface by 3 min, while of the 11 O-ag-positive strains, 9 did not (Fig. ). The O-ag-positive exceptions were YeO3-c-trs8 and YeO3-O28, both being Ail positive and YadA negative. Eight of the above nine O-ag-positive strains did not bind C3b at 15 and 30 min; six of them were Ail negative. Interestingly, the remaining two expressed O-ag and Ail in combination with YadA (YeO3-OC) or OC (YeO3-028). YeO3-trs11, though YadA, O-ag, and Ail positive, had bound some C3b at 15 min and more at 30 min.
Most of the strains strongly deposited the cleavage product of C3b, iC3b, on the cell surface. The strains that did not show deposition of HMW C3 or C3b also showed no or less deposition of iC3b. After 3 min, from the group of O-ag positive mutants the single-positive strains YeO3-c-Ail-OC and YeO3-Ail-OC (22°C) showed some iC3b deposition that increased with time (Fig. ). At this time, YeO3-R1 was an exceptional O-ag-negative strain not displaying iC3b. At 15 min, however, only the Ail-negative O-ag-positive strains expressing either YadA or OC (YeO3-Ail [at 22°C], YeO3-Ail-OC, and YeO3-c-Ail) still showed no iC3b deposition; however, some had taken place at 30 min. At 30 min, no iC3b deposition was visible on the wild-type strain (Fig. ).
Deposition in NHS. (i) HMW C3.
In general, more C3 deposition took place in NHS than in EGTA-Mg-treated serum. Two of the three strains that did not bind HMW C3 at 3 min expressed YadA in combination with Ail (YeO3 and YeO3-OC). YeO3-trs11 was an exception, being the only YadA-, Ail-, O-ag-positive strain that deposited HMW C3. YeO3-c-Ail-OC was the third O-ag-positive strain not depositing HMW C3. The strain with the same phenotype (YeO3-Ail-OC 22°C) showed a little HMW C3 at 3 min. At 15 min, only triple-positive OC mutants (YeO3-trs11 and YeO3-OC) resisted HMW C3 deposition. However, HMW C3 deposition was seen with all the strains at 30 min.
Nine of the 11 O-ag-positive strains showed no C3b deposition at 3 min (Fig. ). The two O-ag-positive exceptions were the Ail- and OC-expressing strains YeO3 and YeO3-O28. YeO3-c-trs8 started showing some C3b at 15 min. YeO3-trs11 and YeO3-OC, both missing OC, were the only O-ag-, Ail-positive strains that did not bind C3b at 15 min. At 30 min, no C3b binding was observed with the four O-ag-positive, Ail-negative strains missing either YadA, OC, or both (YeO3-Ail [at 22°C], YeO3-Ail-OC, YeO3-c-Ail, and YeO3-Ail-OC [at 22°C]). The other single O-ag-positive strain, YeO3-c-Ail-OC, grown at 37°C and expressing shorter O-ag, bound C3b at 30 min (Fig. ). No C3b was deposited on YeO3 and O-ag-expressing triple-positive strains (YeO3-028 and YeO3-OC). The exception was again YeO3-trs11, which bound C3b at 30 min.
In NHS after 3 min of incubation, only the triple-positive, YadA-expressing strains did not display iC3b on the surface (YeO3, YeO3-Ail, and YeO3-OC); again, YeO3-trs11 was an exception, showing some iC3b deposition. At later time points all strains became iC3b positive (Fig. ).