To see if the three tested Y. enterocolitica strains are able to degrade NETs, NET degradation assays were performed. Therefore, blood-derived neutrophils were stimulated for 4 h with PMA to induce NETs and then incubated with washed bacteria (Figure 5-3A) or the supernatant (Figure 5-2, lower row) to quantify degradation of NETs. To investigate whether there is a serotype-specific difference, the data of co-incubation with washed bacteria were normalised to an identical amount of bacteria (1*107 cfu) (Figure 5-3B). MN of S. aureus was used as positive control for NET degradation. As shown in Figure 5-3A, the result of the NET degradation assay showed a significant reduction of NETs mediated by the washed bacteria in comparison to the control (Figure 5-3A). The strongest degradation was observed for serotypes O:8 and O:9. Degradation was also observed when NETs were incubated with the supernatants of Y. enterocolitica serotypes (Figure 5-3A and B). Here, a significant difference was apparent only between serotype O:3 and the other two serotypes. No difference was observed between O:9 and O:8. The data indicated that all three tested Y. enterocolitica strains are able to degrade NETs.
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Figure 5-1. Different Y. enterocolitica serotypes induce NET formation over time.
Primary blood-derived neutrophils were incubated with washed bacteria (MOI 1) for 0.5 h, 1 h, 2 h, 3 h and 4 h. Co-incubation of the cells with serotypes O:9 (A) displayed significant NET release after 1 h, whereas co-incubation with O:8 (B) and O:3 (C) resulted in release of NETs already after 0.5 h. 25 nM PMA was used as a positive control of NET release. Results of 4 independent experiments with each 6 individual images were analysed using one-tailed Student´s t-test compared to untreated control. ns= not significant, * = p<0.05, ** = p<0.005,
*** = p<0.001; PMA treatment was analysed using one-tailed Student´s t-test compared to untreated control. ### = p<0.001.
45 Figure 5-2. All three Y. enterocolitica serotypes exhibit degrading activities resulting in dismantling of NETs. A) Representative fluorescent micrographs of PMA pre-treated neutrophils co-incubated with 0.01 U/ml MN from S. aureus, washed Y. enterocolitica strains or Yersinia supernatants showed clear visible degradation of the NET fibres. NETs were visualised with a primary H2A-H2B-DNA complex antibody and a secondary Alexa 488-labelled goat-anti-rabbit antibody (green). DNA was stained with DAPI (blue).
Figure 5-3. All three Y. enterocolitica serotypes exhibit degrading activities resulting in dismantling of NETs. Incubation of neutrophil-derived extracellular traps with A) all three washed Y. enterocolitica serotypes and B) all Y. enterocolitica serotypes normalised to an identical cfu of 1*107 displayed degradation of the NETs within 1 h; PMA control was adjusted to 100% NET area. Data of 4 independent experiments with each 6 individual images were analysed using one-tailed Student´s t-test compared to PMA control with ### = p<0.001; Differences between the serotypes were analysed using one-tailed Student´s t-test.
ns= not significant, * = p<0.05, ** = p<0.005, *** = p<0.001.
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A possible mechanism of pathogens evading NETosis could be the production or secretion of nucleases. For Enterobacteriacea a NET-degrading nuclease has not been described yet. Interestingly, it has been recently discussed that in case of Y.
ruckeri the secretion of a DNA endonuclease could assist in escaping from NETs through the digestion of the chromatin scaffold (Sallum and Chen, 2010). The authors showed an increase in bacterial intracellular and extracellular endonuclease expression after exposure of the bacteria to different concentrations of an antimicrobial peptide, called cecropin B. However, a specific NET-degrading effect has not been described.
NET degradation by Y. enterocolitica is dependent on Ca2+ and Mg2+ ions.
To further investigate whether the detected degradation capability of Y. enterocolitica was possibly due to a nuclease activity, calcium (Ca2+) and magnesium ion (Mg2+) availability was blocked during the 1 h co-incubation time with the washed bacteria.
For all three tested serotypes, there was significant degradation by the washed bacteria alone (data not shown) in cases of ion availability as well as in the treatment with protease inhibitor (Figure 5-4A), suggesting that no proteases are involved in NET degradation mediated by Y. enterocolitica. Interestingly, treatment with HBSS lacking Mg2+ and Ca2+ ions resulted in complete abolishment of the degradation in comparison to the control with PMA (Figure 5-4B).
Figure 5-4. NET degradation mediated by Y. enterocolitica serotypes O:9, O:8 and O:3 is dependent on Ca2+ and Mg2+ ions. A) Treatment of the washed bacteria with PI showed no influence on the NET degradation. B) Depletion of calcium and magnesium ions resulted in a total lack of degradation ability by the Y. enterocolitica serotypes; Comparison between the serotypes and PMA control were performed using one-tailed Student´s t-test of three
47 independent experiments with 6 individual images each. ns= not significant, ** = p<0.005, ***
= p<0.001.
The observed repressed degradation capability in the absence of Ca2+ and Mg2+ led to the hypothesis that a nuclease is involved in the NET degradation by Y.
enterocolitica. Nucleases are Ca2+ and Mg2+ dependent, using the minerals as enzymatic cofactors to activate their cleavage activity. The availability of these cofactors also plays a role in a different pattern of virulence factors (Dewoody, Merritt and Marketon, 2013). Depriving Ca2+ from the medium in vitro triggers a massive up-regulation of T3SS gene expression along with secretion of T3SS substrates (Straley and Bowmer, 1986; Dewoody, Merritt and Marketon, 2013). This results in a varied set of secreted Yops via the T3SS secretion system (Dewoody, Merritt and Marketon, 2013). In the presence of Ca2+, secretion of early and middle T3SS substrates into the extracellular milieu is readily observed, whereas the late Yops are not released in large amounts until either Ca2+ chelation or contact with a host cell occurs (Dewoody, Merritt and Marketon, 2013). Whether Y. enterocolitica secrete nucleases and/or if early or middle Yops are able to degrade NETs is an issue that requires further investigation.
Y. enterocolitica serotypes O:9, O:8 and O:3 show extracellular nuclease