Lysostaphin – targeting Staphylococcus aureus

Lysostaphin is a zinc endopeptidase that cleaves the pentaglycine cross-bridges present in the bacterial cell wall of S. aureus (see figure 1). It is effective for degrading S. aureus biofilms. For using lysostaphin in our dextran hydrogel is it important that it gets secreted by our bacteria which also produce the adhesin.

Figure 1. Schematic overview of Lysostaphin cleaving the pentaglycine on the cell membrane of S. aureus.

Overview

To achieve our results, we performed three types of experiments:

1. Cloning of the constructs for lysostaphin expression and secretion. We succeeded in making the DNA constructs for the Gibson assembly.
2. Protein expression experiments, in which we ensured that lysostaphin got expressed and secreted. After the Gibson assembly, a double transformation was performed to combine the expression and secretion plasmids for lysostaphin. A western blot confirmed that lysostaphin was secreted in the cell growth medium.
3. Functionality experiments of lysostaphin on plates containing Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). We observed halo formation around our lysostaphin-producing E. coli, but no halo formation around a negative control. These results combined confirm that the eradication of S. aureus is due to the lytic activity of lysostaphin produced by our E. coli, and not by possible remnants of antibiotics.

Cloning

For the lysostaphin expression and secretion, we needed two plasmids. First, for the lysostaphin expression a plasmid containing Lysostaphin-HlyA-His in a pBAD vector was designed (see figure 2). The HlyA tag on lysostaphin is necessary for the secretion of lysostaphin.

Figure 2. Linearized vector of pBAD-Lysostaphin-HlyA-His. The orange part between Lysostaphin and Hlya is the thrombin linker. AraC is the repressor and araO₁ and araO₂ are the operators.

The second plasmid is for the secretion, containing the secretion system HlyB/D. This gene is inserted into two vectors: pTetO and pSTV (see figure 3). Preferably, pTetO was used, since that one has a different inducer than our adhesin protein, allowing for the individual regulation of every protein. However, our supervisors had more experience with pSTV in combination with the secretion system. Therefore we decided to also test this construct for lysostaphin secretion. Our supervisor had already produced the construct pSTV-HlyB/D. Therefore, there is no PCR nor Gibson assembly performed on this construct.

Figure 3. Linearized vector of pSTV-HlyB/D with an lac operator and a linearized vector of pTeto-HlyB/D with an TetR repressor.

As a negative control sample, a plasmid containing only the secretion tag (HlyA) was used (see figure 4). This was necessary to compare it with lysostaphin secretion and functionality. Our supervisor already had the construct pET24a-HlyA. Therefore there is no PCR nor Gibson assembly performed on this construct. This can be combined with the pSTV-HlyB/D as the pTetO-HlyB/D vector.

Figure 4. Linearized vector of pET24a-HlyA containing also a lac operator and repressor.

We successfully performed PCR reactions of pBAD, lysostaphin-HlyA-His, pTetO and HlyB/D. The results of the PCR products can be seen in figure 5.

Figure 5. A. PCR Lysostaphin-HlyA-His with an expected size of 1681 bp. B. PCR pBAD, with an expected size of 3957 bp. C. PCR pTetO with an expected size of 2598 bp. D. PCR HlyB/D with an expected size of 3597 bp

Once the PCR had succeeded, the Gibson assembly could be performed to generate the constructs shown in figure 4. To check if the insert was present, a double-digestion was performed using adequate restriction enzymes. The digestion was performed on plasmids purified from three different colonies of C2987 pTetO-HlyB/D and two colonies of C2987 pBAD-Lysostaphin-HlyA-His. The bands for pTetO-HlyB/D were expected at 1471 and 4724 bp and pBAD-Lysostaphin-HlyA-His at 1450 and 4021 bp. Columns 3, 4 and 5 in figure 5 show that pTetO-HlyB/D and pBAD-Lysostaphin-HlyA-His contain the correct insert.

Figure 6. Digestion of the construct pTetO-HlyB/D (line 1-3) and pBAD-Lysostaphin-HlyA-His (line 4-5). For the construct pTetO-HlyB/D the restriction enzyme NdeI was used and for pBAD-Lysostaphin-HlyA-His the restriction enzymes BamHI and pstIHf were used.

Protein expression

In order to test the expression of lysostaphin prior to performing secretion experiments, the pBAD-Lysostaphin-HlyA-His construct was transformed in BL21(DE3) cells. Protein expression was induced by arabinose. After four hours, cells were harvested and lysed. The protein composition of the lysate was further analysed using SDS-PAGE stained with Coomassie blue. A clear overexpression band at 25 kDa is observed in figure 7. However, the expected size of Lysostaphin-HlyA-His is around 50 kDa, since lysostaphin is 27 kDa and HlyA 23 kDa. We reason Lysostaphin-HlyA-His is performing self-cleavage due to the lysostaphin activity, likely targeting the Thrombin-linker. This is a Glycin-rich peptide.

Figure 7. SDS PAGE gel of pBAD-Lysostaphin-HlyA-His induced by 20 mM arabinose. The samples were taken before the induction and after 4 hours. The 4h - shows the non-induced sample, which was taken as negative control. An over-expression band is visible around 25 kDa.

To test whether lysostaphin could be secreted, both HlyB/D and lysostaphin were expressed by arabinose and IPTG induction in presence of Calcium. After 4 hours, the growth medium (LB) was harvested, concentrated (app. 30X), washed, and analysed using SDS-PAGE. Since the SDS-PAGE stained using Coomassie blue did not show very convincing secretion, a western blot was performed using anti-his primary antibodies in order to bind the His-tag attached to the HlyA secretion tag. Since the pSTV expression system showed the most promising secretion results, this western blot was only performed on the growth medium of cell transformed with pBAD-Lysostaphin-HlyA-His and pSTV-HlyB/D plasmids.

Figure 8. Western blot on cell-free growth medium of induced pBad-lysostaphin-his + pSTV-HlyB/D with anti-his antibody. There are fat secretion bands at ~25 kDa, indicating the cleavage of lysostaphin and its secretion tag. However at the highest concentration (30x diluted), there is a clear band demonstrating the secretion of the full construct.

The western blot shows that the largest amount of lystostaphin-HlyA-His is cleaved at the thrombin linker (figure 8). However, the most concentrated growth medium (30X) also displays a clear band at approximately 50 kDa, corresponding to the full-length lystostaphin-HlyA-His. The western blot thus confirms the secretion of the lysostaphin fusion protein.

Functionality Experiments

Once secretion had been demonstrated, the last step was the verification of the activity of lysostaphin against S. aureus. Those experiments were performed in collaboration with PAMM, the regional center for infectious diseases and pathology of South East Brabant, the Netherlands. We designed the experimental protocols, and the experiments were performed at PAMM, where working with S. aureus was permitted (biosafety regulations).

Three experiments were performed: one with cell lysate, one with cell growth medium and one with cell culture. As a positive control the E. coli cells transformed with the plasmids pBAD-Lysostaphin-HlyA-His and pSTV-HlyB/D was used on plates seeded with S. aureus cultures. pSTV-HlyB/D is chosen for the secretion plasmid since this construct showed the most promising secretion in our E. coli. As a control, plates were seeded with S. agalactiae (HSB). This is a gram positive bacterium which should not be susceptible to lysostaphin. However, it would be affected if traces of antibiotic were still present in the samples. As an extra negative control we used E. coli cells transformed with pET24a-HlyA and pSTV-HlyB/D, which only secrete the HlyA secretion tag. This was added only on plates seeded with S. aureus.

Cell lysates

Cultures of Bl21(DE3) (pBAD-Lysostaphin-HlyA-His + pSTV-HlyB/D) and of BL21 DE3 (pET24a-HlyA + pSTV-HlyB/D) were induced using IPTG and/or arabinose and incubated for 4 h. Further cells were lysed using French press and lysates were washed thoroughly. This last step was done to remove any potential traces of antibiotics, in order to exclude antibiotics as a source of bactericidal activity against S. aureus. S. aureus and S. agalactiae were seeded on separate Mueller-Hinton agar plates and 3 µL and 5 µL of E. coli (pBAD-Lysostaphin-HlyA-His + pSTV-HlyB/D) cell lysate was applied. After an overnight incubation with the cell lysate of our lysostaphin-producing E. coli and HlyA producing E. coli the results in figure 9 were obtained (3 µL top and bottom left; 5 µL top and bottom right).

Figure 9: A. Plate seeded with S. aureus with drops of cell lysate of induced lysostaphin-producing E. coli B. Plate seeded with S. aureus with drops of cell lysate of Lysostaphin producing E. coli. C. Plate seeded with S. agalactiae with drops of cell lysate of Hly-A producing E. coli

Clear halo formation on the plate seeded with S. aureus can be observed, indicating the production and activity of lysostaphin in the cell lysate. No halo formation was observed on the plate seeded with S. agalactiae nor on the plate with the added negative control (HlyA secretion tag only) samples. These results confirm that the results observed are caused by the lytic activity of lysostaphin produced by our E. coli, and not by possible remains of antibiotics or by the secretion tag.

Cell growth medium

Next, we tested the functionality of the secretion system. The plasmids for secretion and production were simultaneously induced as well for the Bl21(DE3) (pBAD-Lysostaphin-HlyA-His + pSTV-HlyB/D) as for the BL21 DE3 (pET24a-HlyA + pSTV-HlyB/D). After four hours of induction, a sample of the growth medium (LB) was harvested, filtered and washed extensively to remove any residual traces of antibiotics. The growth medium was concentrated 30X. S. aureus and S. agalactiae were seeded on separate Mueller-Hinton agar plates to which 3 and 5 µL of E. coli growth medium were applied. After an overnight incubation with the 30X concentrated growth medium of our lysostaphin- and HlyA-producing E. coli’s, the results in figure 10 were obtained (3 µL top and bottom left; 5 µL top and bottom right):

Figure 10. A. plate seeded with S. aureus with 30X concentrated growth medium of induced Lysostaphin producing E. coli. B. Plate seeded with S. agalactiae with 30X concentrated growth medium of induced lysostaphin-producing E. coli . C. Plate seeded with S. aureus with 30X concentrated medium of induced HlyA secretion E. coli.

Clear halo formation on the plate seeded with S. aureus can be observed, containing the growth medium from the E. coli producing lysostaphin, confirming the production, secretion and activity of lysostaphin in the growth medium. No halo formation was observed on the plate seeded with S. agalactiae, but also not on the negative control, which is our E. coli producing only the secretion tag. This confirms that the results observed are caused by the lytic activity of lysostaphin produced by our E. coli, and not by possible remains of antibiotics. This confirms that lysostaphin is successfully secreted in the growth medium by our engineered E. coli (pBAD-Lysostaphin-HlyA-His + pSTV-HlyB/D) and that the lytic activity observed against S. aureus is due to the lysostaphin and not HlyA or other components in the medium.

Co-culture

As an ultimate experiment for confirming the secretion of the lysostaphin construct by E. coli, a co-culture of S. aureus, methicillin-resistant S. aureus (MRSA) or S. agalactiae with E. coli BL21 (DE3) on Mueller-Hinton agar plates was performed. The cultures were first washed extensively with LB to remove any residual traces of antibiotics, before being applied to the plates. The plates were covered with our inducers, IPTG, arabinose and CaCl₂ to induce E. coli to express lysostaphin-HlyA-His or HlyA and the HlyB/D secretion proteins. First S. aureus or Streptococcus agalactiae were plated on the inducer-agar. Subsequently, drops of uninduced BL21 (DE3) cultures were put on top of this film. The results after an overnight incubation can be seen in figure 11.

Figure 11. a: S. aureus plate co-cultured with E. coli with induced lysostaphin expression. b: S. agalactiae plate co-cultured with E. coli with induced lysostaphin and HlyB/D expression. c: S. aureus plate co-cultured with E. coli with induced HlyA and HlyB/D expression. d: MRSA plate co-cultured with E. coli with induced lysostaphin and HlyB/D expression. e: MRSA plate co-cultured with E. coli with induced HlyA and HlyB/D expression and S. aureus.

Once again, clear halo formation was observed on the S. aureus plate. The MRSA plate was also clearly affected by E. coli producing lysostaphin. The production, secretion and activity of lysostaphin were thus confirmed. No halo formation was observed on the S. agalactiae plate nor on the negative control. This confirms that lysostaphin is successfully secreted by our engineered E. coli and that the lytic activity observed against S. aureus and MRSA is due to the lysostaphin and not due to HlyA or other components that are produced by our E. coli.

Conclusion

Through a series of experiments performed in collaboration with PAMM, we confirmed the production and the secretion of lysostaphin by living E. coli. Importantly, the experiments show that lysostaphin effectively kills S. aureus and MRSA.

We observed the cleavage of our lysostaphin construct from its secretion tag. Although this was unexpected, it can be explained by the fact that lysostaphin cleaves pentaglycine bridges. A similar structure is also present in the thrombin linker (tetraglycins). As lysostaphin still got secreted and was demonstrated to be functional, we believe the cleavage is not problematic for our purpose.

Outlook

For further experiments we will make a plasmid containing both the pBAD-Lysostaphin-HlyA-His and pSTV-HlyB/D genetic modules. This is necessary since the vector pSTV shares the same antibiotic resistance as the adhesin protein. The production of a plasmid merging the secretion and the lysostaphin production functions would allow the cotransformation with the plasmid for adhesin production. The primers have already been designed, but due to a lack of time we did not succeed to generate the merged plasmid.

Furthermore, we would like to expand our system to the production of other bacteriocins, such as Pyocin S5. A gene block containing the pyocin sequence was already ordered, but it has not yet been integrated into the system.