Complementation experiments of various IreK constructs

To test the contribution of either the kinase or PASTA domains of IreK in E. faecalis, various mutants and constructs were designed and synthesised using the TetH-

MIC Cefotaxime (µg mL-1₎ Concentration of anhydrotetracycline 0 ng mL-1 5 ng mL-1 10 ng mL-1 20 ng mL-1 25 ng mL-1 OG1RF Tn1549 512 512 512 512 512 OG1RF::Tn1549 ΔireK 8 8 8 8 8 OG1RF::Tn1549 ΔireK TetH-ireK (FL) 256 512 512 512 1024 MIC Ceftazidime (µg mL-1₎ Concentration of anhydrotetracycline 0 ng mL-1 5 ng mL-1 10 ng mL-1 20 ng mL-1 25 ng mL-1 OG1RF Tn1549 2048 2048 2048 2048 2048 OG1RF::Tn1549 ΔireK 32 32 32 32 32 OG1RF::Tn1549 ΔireK TetH-ireK(FL) 128 1024 1024 2048 2048

ireK(FL) plasmid (pCWT050) as the backbone vector. These constructs were electroporated into OG1RF::Tn1549 ΔireK strains and selected on Erm30 plates. To gain an

understanding into the contribution of each domain, the enhanced cephalosporin resistance was phenotypically tested for each construct and compared to OG1RF::Tn1549, OG1RF::Tn1549 ΔireK and Tn1549 ΔireK TetH-ireK(FL) (Table 3.10). Each complementation strain was induced with 20 ng mL-1 _{anhydrotetracycline. Ceftazidime}

was used to test each of the different constructs as it had the strongest MIC that was in a detectable range and was thought to give the largest difference if any between each construct using the micro-dilution MIC method.

Table 3.4: Complementation of different ireK constructs in the complementation plasmid under 20 ng mL-1 _{anhydrotetracycline in Iso-sensitest media. (Experiments performed in duplicates). Green – KD, Blue}

– TM, Red – E. faecalis PASTA, Yellow - Substituted PASTA.

Schematic of Construct Construct Description (OG1RF) MIC Ceftazidime (µg mL-1₎

Tn1549 2048

Tn1549 ΔireK 32

Tn1549 ΔireK TetH-ireK (FL) 2048

Tn1549 ΔireK TetH-ireK (No Pasta) 128

Tn1549 ΔireK TetH-ireK (K41R) 32

Tn1549 ΔireK TetH-ireK (No Kinase) 32

Tn1549 ΔireK TetH-ireK (PASTA 1-5 minus

peptide)

2048

Tn1549 ΔireK TetH-ireK (PASTA 1-4) 2048

Tn1549 ΔireK TetH-ireK (PASTA 1-3) 2048

Tn1549 ΔireK TetH-ireK (PASTA 1-2) 1024

Tn1549 ΔireK TetH-ireK (PASTA 1) 1024

Tn1549 ΔireK TetH-ireK (PASTA 4-5) 2048

Tn1549 ΔireK TetH-ireK (S. aureus PASTA) 1024

When testing each of the TetH inducible constructs containing either a different mutant or truncation, the most significant differences were apparent when a whole domain was truncated (Kinase (KD) or PASTA). The truncation of the KD showed a large decrease in MIC that mirrored Tn1549 ΔireK levels at 32 µg mL-1. _{This illustrates the role}

of the kinase in phosphorylating other protein targets to cause enhanced cephalosporin resistance. Of particular note in Table 3.10 is the K41R mutation in the FL IreK construct. Initial characterisation of the eSTK in B. subtilis revealed that a mutation of residue K40 to K40R removed the enzymatic activity of the protein (Madec et al., 2002). This residue was constructed following alignment of the B. subtilis study and the K40 reside was aligned and homologous to the IreK amino acid sequence (K41). This mutation was also constructed in the Tn1549 ΔireK TetH-ireK (FL) and demonstrated how critical this amino acid residue is on enzymatic activity as it caused the FL protein to become inactive in the enhanced cephalosporin phenotype experiment.

Removal of all the PASTA domains (Tn1549 ΔireK TetH-ireK (No Pasta)) resulted in a sharp decrease in MIC from 2048 to 128 µg mL-1 _{when compared directly with}

OG1RF::Tn1549 and Tn1549 ΔireK TetH-ireK (FL). Although the removal of all the PASTA domains does not result in a Tn1549 ΔireK MIC phenotype (32 µg mL-1 _{ceftazidime), this}

may be attributed to the ability of the kinase domain to self oligomerize independently of the PASTA domains and that the role of the PASTA domains is the concentrate the kinase domain of the protein in a particular location in the E. faecalis cell. In a study comparing the role of the IreK homologue in S. aureus PknB, two virulent strains were compared, including COL and USA300 (Tamber et al., 2010). Interestingly COL had a point mutation on the gene for PknB that resulted in a stop codon being introduced resulting in the truncation of the PASTA domains. When comparing the antibiotic resistance between both strains, it was suggested that the kinase alone was sufficient for antibiotic resistance.

Truncation of each individual PASTA domain resulted in a phenotype that was similar to OG1RF::Tn1549 MIC levels to ceftazidime (1024-2048 µg mL-1_{) and that a single}

PASTA domain (Tn1549 ΔireK TetH-ireK (PASTA 1)) was sufficient to complement to WT levels. Therefore, the number of PASTA domains present in E. faecalis is not directly linked to the enhanced cephalosporin resistance observed in this study and indicated that they may have a separate role. A recent paper has suggested that the PASTA domains

may act as a “molecular ruler” and monitor the thickness of the PG in growing cells. This was demonstrated in S. pneumoniae and the removal of each PASTA domain resulted in a decrease in PG thickness (Zucchini et al., 2018). It has also been recently demonstrated that in E. faecalis OG1 WT strains with various PASTA chromosomal truncations, that there is not much of a change in MIC against ceftriaxone with each truncation and is consistent with the phenotype observed here (Labbe and Kristich, 2017). In a previous study looking at the IreK homologue in E. faecium, a long C-terminal S/T terminal peptide is present and was suggested to interfere with PASTA oligomerisation (Desbonnet et al., 2016). This motif is also present in E. faecalis but is much shorter. A truncation of this Ser/Thr terminal peptide was also constructed in IreK but showed no difference in MIC.

To explore the role of PASTA domains in the context of enhanced cephalosporin resistance further, the PASTA domains from E. faecalis were substituted with either the PASTA domains from S. aureus PrkC (containing 4 PASTA domains) or B. subtilis PrkC (containing 3 PASTA domains). Although the S. aureus PASTA domains did not show a large difference in MIC compared to E. faecalis IreK PASTA domains (1024 µg mL-1_{), there}

was a modest difference between the B. subtilis PASTA domains in IreK (512 µg mL-1_{). It}

has been suggested and shown in a different study that PASTA domains can bind PG directly and upregulate a cellular response when changes in the extracellular environment are detected (Shah et al., 2008). The major difference between E. faecalis and S. aureus is the PG cross linking stem (L-ala2 and G5 respectively) and the major

difference between E. faecalis and B. subtilis is amidation in the second position, the 3rd

position in the pentapeptide stem (Lys and Dap respectively) and therefore no crosslinking stem is present in B. subtilis.

There are also modifications of the glycan strands that can occur between each species including N-deacetylation (B. subtilis) and O-acetylation of the MurNAc residue (E. faecalis and S. aureus) (Vollmer, 2008). Therefore, the difference in MIC observed in this experiment with the PASTA domains substitutions might be due to the variation in the PG between each species.

3.9. Localisation of IreK in inducible complementation