DIARREA AGUDA

At the time that this investigation was undertaken a WTA biosynthetic pathway was well established (Swoboda et al., 2010) with all steps having been assigned their proteins (Refer to Chapter 1.9.1, Figure 1.10). However the final step in WTA

synthesis, the transfer of the cell wall polymer from their lipid-linker precursor to the cell wall peptidoglycan remained uncharacterised. It was suggested that the proteins responsible for this step were the uncharacterised LytR-CpsA-Psr (LCP) proteins, SA2103, SA1195 (MsrR) and SA0908, in personal communications with R. Daniel and the B. Bergi-Bächi group. This was later supported by evidence within B.subtilis that LCP proteins carried out the linkage to the peptidoglycan (Kawai et al., 2011) and they were subsequently published as the hypothetical WTA ligases (Dengler et al., 2012; Over et al., 2011). These proteins along with TarO, the first step of the WTA

biosynthetic pathway, were chosen to be investigated. In B.subtilis the WTA

biosynthetic machinery has been shown to form a complex network of interactions through yeast and bacterial two hybrids (Formstone et al., 2008), thus these four proteins were felt to be adequate to suggest if the WTA biosynthesis proteins interact with the divisome.

The topology of the LCP proteins was unknown so their protein sequences were obtained from NCBI and the topology predicted using ConPred II (Arai et al., 2004).

S.aureus TarO, SA1195 and SA0908 were all predicted to have at least one

transmembrane helix with an Nin-Cout topology. SA0908 was predicted to be soluble

(Table 3.1). As members of the LCP family, SA2103, SA1195 and SA0908 were expected to have a short intracellular, a transmembrane domain and a large extracellular region containing the LCP domain (Hübscher et al., 2008; Kawai et al., 2011). Therefore N- terminal fusions of each protein are necessary to position the adenylate cyclase fragments in the cytosol.

Penicillin binding protein 4 (PBP4) is required for the synthesis of the characteristic highly cross-linked peptidoglycan of S.aureus and localises to the septum, however in the absence of WTA synthesis it becomes dispersed throughout the entire cell

84 membrane and is unable to function normally (Atilano et al., 2010). This interesting observation suggests an interaction between PBP4 and the WTA biosynthetic

machinery, despite a negative result previously seen between PBP4 and TarO (Atilano

et al., 2010), and therefore was included within this investigation. The crystal structure

of PBP4 has shown that it consists of two domains; an N-terminal domain containing its transpeptidase domain and an all β-sheet C terminal domain that is adjacent to the trans side of the cytoplasmic membrane (Atilano et al., 2010; Navratna et al., 2010; Scheffers and Pinho, 2005).

To fully investigate the interactions between the proteins of interest and the cell division machinery, a list of 22 S.aureus proteins were chosen. The list encompasses all aspects of the division process, from DNA segregation and correct septum placement, through to late division proteins involved in septum biosynthesis and synthesis of peptidoglycan-associated components. Homologues of proteins involved in B.subtilis elongation were also included, since S.aureus does not undergo any cell elongation, these proteins may play an alternative role in division of cocci (Steele et al., 2011). 3.2.2 Construction of BACTH plasmids

The genes of interest were amplified by PCR and cloned into pKT25 and pUT18C to create in-frame protein fusions to the C-terminus of T25 and T18 (Figure 3.1). Ligation products were used to transform electrocompetent E.coli TOP10 with selection on LB kan 50μg ml-1 or amp 100 μg ml-1. Positive clones were identified by colony PCR using the same primers used to amplify the genes of interest (section 2.9.3, data not shown) and by plasmid extraction, restriction digestion with EcoRI and BamHI, and

electrophoresis on a 1% (w/v) agarose gel (Figure 3.2). DNA bands of the correct size were seen for each plasmid (Table 3.2). Plasmids were sequenced by the University of Sheffield Core Genomics Facility to exclude the possibility of errors having been introduced during PCR (data not shown). The translated plasmid sequences showed 100% identity to predicted amino acid sequences, although a synonymous mutation was seen in the third position of the codon in the case of T25-TarO twice and T25- SA0908 once.

85 GENE Putative Function Nucleotide Length (bp) Amino acid length Mass (kDa) Predicted Topology TarO UDP-N-GlcNAc: UDP-P GlcNAc 1-P transferase 1056 351 38.5 SA2103 Putative LCP; cell envelope- related transcriptional attenuator domain 948 316 34.7 SA1195 (MsrR) Peptide methionine sulfoxide reductase regulator; Influences lytic behaviour(Deng ler et al., 2011) 984 327 37.0

SA0908 Putative LytR; transcriptional

regulator

1218 405 45.7 Predicted as soluble

Table 3.1 Results obtained from bioinformatic search of proteins of interest.

Table showing the results obtained from an NCBI search of TarO, the first protein in the WTA biosynthetic pathway, and proteins suggested to be involved in the attachment of WTA, the final step in the biosynthesis of WTA. Putative function, nucleotide length and amino acid length were all obtained from NCBI

86 3.2.3 E.coli BTH101 as a reporter strain for the detection of protein-protein

interactions

E.coli BTH101 is a non-reverting adenylate cyclase deficient reporter strain with high

complementation efficiency. The frequency of spontaneous Lac+ revertants due to cAMP/CAP independent promoter mutations is 10-8, making BTH101 a suitable strain for detection of protein-protein interactions seen by the reconstruction of the separated adenylate cyclase (Karimova et al. 1998). E.coli BTH101 was transformed with one of the constructed plasmids and made electrocompetent. Plasmids carrying fusions of T25 or T18 to a number of S.aureus cell division and other proteins (FtsZ, DivIB, DivIC, EzrA, FtsA, FtsL, FtsW, PBP1, PBP2, PBP3, PBP4, ParC, ParE, YneS, YpsA, GpsB, SepF, ZapA, Noc, RodA, MreC, MreD, ypfP, LtaA, LtaA, DivIA, DnaK) were then transformed into the electrocompetent BTH101 containing one of the T18- or T25- constructed plasmid.

3.2.4 Investigating physical interactions with S.aureus WTA biosynthesis proteins of