Identification of hyaluronic acid associated genes and the has operon

HA production in S. equi species involves the following five enzymes, hyaluronan synthase (hasA), UDP-glucose dehydrogenase (hasB), UDP- glucose pyrophosphorylase (hasC/galU), (hasD/glmU) and

phosphoglucoisomerase (hasE/pgi) following the biosynthetic pathway presented in Figure 40 (Chapter 4). Hyaluronan synthase is responsible for assembling the HA chain and the other enzymes are responsible for

producing the two precursors (Chen et al., 2009b). Previously it has been described that the has operon of S. equi subspecies differs between

zooepidemicus and equi in that all five genes are encoded within a single

contiguous operon in zooepidemicus but in equi the operon contains only

hasA, hasB and hasC, with hasD and hasE being organised within a

separate operon (Blank, Hugenholtz and Nielsen, 2008). Unfortunately in all of the genes of interest the beginning of the has operon was at the end of contigs and so the structure of the operon could not be confirmed as

previously described. Due to the fragmentation of the DNA during paired- end sequencing, genes may be lost and operons may be split between contigs which prevents the analyses of operon structure (Alkan, Sajjadian and Eichler, 2011; Denton et al., 2014). Completion of the genome would require further rounds of sequencing which unfortunately the budget for this project would not allow.

3.4.3. Gene discrepancies between phenotypes

Whilst no obvious discrepancies were observed in the genomes of the two phenotypes of SE40327 in terms of genes involved directly in HA

production a number of variations were observed in other genes. Deciding which genes were of interest and confirming the variation between

phenotypes was challenging, however three genes were eventually identified and investigated further.

80 3.4.3.1. Collagen-like surface protein

This gene was selected for investigation initially due to the fact it is located on the same contig and upstream of the has operon.

Following further analysis it was discovered that the sequence

matched to an LPXTG motif. LPXTG motifs are commonly found in surface proteins of Streptococci and are recognised by sortases during the covalent attachment of complete surface proteins such as

virulence factors like M protein to the cell wall (Scott and Barnett, 2006). It has previously been observed that in mutants deficient in the LPXTG motif surface proteins fail to associate with the cell wall, highlighting its importance in the sorting and arrangement of surface proteins (Novick, 2000). The nucleotide sequence of the gene from 40327MV matches 100% to the collagen-like cell surface-anchored protein sclH of S. equi subsp. equi (accession: VEH29797.1). The lack of identifiable start codon or ribosome binding site in

40327NMV suggests that transcription of this gene may have been lost in this phenotype. Another enzyme involved in the placement of surface proteins is foldase which is discussed in chapter 5.

3.4.3.2. M protein trans-acting positive regulator Mga

Mga is a regulator of multiple genes that functions to activate the transcription of virulence factors in response to environmental stimuli (McIver, Thurman and Scott, 1999). The introduction of aromatic rings into a protein by amino acid substitution is likely to promote changes to the structure, stability, flexibility and hydrophobicity. As the substitution appears to have occurred within the DNA binding helix-turn-helix region it is possible this could impede DNA binding and regulation of transcription. “Phase-locked” variants of

Streptococcus with respect to M protein and HA capsule were often

observed to have accumulated deletions in the mga genes (Cleary et

al., 1998). Whilst the evidence points to HA production being

regulated by other means than Mga (Falaleeva et al., 2014; Velineni and Timoney, 2018), previously it has been observed that S. pyogenes strains maintained in prolonged stationary phase (3-5 days) present with ceased or reduced expression of multiple virulence factors

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including Mga, M protein and capsule (Leonard, Woischnik and Podbielski, 1998). Mga is a transcriptional regulator and it has been observed that it possesses more than one DNA binding domain which has lead to speculation that it is capable on acting on numerous promoter targets (McIver and Myles, 2002). Whilst none of these observations indicate a direct link between capsule production and Mga regulation, the observation of this mutation between mucoid and non-mucoid phenotypes in conjunction with the accepted notion that capsule is a virulence factor could be considered cause for further investigation into the effects of Mga function on phenotype with respect to capsule. The majority of research into the mga operon and regulon has been conducted in S. pyogenes however a comparison of the operons indicates that the role of this regulator may differ slightly in S. equi.

3.4.3.3. RNA polymerase sigma-70 factor family

The missing stretch of sequence at the beginning of the gene in 40327NMV does not appear to affect either of the regions identified in pfam as promoter recognition or binding regions. Sigma factors provide yet another mechanism for pathogenic bacteria to respond to changes in environmental conditions such as pH and temperature and also have been found to have a role in the transcription of virulence factors (Opdyke, Scott and Morgan Jr, 2001). Members of the sigma- 70 family of sigma factors associate with RNA polymerase in order to direct the enzyme to the relevant promotor sequences and thus is essential for transcription initiation (Paget and Helmann, 2003). Whilst they have been associated with regulation of virulence factors, sigma-70 family includes the “housekeeping” sigma factor as well as members specifically associated with stress responses (Kazmierczak, Wiedmann and Boor, 2005) and capsule production has been

associated with response to stress stimuli such as pH changes and oxidative stress (Cleary and Larkin, 1979; Liu et al., 2008; Henningham et al., 2015; Chiang-Ni et al., 2016). It has been

claimed that a promoter motif for sigma-70 factor has been identified upstream of the hasE/pgi gene in S. equi species, speculating that this

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gene is under the regulation of more than one operon due to its essentiality in other cellular processes (Blank, Hugenholtz and Nielsen, 2008). Thus it is not illogical to speculate that sigma factors play a role in regulation of capsule production. Whether the loss of sequence at the beginning of the RNA polymerase sigma-70 factor gene in 40327NMV plays a role in loss of HA production is uncertain since as previously mentioned the loss has not occurred within

conserved regions. Further investigation in this case is outwith the scope and resources of this project.