PBPs as part of the cell wall synthesis complex

Some PBPs are unique and essential, whereas others demonstrate a level of functional redundancy within the cell. The interaction of a range of PBPs for localisation as well as functional and regulatory reasons has been shown. Cell wall metabolism during the cell cycle can be generally divided into two stages in rod shaped bacteria and the pneumococci; elongation and division, requiring tight temporal and spatial control of a range of essential proteins (Macheboeuf et al.,

2006) and increasingly recognised as involving two separate large multi-subunit

complexes (Zapun et al., 2012). Variation exists in the mechanisms due to the

different shapes of bacteria (Pinho et al., 2013), and the components of spatially distinct division and elongation complexes are best understood in the Gram-negative

model organism Escherichia coli (Typas and Sourjik, 2015).

The tightly regulated polymerisation of FtsZ (bacterial tubulin homologue) at the septum forms the Z ring and initiates cell division (Bi and Lutkenhaus, 1991;

Errington et al., 2003) by recruitment of other members of the septal machinery to

the division site, including the septal PBPs. In rod shaped bacteria, the PBPs involved in peripheral peptidoglycan synthesis co-localise with an actin-like cytoskeleton containing MreB in patches that move along the long axis of the cell

powered by peptidoglycan synthesis (Domínguez-Escobar et al., 2011; Garner et al.,

2011; van Teeffelen et al., 2011). Cocci do not possess an MreB homologue and

therefore the mechanism of peripheral cell wall synthesis is unclear (Pinho et al.,

2013).

Immunoprecipitation, biochemical, immunofluorescence and genetic experiments in

E. coli, Bacillus subtilis and Streptococcus pneumoniae have shown cell division and elongation to involve at least one Class A and Class B PBP, along with lytic transglycosylases and other cell wall proteins (Massidda et al., 2013; Popham and Young, 2003; Typas and Sourjik, 2015).

This thesis focuses on the Gram-positive pathogens Staphylococcus aureus and S.

the role of PBPs in the cell wall synthetic machinery of these bacteria. Illustrated in Figure 1.8.

1.7.2.1 Staphylococcus aureus cell wall synthesis machinery

Spherical cocci such as Staphylococcus aureus build their cell wall using a single

basic machinery that can be transient in some cases (Pinho et al., 2013).

Staphylococcus aureus has an exceptionally minimal mechanism of cell wall

synthesis which occurs only at the septum (Kuru et al., 2012; Pinho and Errington,

2003) and requires only four PBPs (and one monofunctional transglycosylase), with an additional acquired transpeptidase in methicillin resistant strains. One each of Class A HMW, Class B HMW and LMW PBPs; PBP2, PBP1 and PBP4 localise at the septum (Pinho and Errington, 2003), and the localisation of the fourth HMW Class B PBP3 is not known to date. PBP1 is recruited to the mid-cell by an as yet

unidentified divisome protein (Pereira et al., 2007; 2009), and PBP2 is recruited by

its Lipid II substrate (Pinho and Errington, 2005). PBP4 as a LMW PBP is unusual in having transpeptidase activity and is responsible for forming highly cross-linked peptidoglycan. It is recruited by an intermediate of the wall teichoic acid (WTA) synthesis (pathway) (Atilano et al., 2010) after the initiation of peptidoglycan synthesis by PBP1 and PBP2.

1.7.2.2 Streptococcus pneumoniae cell wall synthesis machinery

The ovoccocal shape of pneumococcal cells suggests that the cell wall is formed by successive elongation and division processes, and therefore is likely to involve a distinct elongasome and divisome similar to that of rod shaped bacteria such as

E. coli. However, the peripheral machinery in streptococci is adjacent to the septum

instead of in the sidewall (Pinho et al., 2013), which may more resemble the recently

identified FtsZ dependent preseptal elongation of E. coli (Typas et al., 2012). The current evidence suggests the presence of one large complex containing both sub-

machineries, which assembles at the midcell (reviewed excellently by Massidda et

causing the cell to elongate, and the septal machinery synthesises the cross wall by

following the leading edge of the septum (Pinho et al., 2013). Recent high-resolution

microscopy of live cells following the incorporation of fluorescent probes into the growing cell wall has suggested that the apparatus may separate into two separate machineries in late cell division (Cadby and Lovering, 2014; Tsui et al., 2014), which would support this two state model. It is clear that the mechanisms of cell wall

synthesis in ovococci such as S. pneumoniae are not fully understood, but new

methods of observing live cells (Tsui et al., 2014) show real promise for future work.

The current knowledge of the role of PBPs in the pneumococcal cell wall identifies that the Class B transpeptidases PBP2x and PBP2b are found in the septal and

peripheral sub-machinery respectively (Massidda et al., 2013), however PBP2x may

play a more exceptional role in an unusual form of septal closure as it locates separately from all other peptidoglycan synthesis proteins in the mid to late stages of

cell division (Tsui et al., 2014). This may be mediated through the extracellular C-

terminal PASTA domains of PBP2x (Peters et al., 2014). The bifunctional Class A

PBP1a has been observed in both the peripheral and septal machinery (Land and

Winkler, 2011; Massidda et al., 2013), and it is believed to be important in creating

the septum. The Class C carboxypeptidases are located across the entire cell surface (Barendt et al., 2011). The mechanism of PBP localisation is not well understood, but is believed to be substrate dependent (Hasper et al., 2006; Pinho et al., 2013) and mediated by the trimming activities of PBP3 restricting the correct substrate to the mid-cell (Morlot et al., 2004). Recent work has identified the importance of the StkP/DivIVA/GpsB triad in fine-tuning septal and peripheral peptidoglycan

biosynthesis and therefore maintainance of the oval shape of S. pneumoniae (Fleurie

et al., 2014). This is in keeping with the theory of a single large complex with sub- machineries.