NIVELES MEDIOS
10. Lineamientos sobre traslados de párvulos 14 :
is coiled into a ~50nm wide cable which is then attached to the existing cell wall (Hayhurst et
al., 2008).
A
B
21 peptidoglycan is crosslinked (Wilkinson, 1997). Cross-linking between adjacent stems occurs via linkage of the ε-amino group of the dibasic amino acid to the α-carboxyl group of the terminal D-alanine of tetrapeptides. Although rarely, a 3-3 cross-linkage can occur between two dibasic amino acids. The chemical structure of peptidoglycan is outlined in Figure 1.4.
The glycan chain length varies between organisms, S.aureus have relatively short chains at approximately 6 disaccharides long (Boneca et al., 2000). The C6 group of the MurNAc may be modified by O-acetylation, N-glycosylation or de-N-acetylation
(Vollmer, 2008)in varying degrees and combinations depending on species. These modifications have been shown to provide resistance to lysozyme and other autolysins, and in the case of O-acetylation is associated with pathogenesis and modulation of the host immune response. The arrangement of the glycan strands has undergone much debate and two possible models exist; the glycan strands are
arranged in the plane of the cytoplasmic membrane (de Pedro et al., 1997; Koch, 1998), or the glycan strands are arranged perpendicular to the cytoplasmic membrane (Dmitriev et al., 2004, 2003) (both models are addressed in figure 1.5). The glycan chain lengths of S.aureus are short enough to permit either configuration (disaccharide length ~6 at 1.03nm per disaccharide (Carlstrom, 1957); Peptidoglycan layer width 20- 35nm). However, recent publications have suggested a poorly ordered planar
orientation is adopted by the Gram-negative organisms E.coli and C.cresentus, as shown by electron microscopy, neutron scattering and atomic force microscopy (Gan
et al., 2008; Vollmer and Höltje, 2004; Wang et al., 2012). Furthermore, the rod-
shaped Gram-positive B.subtilis exhibits a more complicated architecture of
peptidoglycan cables wrapping around the cell cylinder (Figure 1.5C) (Hayhurst et al., 2008).
1.6 Peptidoglycan synthesis.
Peptidoglycan is synthesised in three key stages: synthesis in the cytoplasm of a monosaccharide pentapeptide; assembly of the disaccharide-pentapeptide monomer unit on the inner surface of the cytoplasmic membrane and translocation of the monomer to the periplasm; and finally transglycosylation of the monomer unit into a
22 glycan polymer, and transpeptidation into the sacculus (Typas et al., 2012). Rod- shaped organisms alternate between two modes of cell wall synthesis; elongation, where peptidoglycan occurs in a potential helical pattern along the lateral cell wall (Daniel and Errington, 2003); and septal growth, where synthesis occurs at the septum leading to the formation of the septal disc (Pinho and Errington, 2003). Cocci offer a simpler model of cell wall synthesis, because the FtsZ-dependent cell wall synthesis is predominant and can account for the synthesis of the entire new hemisphere of each daughter cell. It is important to note the difference between the two types of cocci: true cocci, such as staphylococci, which are truly round; and ovococci, such as enterococci, which are elongated ellipsoids. Ovococci and true cocci do not have the same mechanisms of cell wall synthesis during the cell cycle (discussed in Figure 1.6, alongside rod-shaped organisms).
The enlargement of the multilayered sacculus has been proposed to occur via a three- for-one mechanism of ‘inside-to-outside’ growth (Höltje and Heidrich, 2001; Höltje, 1998). An inside-to-outside model for the flux of cell wall material suggests that the cell wall inner layer contains newly synthesized peptidoglycan. This peptidoglycan is introduced in a ‘three-for-one’ manner, where one glycan strand in the sacculus is replaced by a nascent triplet of cross-linked glycan strands and pulled into plane of the sacculus under turgor pressure. As the cells grow, the new cell wall passes outwards and stretches, becoming the middle stress bearing zone. The outer zone consists of old, partially hydrolyzed peptidoglycan awaiting solubilisation (Höltje and Heidrich, 2001; Höltje, 1998; Pooley et al., 1978). As S.aureus grows exclusively by division, they have a single peptidoglycan synthesis machinery, which is coordinated by FtsZ during division (Atilano et al., 2010; Pereira et al., 2007; Pinho and Errington, 2005, 2003). AFM has identified that S.aureus forms a thick band of material, which exhibits a corrugated ‘piecrust’ texture, around the cell in the plane of division (described through the cell cycle in Figure 1.7A) (Turner et al., 2010). This ‘piecrust’ rib forms before the centripetal synthesis of the septal disc. Once the septal disc is complete the cell splits and produces two pseudo-hemispherical cells. The thick ‘piecrust’ splits into two ribs which serve to brace the cell, preventing collapse to a smaller energetically
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Figure 1.6 Peptidoglycan dynamics in cocci and ovococci
A Rod-shaped. Most rod-shaped organisms elongate by dispersed helical insertion of
peptidoglycan (MreB-directed in E.coli and Mbl-directed in B.subtilis). A short phase of FtsZ driven elongation precedes division. Divsision occurs rapidly by constriction or septum formation. Daughter cells separate and initiate elongation again.
B True cocci. Peptidoglycan synthesis occurs at septation. The division ring is initiated. The
septum then closes centripetally, like the iris of a camera. A complete cross-wall forms, dividing the cell into two hemispherical daughter cell compartments. Daughter cells separate, the septal cross-wall becomes the new cell wall hemisphere and division is initiated on the next orthogonal plane.
C Ovococci. An annular outgrowth of the cell wall termed an equatorial ring, demarks the
initiation site of the new cell wall. Synthesis of an invaginating cross-wall is initiated and the equatorial ring is split in two and the rings are driven apart by peripheral wall synthesis. The equatorial rings approach the mid-cell of the forming daughter cells. Peripheral extension switches to constriction. The annular cross-wall closes forming a new cell pole. Peripheral growth initiates in the daughter cells.
A
B
C
Elongation Division Elongation machinery Division machinery Elongation via division machinery Division Division24 favourable shape, and forcing the new cell wall to stretch as the cell grows in size. To allow this growth the peptidoglycan is remodelled by irreversible autolysis of covalent bonds (observed by AFM as a transition from centric ring architecture to a knobbly architecture) within long glycan strands. This makes the cell wall more elastic by sharing the stress bearing function between the glycan strands and more flexible peptide stems and allowing the expansion from hemisphere to spherical (Figure 1.7B) (Wheeler R., 2012). The ‘piecrust’ ribs are retained after division and have been proposed to encode enough information for the cell to ‘remember’ previous division planes (Figure 1.7A). The most recent division plane will be seen as a whole rib, the division before as a half rib and the third most recent division as a quarter rib. This rib is uniquely bounded by two T junctions which may allow the cell to mark this plane for the next division (Turner et al., 2010). The question of how the division machinery are recruited to this nascent septal ring is still not understood but has been hypothesised as a function of membrane distortion due to the ‘piecrust’ ribs (Wheeler R., 2012) which may be recognised by DivIB (Bottomley, 2011).
1.7 Peptidoglycan hydrolysis
For a cell to continue growing and dividing peptidoglycan must be remodelled and hydrolysed at specific times and specific sites. This hydrolysis of either the glycan or peptide chain is carried out by a group of enzymes known as peptidoglycan hydrolases (Vollmer et al., 2008). Some of the physiological roles of peptidoglycan hydrolysis include cell growth, cell-wall turnover, peptidoglycan maturation, cell division, separation and pathogenicity (Foster, 1995; Stapleton et al., 2007; Vollmer et al., 2008). It is also involved in more specialised functions; differentiation to endospores (Errington, 2003), assembly of macromolecular trans-envelope complexes (Hirano et
al., 2001; Koraimann, 2003), cross-species and inter-species competition (Ellermeier et al., 2006; Russell et al., 2011), competence (Ahn and Burne, 2006; Eldholm et al., 2010)
and biofilm formation (Vollmer et al., 2008). In addition, peptidoglycan hydrolysis releases turnover products which serve as signalling molecules for recognition of bacteria by other organisms and, in some bacteria, for the induction of β-lactamase (Jacobs et al., 1997; Vollmer et al., 2008).
25
Figure 1.7 Growth of peptidoglycan and structural inheritance of division planes in S.aureus A) Location and remodelling of piecrust and rib features through the division cycle, resulting in
T junctions and cross sections. The quarter rib is a distinctive feature (i) and a new piecrust is formed in this plane (ii), it is then split in two as the cell divides (iii). This leads to a revised rib pattern that specifies the next round of division (Taken from Turner et al., 2010).