Alternative Host Clearance Mechanisms and Future Projects

Clearance of the choline-free bacteria is most likely a process that is cooperatively achieved by several alternative immune effector mechanisms.

For example, the choline-free organisms may produce a less protective capsule while growing in vivo. Although the presence of capsular polysaccharides in choline-free bacteria was demonstrated [41], the amount was never quantitatively assessed. With a weaker capsule choline-free pneumococci may be more susceptible to other host

A weaker capsule production may occur if the expression of capsular polysaccha-rides and their amount on the pneumococcal surface is directly correlated to the presence of choline and/or the teichoic acid pathway. As already described in the introduction, the synthesis of these two polysaccharides (capsule and TA) is very much alike and includes common chemical precursor molecules (e.g. UDP, and other Nucleotid-sugar-precursors...). Since neither of the pathways is presently fully under-stood there may even be more overlapping molecules and or protein catalysts in-volved.

For instance it is conceivable that chemically different components of the cell surface are delivered through shared transport ligands, e.g. bactoprenol molecules that have to be released and recycled during teichoic acids synthesis to enable the subsequent transport of capsular polysaccharides or peptidoglycan chains. It seems that the cru-cial step in the recycling process of these precursor molecules is coupled to the activ-ity of the TacF flippase and the transport of teichoic acids to the surface. Although the transport of free teichoic acid chains can be achieved by the choline-independent mutant bacteria, the flipping catalyzed by the mutant TacF might occur at a lower rate. This may slow down polysaccharide-synthesis and lead to a bacte-rium with a weaker capsule. It needs to be mentioned that the presence of the cap-sule is not essential for the viability of the bacterium in vitro, which is demonstrated by a variety of unencapsulated, rough laboratory strains.

In contrast to the non-capsular polysaccharide synthesis pathway, which is not es-sential for growth in vitro, the very similar peptidoglycan synthesis pathway is essen-tial for pneumococcal viability. Comparable to the synthesis of capsular polysaccha-rides and TA, this third pathway for peptidoglycan synthesis may also use this common carrier lipid molecule. Therefore it was speculated [139] that a block of the choline-dependent regeneration of the common precursor from the TA pathway in a wildtype bacterium, will also lead to a halt of peptidoglycan synthesis, which may be responsible for the lethal effects on the cell.

Future work could focus on the evaluation of this choline-dependent capsule expres-sion. If the above mentioned theory was true, it would be interesting to test the viru-lence of other available choline-independent bacterial mutants that posses alternative tacF flippases (501, JY2190). Maybe the TA turnover kinetics of these tacF mutants

Discussion

enable a faster precursor recycling, resulting in a more protective capsule and altered virulence. This would mean that different tacF alleles can be used as a tool for titering capsular expression. It would be very interesting to quantitatively asses the produc-tion of capsular polysaccharides in various choline-independent mutants. It is also possible that choline in the cell wall has a direct regulatory effect on synthesis of cap-sular polysaccharides. Northern Blots would reveal differences in cps locus transcrip-tion profiles with respect to choline availability in the growth medium.

Another important variable for bacterial clearance by the host could be the unknown status and/or localization of choline binding proteins (CBPs) in the choline-free mu-tant. Several members of this family of proteins were shown to be associated with virulence [29,140,141] or immune protective potential [142]. As a consequence of CBP deprivation the autolytic system is impaired and choline-free pneumococci grow in long chains. These chains may present a greatly increased target size for phago-cytic cells of the host. However, mimicking chain formation of choline containing bac-teria by mutating genes lytA and lytB did not result in such a drastic loss of virulence in a murine sepsis model, demonstrating that the avirulence of the choline-free bacte-ria cannot be exclusively linked to the growth in long chains [41]. It is rather possible that the loss of the choline residue and a change in the surface charge might also have an effect on phagocytosis. For instance, phagocytic scavenger receptors rec-ognize negative charges. Binding studies between cholinated pneumococcal teichoic acids and scavenger receptor-I suggested that the positive choline charge actually interferes with the binding [67] and could counteract phagocytosis. This might explain the enhanced clearance of choline-free bacteria as well.

To understand the effect of choline in the cell wall on phagocytosis a variety of ex-periments could be done. Studies on splenectomized animals revealed that the spleen is of crucial importance for the removal of S. pneumoniae from the host. In a first setup the cemetery of choline-free bacteria should be determined by microscopi-cally localizing labeled bacteria in the spleen. From the anatomy of the spleen one could infer which phagocytic cell type is mainly involved in the clearance. The use of knock-out mice, deficient in phagocytic receptors SIGN-R1 or MARCO might even give further information about the specific receptor-pathogen interactions.

Subse-rial surface epitope. One could purify cholinated and unsubstituted WTAs and LTAs and coat latex beads with these components. Applying these coated beads on cell culture phagocytes (e.g. macrophages) would give an understanding whether cho-line-free TA are better ingested by the eukaryotic cells. A similar phagocytosis in vitro approach was already used for lipid components of mycobacteria [143].