As most bacterial species do not have the capacity to synthesize phosphatidylcholine (PC), the reason for the presence of PC in some bacterial species has remained mysterious. It was originally proposed that bacteria with large amounts of internal membranes, such as photosynthetic bacteria, have a requirement for this lipid (Hagen et al., 1966
). Interestingly, several species that interact intimately with host cells have significant PC content in their membranes, so this lipid may play an important function in allowing these microorganisms to interface with their hosts (Lopez-Lara and Geiger, 2001
; Goldfine, 1984
). There had been little information supporting this hypothesis, and each species having PC may synthesize the lipid for a different reason. The original observation supporting its importance was the demonstration that mutations depressing PC synthesis in the nitrogen fixing Bradyrhizobium japonicum
cause reduced root nodule occupancy (Minder et al., 2001
). Similarly, Brucella abortus
mutants defective for synthesis of PC show unexplained defects in cell interactions and virulence (Comerci et al., 2006
; Conde-Alvarez et al., 2006
). The clearest connection between a molecular defect and a phenotype is the case of A. tumefaciens
, in which loss of virulence in PC-defective mutants results from lowered transcription of the genes encoding the T DNA transfer apparatus, a result very different from what we observed with the Dot/Icm complex (). There is no mechanism, however, that links membrane composition to transcription of this locus (Wessel et al., 2006
). The experiments described here are consistent with an important role for PC in the virulence of an intracellular pathogen. Many of the defects observed could be ascribed to loss of the flagellin protein, which unlike the A. tumefaciens
example, occurs at the post-transcriptional level.
The low yield of bacteria after infection of macrophages by PC- mutants appears to be the consequence of two related defects: poor initiation of intracellular replication and a defect in the ability of the bacteria to associate with macrophages. Once initiation of replication occurs, however, the rate of replication of these mutants did not appear strongly altered by the absence of PC. This small effect on replication rate is somewhat surprising given the increased association with LAMP-containing compartments for mutants relative to wild type. Having a lowered initial level of infection, however, could be the result of both an uptake defect as well as loss of viability of the improperly targeted bacteria. Therefore, the viable PC- mutants seen at early timepoints may be primarily those that have targeted properly and which will eventually replicate. Furthermore, different initial doses of infected macrophages results in nonidentical monolayer conditions that can greatly affect the rate of bacterial intracellular replication. For instance, we have observed that at low MOI, there is a detectable cytokine response that is a function of bacterial infectivity levels (M. Liu and R. Isberg, data not shown). This response, in which monolayers having lower numbers of viable bacteria result in less restriction of L. pneumophila growth, makes it difficult to directly correlate replication rates to targeting defects.
There is a large body of work indicating that L. pneumophila
entry into post-exponential phase allows expression of functions required for productive growth in macrophages (Byrne and Swanson, 1998
). We found that the loss of PC interfered with proper function of a subset of activities associated with this state, such as cytotoxicity, motility, cell association and intracellular growth (Molofsky and Swanson, 2004
). These defects did not appear to be a result of an inability to respond to signals resulting from nutrient depletion, as cultures of wild type strains and PC- mutants showed identical induction of both translocated substrates and the flagellin structural gene after entry into post-exponential phase. The absence of PC, however, did result in the loss of flagellin protein even though the flaA
structural gene transcript was strongly induced in post-exponential phase (). As lack of flagella on the bacterial cell surface reduces both bacterial binding to macrophages as well as cytotoxicity (Molofsky et al., 2005
), the aberrant cellular interactions observed in PC- mutants may largely result from an inability to maintain high steady state levels of the flagellin protein. The molecular basis for the absence of this protein is unclear. Presumably, lack of PC prevents post-exponential phase assembly of the flagellar complex, with the result that unassembled subunits are degraded. PC may be required for assembly of this complex in L. pneumophila
because the events involved in production of flagella all occur in nondividing bacteria, potentially raising problems not observed during exponential phase assembly. PC has many characteristics that may alter protein interactions. For instance, it has been shown to antagonize folding of the LacY protein, indicating it may control folding rates of inner membrane proteins (Bogdanov et al., 1999
; Goldfine, 1984
The second explanation for the role of PC is that the choline head group directly interacts with target cells. In particular, some mucosal pathogens are believed to use bacterial surface molecules that have been modified by phosphorylcholine as ligands for host cell receptors (Swords et al., 2000
; Cundell et al., 1995
). The PC head group could act as a source for such modifications. The primary evidence that bacterial phosphorylcholine is recognized by mammalian receptors are studies indicating that an antagonist of platelet activating factor receptor (PAF-Ra) interferes specifically with adhesion of phosphorylcholine-modified bacterial ligand (Swords et al., 2000
; Cundell et al., 1995
). As PAF has a phosphorylcholine group, it is attractive to speculate that mammalian receptors that recognize the PAF-Ra antagonist also recognize phosphorylcholine-modified bacterial cell surfaces. Similar to results in Streptococcus pneumoniae
and Haemophilus influenzae
, we found that the PC-dependent cell adhesion of L. pneumophila
could be blocked by the addition of a PAF-Ra, with adhesion of a mutant lacking PC being totally resistant to the action of the antagonist (). Mammalian cells may directly recognize an L. pneumophila
ligand that contains either the phosphocholine headgroup or a derivative of PC. Consistent with this model, we found that the efficiency of bacterial association with macrophages was a direct function of the PC concentration, as derivatives that had higher PC levels than wild type also associated with macrophages more efficiently than wild type, as if a PC derivative were limiting for cell association.
The clear preference of L. pneumophila for using PcsA, rather than the PmtA pathway must be explained. One possibility is that the products of the PmtA pathway negatively impact either bacterial viability or the ability of the organism to interact with the host. Unlike PcsA, which only produces PC as a product, the PmtA pathway results in low-level synthesis of the monomethylated and dimethylated derivatives of phosphatidylethanolamine, which have uncertain roles in membrane biology. These lipids could interfere with either adhesion of the cells or assembly of membrane proteins. A second explanation for the predominance of the PcsA pathway is that the synthase is a direct sensor of environmental conditions, using choline availability as an indicator of the status of the locale in which the bacterium is found. Limiting choline concentrations may be a signal for the bacterium to either slow its growth or make compensatory changes in lipid side chain composition to accommodate environments in which choline is limiting. The presence of large pools of choline could be a signal for the bacterium that host cells are available for intracellular replication, allowing the proper assembly of envelope components critical for host cell interaction.
Although it may be counterintuitive that the bacterium relies on an extracellular source of precursor rather than utilize its own biosynthetic pools, the fact that interaction with hosts is a central aspect of the biology of L. pneumophila must be an important determinant of how this preference for PcsA arose. PC synthesis appears to be a critical sensor of the nature of the environment surrounding the microorganism. Biosynthetic precursors found in lung tissue, therefore, can modulate virulence of this organism by controlling the ability of the organism to incorporate PC into its cell envelope. Investigating how the environment controls the PC content in cells and how this phospholipid modulates host cell interaction are of central importance for understanding the pathogenesis of L. pneumophila.