Crucial to the ecology and subsequent disease-causing capability of L. pneumophila
is its unique relationship with protozoa. The ability of L. pneumophila
to replicate within amoebae allows the pathogen to persist in aquatic environments and also replicate in mammalian cells. Many processes important for subsequent environmental persistence are also principal virulence processes. Despite a high number of strain-specific genes among different isolates of L. pneumophila
serogroup 1 (6
), all strains share the core genes required for their unique intracellular life cycle.
In this study we established that the gene encoding a putative L. pneumophila-specific adenylate cyclase, LadC, is among a cohort of genes important for initial infection of epithelial cells, macrophages, and A. castellanii. The differences in bacterial numbers recovered from cells infected with wild-type L. pneumophila or the ladC mutant were most obvious at 3 h after infection. When we examined the initial interaction between THP-1 macrophages and derivatives of L. pneumophila, we found that the ladC mutant adhered in smaller numbers to the host cell than wild-type L. pneumophila and that this was already evident at 15 min after infection. Despite the reduction in total numbers of the ladC mutant interacting with host cells, the proportion of cell-associated ladC mutant bacteria internalized by THP-1 cells was equivalent to that of the wild-type strain. Consistent with the absence of a defect in bacterial uptake for the ladC mutant, there was no difference in trafficking of the LCV between wild-type L. pneumophila and the ladC mutant, as measured by acquisition of the late endosomal marker LAMP-1 (data not shown). The adherence defect of the ladC mutant in vitro also conferred a colonization defect in vivo upon infection of A/J mice. This confirmed that ladC makes an important contribution to the virulence of L. pneumophila in a respiratory infection model, presumably by influencing initial contact between the bacterium and host cell.
Amino acid sequence analysis predicted that the putative LadC protein was capable of converting ATP to cAMP. cAMP is one of the most ubiquitous signaling molecules in both prokaryotes and eukaryotes and allows an organism to respond rapidly to a variety of stimuli. In prokaryotes, cAMP traditionally mediates biological change via regulation of transcription through its interaction with CRPs. However, here microarray studies demonstrated that inactivation of ladC
had no influence on L. pneumophila
transcription either in vitro or during infection of A. castellanii
. This was surprising, as we demonstrated using fractionation and immunoblotting as well as PhoA fusions that LadC localized to the bacterial inner membrane. Given that our microarray studies suggested that the predicted product of ladC
was not involved in the regulation of transcription, LadC may aid virulence by modulating protein-protein interactions, signal transduction, and protein activity. cAMP is also able to bind to and activate a number of proteins not involved in transcriptional regulation and signal transduction. This is not a well-studied mechanism of cAMP function in bacteria; however, there are many examples of posttranslational modifications of protein function in eukaryotes by cAMP. Classical examples of this include the activation of protein kinase A leading to protein phosphorylation, regulation of ion channels, and, more recently, activation of the Ras-like GTPase Rap1 (14
The behavior of the ladC mutant complemented with ladCN430A/R434A is another intriguing aspect of this study. Clearly this putative catalytically inactive form of ladC was unable to complement the attenuated ladC mutant, suggesting that production of cAMP is crucial for LadC function in virulence. However, this strain demonstrated a severe replication defect in THP-1 cells and A. castellanii, and interestingly, this attenuation was not due to a true dominant negative phenotype, as wild-type L. pneumophila carrying ladCN430A/R434A did not show diminished replication in A castellanii. In addition, this phenotype was observed only for full-length ladCN430A/R434A, as complementation of ladC::Km with pLadC1-296, pCYC, and pCYCN430A/R434A did not confer a similar replication defect. The presence of an inactive enzyme may have resulted in aberrant signaling and/or interfered with the balance of ATP and cAMP and in so doing disrupted the function and signaling roles of the four other putative adenylate cyclases present in L. pneumophila. However, at this stage the reason for the increased attenuation of the ladC mutant complemented with ladCN430A/R434A is unknown.
Analysis of the L. pneumophila
genome revealed no close CRP homologues; however, there are five genes encoding predicted proteins with conserved cAMP binding domains. Two of these, lpp2063 and lpp2777 (also termed legN
, delineating homology to eukaryotic motifs [12
]), encode putative proteins with conserved cyclic nucleotide monophosphate binding domains and may act similarly to traditional prokaryotic CRPs. There are another three genes encoding putative proteins that contain a cyclic nucleotide monophosphate binding domain, which may indicate other roles for cAMP in L. pneumophila
. These are lpp0611, a putative flavin adenine dinucleotide-dependent oxidoreductase; lpp3069, a putative sulfate transporter; and lpp1482. lpp1482 contains a conserved CaaX amino terminal protease domain, where “a” represents aliphilic amino acids. CaaX proteases play an important role in eukaryotes, cleaving the aaX amino acids from C-terminal farnesylated CaaX motifs (52
). This modification is one step within CaaX processing that renders proteins, such as Rab GTPases, hydrophobic at their C termini, allowing membrane association (18
). Of these, lpp2063, lpp2777, lpp3069, and lpp1482 are also upregulated two- to fourfold in the transmissive phase of L. pneumophila
growth, similar to the case for ladC
Several similarities can be drawn between the intracellular pathogens L. pneumophila
and Mycobacterium tuberculosis
in relation to their adenylate cyclase repertoire and their ability to replicate inside mammalian cells and prevent phagolysosome fusion (10
). M. tuberculosis
strain H37Rv encodes 16 class III cyclases, 4 of which have conserved HAMP domains, similar to the case for LadC (47
). Many of the M. tuberculosis
adenylate cyclases have undergone biochemical characterization, although their role in virulence remains unclear (47
). It is known that cAMP can influence transcription within M. tuberculosis
, and 10 putative CRPs have been identified from the genome sequence (41
). However, the wide array of both adenylate cyclases and putative cAMP binding proteins suggests more diverse action of cAMP in M. tuberculosis
than in L. pneumophila
). In this study, recombinant full-length LadC and recombinant protein encompassing just the CycC domain of LadC were analyzed repeatedly for adenylate cyclase activity under a variety of conditions in vitro (data not shown). However, these attempts at the biochemical characterization of LadC were unsuccessful, and we concluded that we were unable to mimic the biological conditions where LadC is active.
This study has demonstrated that the putative adenylate cyclase LadC is involved in the ability of L. pneumophila to infect a broad range of hosts, specifically through initiating an interaction between the bacterium and host cell. The mechanism by which this is achieved remains unclear; however, it appears that under the conditions examined here, it is not accomplished via the traditional role of cAMP as a second messenger to regulate bacterial transcription. We also did not detect any obvious differences in LPS staining or in protein secretion and outer membrane protein profile between wild-type L. pneumophila and the ladC mutant (data not shown), although a sensitive mass spectrometry approach may yet identify variations in LPS profile or protein abundance that could account for the adherence defect of the ladC mutant. Above all, further investigation of the five conserved adenylate cyclases of L. pneumophila and the various roles of cAMP during host cell infection will help to clarify this complex signaling system and its intrinsic link to pathogenesis.