Despite Leishmania META1's well known association with virulence, its functions have remained poorly understood. We have attempted to elucidate the functions of META1 by identifying and examining its known homologs in other organisms. We have observed an unusual phyletic relationship between bacterial HslJ and trypanosomatid META1 which highlights LGT as the most rational explanation for the origin of META1 sequences in trypanosomatids.
Analyses of several pathogen genomes suggest a widespread occurrence of LGT [20, 22, 44–49]. These studies have relied on combinations of aberrant nucleotide composition or sequence similarity searches and phylogenetic analyses. In our study we have included all of these approaches: sequence similarity, overall GC content bias, GC content bias at 1st and 3rd codon positions and codon usage bias of META1 over whole genome of L. major and phylogenetic distribution pattern. All of the individual analyses are consistent with the notion of META1 being laterally transferred from bacteria into trypanosomatids (Figures 2 and 3
). In the analyses of such LGT events it is crucial to factor in multiple lines of evidence . By itself, the phylogenetic tree may be open to additional interpretations such as a "reverse" LGT flow from trypanosomatids into bacteria or a retention of META1 in trypanosomatids, from a common ancestor (with bacteria) with a loss from other species. However, the consistent topology of the phylogenetic tree, rooted in multiple manners (Figure 3 and Additional file 10
) taken together with the striking anomalous nucleotide composition of META1 in Leishmania make it unlikely that the LGT flow of META1 was from trypanosomatids into bacteria. Or, that META1 was retained within Leishmania from a common ancestor: if META was persisting from an ancient common ancestor, its GC composition would have been assimilated into the trypanosomatid genomes. However, we cannot exclude a possibility that META1 was present in an ancient common ancestor, was lost during the evolutionary emergence of eukaryotes and at some point, regained by trypanosomatids through a LGT from bacteria. Clearly, more definitive answers to such events will emerge as the genome sequence information coverage of various taxa expands.
Analyses of anomalous nucleotide composition is quite effective in the detection of recent LGT events but becomes handicapped as the time scale of the event in question increases typically because the gene in question has more time to adjust to the host genome . Given the diversity of bacterial species that have the META domain and the limited occurrence of META only in trypanosomatid eukaryotes, it is most likely that the transfer of META1 into trypanosomatids was an ancient event. In this regard, it is striking that the anomalous nucleotide composition of Leishmania META1 continues to be easily discernible.
Maintenance of the foreignness of a laterally transferred gene in the context of its recipient genome could reflect either a recent transfer event or a selective pressure to maintain certain features. Given the occurrence and conservation of META1 sequence throughout trypansomatids, it is most likely the transfer of META1 from bacteria was an early event into a trypanosomatid ancestor. An organism always maintains certain evolutionary constraints on its essential genes, in order to prevent loss of function caused due to mutations. Thus, a gene under purifying selection will evolve at a slower rate than a gene under positive selection. Upon comparison of META1 with its bacterial homologs, we found that META1 is under strong purifying selection, therefore, highlighting the importance of function of this gene for the organism (Figure 4).
Selection constraints on the META1/HslJ pair also underline the possibility of having similar properties. Our results have highlighted some of the properties shared by HslJ and META1. hslJ transcript up-regulation was found to be associated with pathogenicity in E. coli  and recovery of heat injured Salmonella enteritidis . In comparison, we observed that META1 expression too is regulated in developmental stages in L. donovani, consistent with earlier reports . We have also shown that attenuated Leishmania have lower META1 expression than virulent cells, consistent with a role for META1 in Leishmania virulence. In Leishmania, META1 expression is inducible by both temperature and acidification, with the temperature-mediated change being more significant (Figure 5
). This observation underscores the heat-inducible property of META1, like its bacterial homolog HslJ .
HslJ has been associated with increased resistance against a gyrase inhibitor novobiocin . We examined in Leishmania if META1 expression is associated with novobiocin resistance in virulent & attenuated lines of Leishmania as well as in META1 overexpressing lines. In the above mentioned conditions, we found no such correlation (data not shown). Clearly, not all functions are common between META1 and HslJ. Recently, L. amazonensis META2 that has 3 META domains and a C-terminal calpain-like domain was reported to be implicated in novobiocin resistance . This raises the possibility that our observations may be explained by META2 complementing for novobiocin resistance independent of META1 expression levels.
The 3D-modeling studies of META1 allowed us to identify an additional structural homolog of META1, the secretin pilot protein of Shigella flexneri, MxiM (Figure 6A
). These three proteins: META1, HslJ and MxiM share a strikingly similar fold, in spite of a very low sequence identity. The essential features about the structural similarity of these proteins highlighted upon superposition are: a common cracked barrel motif and a set of conserved hydrophobic residues in the central pocket of the protein (Figure 6B
). Such a structural homology between META1, HslJ and MxiM suggests a possibility of similar function of these proteins. Many proteins with similar function have been known to retain similar structural folds despite very low sequence identity and ambiguous secondary structure prediction [54, 55]. The hydrophobic cavity in MxiM is known to bind lipid moieties of bacterial membranes. However, unlike MxiM, the putative cavity of META1 has at least one charged amino acid suggesting that the ligand of this putative pocket may be different from MxiM (Figure 6B). It is possible that these proteins participate in similar activities, with certain residues in the cavity defining specificity of the ligand.
Changes in levels and predicted structure of META1 affect the quantum of extracellular activity of the well characterized marker for Leishmania secretory processes, SAP (Figures 7 and 8
). A comparison of proportion of intracellular v/s total SAP activity shows that overexpression of either WT or mutant META1 changes the extracellular SAP secretion (Figure 8B
). However, while ectopic expression of L80F mutant and WT reduces extracellular SAP activity, the L58F mutant META1 caused an increase in extracellular SAP activity. The L58F mutation appears to act as a loss of function event: there is more META1 protein but with a reduced negative effect on SAP activity. The proximity of this 58th position to the entrance of the putative hydrophobic cavity (Additional file 7
) suggests impairment in interactions with the probable ligand of the putative cavity that may interfere with META1's function, in turn interfering with META1's ability to suppress SAP secretion. Additionally, in L. donovani, L58 epitopic region seems to be highly antigenic: when lysates of cells overexpressing the different forms of META1 were probed with polyclonal META1 antisera on a western blot, the L58F reactivity was extremely low compared to WT overexpression control. However, when the same proteins, all of which have GFP tagged to their C-termini, were probed with polyclonal GFP antibody, cell lysates exhibited equivalent amounts of GFP-tagged proteins (Additional file 6).
The L80F mutation dominates over L58F mutation in case of the double mutant (L58,80F), as seen by its overall effect on SAP activity (Figures 8A and 8B
). Contrary to the L58 position, L80 lies in the core of the cavity: this change in position may also explain the altered consequences of the two mutations. Overall, our results on META1 mutagenesis in its putative hydrophobic cavity are consistent with a role for META1 in secretory processes in Leishmania and that this cavity is important for META1's function. However, the double mutant has an altered growth (Figure 8C
), suggesting that META1 may also be participating in events other than secretion.
Overall, the consequences of META1 mutations on extracellular SAP activity are consistent in both L. donovani and L. major: L58F overexpression leads to increased SAP activity; L80F has an equivalent effect on SAP activity to WT META1 and L80F dominates over L58F in the double mutants (Additional file 8
). Additionally, the L58F,80F double mutant has growth effects in both species (Figure 8C and Additional file 8
). However, we did observe some differences between the two Leishmania species. One, the extent of effect on SAP activity is greater in L. donovani. Second, L80F has distinct effects in L. major. In L. major, L80F and L58,80F mutant META1 protein expression was not seen while L58F mutant protein is equivalent in expression as the overexpressed WT (Additional file 8
). In spite of the presence of META1-GFP fusion transcripts in both L80F and L58,80F (Additional file 8
), there is barely any detectable amount of mutant protein suggesting that the mutant protein is possibly getting degraded. These observations reiterate the fact that L80F mutation is dominant in L58,80F mutation. Since L80 lies in the core of the cavity which might be important for ligand binding (Additional file 7
), it is possible that mutation at this site has disrupted ligand binding resulting in an unstable META1 conformation, leading to its degradation.
It is increasingly clear that LGT is an important mechanism in the evolution of eukaryotes [50, 56]. An interesting finding has been the observed transfer of gene sets for metabolic pathways [22, 57]. This is particularly of importance in the case of unicellular eukaryotes: LGT from bacteria allows the acquisition of new abilities such as exploitation of ecological niches, infective abilities and metabolic capabilities. A practical advantage of such knowledge is that such LGT acquired genes are more likely to be conducive targets for drug development since the host eukaryote typically lacks these genes [50, 57]. Furthermore, over time, LGT event has played a significant role in the subsequent evolution of the META1 gene family in trypanosomatids, as can be seen from the occurrence of a number of paralogs in Trypanosomes and a separate gene in Leishmania, META2, which has three META domains.
It is conceivable that, for Leishmania, at least one of the advantages of the LGT mediated acquisition of META1 was additional modulation of secretory processes. Secretion can be an important process for a pathogen seeking to modulate its host's responses via the export of effector molecules/virulence factors. Leishmania secrete various bioactive molecules that are involved in pathogenesis . Recently, a novel exosome-based pathway was identified as a general mechanism of protein secretion by Leishmania that is involved in pathogen-to-host communication and export of exosomal cargo into host macrophages . Furthermore, the exosomal cargo influences myeloid cells and is immunosuppressive . Our data is consistent with the correlation of META1 levels associated with Leishmania virulence. This work adds new information on functional role for META1 in secretory processes in Leishmania. Additionally, it identifies a domain within META1 that may be critical for its functions.