Mycobacteria have MPLA activity that is associated with putative cutinase. Though MPLA activity has been alluded to in a few publications, this is the first paper to definitively confirm this activity using radiolabeled 14C and the first paper to associate this activity with a specific mycobacterial protein.
We present the evidence that associates mycobacterial MPLA activity with mycobacterial cutinase. First, the presence of a cutinase motif correlates with MPLA activity, as the motif and the activity are found in all mycobacteria tested. Second, the cellular localization of M. tuberculosis MPLA activity correlates with the localization of cutinase activity, as both are found in the cell membrane and cell wall. Third, purification of MPLA activity from M. smegmatis yielded a protein identified as a putative cutinase by mass spectrometry, and this purified MPLA has cutinase activity by NPB and NPP assays, while known snake venom PLA does not. Fourth, we report that known cutinases have PLA activity, and thus it is reasonable that a protein purified for its MPLA activity is a putative cutinase. Fifth, partially purified MPLA and known cutinases, but not snake venom PLA, share another previously undescribed group of substrates, the Tweens.
Furthermore, it is logical that cutinases have PLA activity. The alpha-beta hydrolase fold superfamily encompasses many enzymes. Cutinases are the smallest members of this family, and in our analysis they bear particular resemblance to the group VII PLA2
s, and to the other PLA2
s using a catalytic serine, though without the C2
domain or a cap over their catalytic domain. However, for cutinases to be established as PLAs, certain criteria put forth in the literature must be fulfilled (34
). We will pursue this once we have exogenously expressed protein. Though PLA activity may not be the primary activity of all cutinases, PLA activity is clearly a part of the cutinase repertoire. In pathogens that do not encounter cutin, the presence of these enzymes likely represents an evolutionary divergence, and activity on other substrates is likely to be more important. Our data add a broad range of phospholipids and the Tweens to the list of cutinase substrates. Of particular interest is partially purified MPLA's activity on sphingomyelin, a phospholipid not known to exist in mycobacteria (27
The activity of these enzymes on Tween is also intriguing, as Tween is widely added to mycobacterial culture medium to prevent cell clumping. A longtime debate has been the concern over Tween as a viable carbon source and the influence of Tween on growth (21
), antimicrobial susceptibility (45
), and the mycobacterial cell wall (22
). The ability of cutinases to cleave these compounds, releasing FAs, provides insight into possible uses of Tween by mycobacteria and the possibility that Tween could alter experimental results, especially in studies of mycobacterial lipids (43
). We are pursuing further investigations in this regard.
Though the MPLA activity is not specific to pathogenic mycobacteria, the difference in the locations of the activity—in the cell membrane and wall in M. tuberculosis
H37Rv and additionally in the culture supernatant in strains of M. marinum
and M. smegmatis
—is intriguing and may suggest an environmental adaptation. Extracellular secretion may provide an advantage for an environmental organism that degrades plant matter, while for an intracellular pathogen this may be a disadvantage due to toxicity. We cannot explain why the cutinase proteins published as being in the culture supernatant are not active under our MPLA assay conditions. Either these enzymes were inactivated in processing, are inhibited, or need a certain cofactor, or not all proteins with a cutinase motif have PLA activity. Notably, it has long been known that esterase activity is absent from the culture supernatant of M. tuberculosis
, in contrast to other mycobacteria, as this property was used as a tool to differentiate them; our results are consistent with this observation (9
The low turnover rate for NPB by partially purified MPLA compared to that of known cutinases may reflect damage to MPLA during the purification process, conditions which are not optimized, or a difference in substrate specificity. Formal kinetic studies will be undertaken when purified recombinant protein is available, as well as kinetic studies on other substrates.
While a few other bacteria have cutinases, the majority of organisms found with the motif are fungi. No proteins with a cutinase motif have been identified in humans. Though cutinases are important in phytopathogens, which can occasionally cause opportunistic infections, this is the first publication on cutinases in a strictly mammalian pathogen. The cutinase motif is prevalent in environmental as well as pathogenic strains of mycobacteria. Though M. tuberculosis
complex organisms have retained seven open reading frames annotated as cutinases, it is likely that evolutionarily the activities of these enzymes have diverged. Though individually the M. tuberculosis
cutinase genes are not essential, according to Himar1-based transposon mutagenesis in H37Rv (15
), their redundancy implies an important role. As there is no rationale for cutinases in mammalian hosts, we propose that these enzymes act on other FA-containing substrates.
Mycobacteria including the M. tuberculosis complex have MPLA activity, and this activity is associated with proteins annotated as mycobacterial cutinases. Known cutinases from F. solani and T. lanuginosus have PLA activity as well, supporting this association. The redundancy of cutinases in the M. tuberculosis genome suggests an important role, and as this pathogen should not encounter cutin, their activity must be directed at alternative substrates, and possibilities include an array of phospholipids and Tweens. The activity of these enzymes may contribute to virulence through their likely modification of mycobacterial and environmental lipids, either for cell wall remodeling or for carbon scavenging.