 parent with apo-hLf in the presence and absence of recombinant PrtA (P = 0.001). The killing of the EF3030 parent with 100 μg/ml (1.25 μM) apo-hLf in the absence of added rPrtA is shown for comparison. The addition of 50 μg rPrtA to the PrtA mutant strain completely restored the strain's ability to be killed by apo-hLf, to the level of that seen with the EF3030 parent. Controls were incubated with the same concentration of PrtA in the absence of apo-hLf. No killing resulted from the incubation of the PrtA mutant bacteria with rPrpA in the absence of apo-hLf (data not shown). (B) Incubation of apo-hLf with rPrtA for 0.6, 1.0, and 16 h prior to incubation with EF3030. Prior incubation of apo-hLf with rPrtA increased the bactericidal activity of apo-hLf when it was added to EF3030 (P = 0.001).
Ability of incubation with rPrtA to increase the bactericidal capacity of a solution containing apo-hLf.LLP are known to be much more bactericidal on a molar basis than apo-hLf (31). Thus, if the killing of pneumococci by apo-hLf is mediated by a fragment of apo-hLf (LLP) that is cleaved from apo-hLf by PrtA, then the prior incubation of apo-hLf with rPrtA should increase the bactericidal capacity of apo-hLf. For this study we treated EF3030 with a subbactericidal concentration of apo-hLf or with the same concentration of apo-hLf after it had been digested for various time points with rPrtA. A final concentration of 1.25 μM apo-hLf was incubated with 50 μg/ml of rPrtA for 0.6, 1, and 16 h. Bacteria were then treated with a 1.25 μM concentration of this treated apo-hLf. As a control, EF3030 was also treated with a total of 1.25 μM native apo-hLf in the absence of rPrtA. It was observed that the apo-hLf that was incubated with rPrtA for the longest period of time (16 h) was the most bactericidal, resulting in 0.65 logs more killing than apo-hLf alone (P = 0.001), a finding consistent with the release of active LLP from apo-hLf. As in Fig. 9A, the incubation of EF3030 with PrtA in the absence of apo-hLf did not result in detectable killing (data not shown).
Identification of the cleavage site of PrtA in lactoferrin.We attempted to identify the site in lactoferrin that is cleaved by PrtA. All of the bactericidal peptides of lactoferrin are known to be derived from the N-terminal end of the protein and are contained in the N-terminal 47 amino acids (36, 51). It was therefore hypothesized that cleavages of lactoferrin that would include all or most of the first 47 amino acid were responsible for yielding the peptide(s) of apo-hLf that is bactericidal to S. pneumoniae. A total of 10 μg/ml of apo-hLf was incubated with or without 10 μg/ml of PrtA in a 50 mM Tris-HCl (pH 7.5) and 50 mM CaCl2 buffer at 37°C for 2 h. Untreated apo-hLf and PrtA-treated apo-hLf were trypsinized and subjected to LC/MS. Results of LC/MS identified a cleavage site between Val78 and Ala79 of the tryptic peptide LRPVAAEVYGTER, thus generating the peptide AAEVYGTER. To confirm this cleavage, trypsin-generated peptides of apo-hLf and PrtA-digested apo-hLf were analyzed by LC-multiple-reaction ion monitoring (MRM)-MS. Individual channels were set up to monitor the parent tryptic peptide, LRPVAAEVYGTER, and the PrtA-cleaved peptide, AAEVYGTER. The molecular ions for each peptide were selected in the first quadrupole and fragmented in the second quadrupole. Specific fragment ions unique to each peptide were selected in the third quadrupole (Table 3). Results of MRM analysis indicated that PrtA cleaved a 78-amino-acid fragment from the N terminus of apo-hLf (Fig. 10 B), whereas no such product was identified in the absence of digestion with PrtA (Fig. 10A). This was the only major cleavage site found in the protein under these conditions. Scanning of the C-terminal region of lactoferrin did not show any evidence of additional cleavage. Even though the cleavage did not occur in the expected first 47 amino acids, which could have yielded the traditional lactoferricin peptide, in the N-terminal region, the fragment obtained by cleavage between residues 78 and 79 includes the first 47 amino acids of apo-hLf, where the bactericidal activity of lactoferrin has been show to reside (19). It is possible that once this fragment is released, it undergoes a heterogeneous pattern of cleavage, which produces many different bactericidal fragments but no major cleavage sites that we were able to identify.
A total of 10 μg/ml of apo-hLf was incubated with or without 10 μg/ml of PrtA in a 50 mM Tris-HCl (pH 7.5) and 50 mM CaCl2 buffer at 37°C for 2 h. MRM analysis of the cleavage of apo-hLf by PrtA was done. Results of LC/MS identified a cleavage between Val78 and Ala79 of the tryptic peptide LRPVAAEVYGTER, thus generating the peptide AAEVYGTER. To confirm this cleavage, MRM of PrtA/trypsin-generated lactoferrin peptides was set up using the parent peptides LRPVAAEVYGTER and AAEVYGTER, with trypsin-derived BSA peptide as an internal standard, and the appearance of the daughter ions derived from the corresponding parental ions was monitored. Results of MRM analysis indicated the cleaved product in lactoferrin samples treated with PrtA (B), whereas no such product was identified in untreated samples (A).
These studies have addressed the hypothesis that the killing of pneumococci by apo-hLf is dependent on the cleavage of an N-terminal LLP (peptide) from apo-hLf by pneumococcal proteases, primarily PrtA, and that it is this cleaved LLP (or a smaller LLP derived from it) that actually kills the pneumococci. This hypothesis is supported by our observations, as follows: (i) serine protease inhibitors block killing by apo-hLf but not by LLP, (ii) incubation of apo-hLf with live pneumococci or the pneumococcal protease PrtA results in protease-dependent removal of ∼8 kDa, (iii) pretreatment of apo-hLf with PrtA makes the mixture of apo-hLf and PrtA severalfold more bactericidal, (iv) mutant pneumococci lacking PrtA are poorly able to degrade apo-hLf and are not efficiently killed by apo-hLf, (v) rPrtA can complement the prtA mutation and restore susceptibility to apo-hLf, (vi) holo-hLf, which is not bactericidal, does not exhibit a serine protease-dependent loss of 8 kDa in mass, and (vii) a major PrtA cleavage site in apo-hLf between amino acids 78 and 79 removes the N-terminal 78 amino acids (∼8 kDa) of apo-hLf, which includes the sequences of the known bactericidal LLP.
It is well established that the LLP from apo-hLf that are produced in the lab by enzymatic attack or weak acid hydrolysis can be much more bactericidal than apo-hLf itself (31, 39, 49, 54). Using two synthesized LLP, we have confirmed that LLP can be more bactericidal to pneumococci than apo-hLf (22). It has been proposed that these antibacterial peptides act at or through the bacterial membrane (1, 4, 16, 27). It seems likely that these short peptides should be able to transit the cell wall and reach the bacterial membrane much more efficiently than intact apo-hLf. These considerations led us to originally hypothesize that killing by apo-hLf is dependent on its cleavage and the consequent release of anti-bacterial LLP.
Holo-hLf has been shown to generally lack bactericidal activity against pathogens and to be less readily cleaved by protease than is apo-hLf (9), and our present findings indicate that apo-hLf is more readily cleaved by incubation with pneumococci than is holo-hLf. It is thought that apo-hLf is more structurally flexible than holo-hLf, allowing its N1 lobe to exist in an open conformation, which in turn may contribute to the greater susceptibility of apo-hLf than holo-hLf to proteolytic cleavage (2, 8, 9).
In earlier work, we demonstrated that PspA, which binds apo-hLf, could block killing by apo-hLf and two LLP (31). In this study we observed that rPspA failed to block cleavage of apo-hL