Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin

Abstract

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.Streptococcus pneumoniae is a major inhabitant of the human upper respiratory tract and can cause pneumonia, sepsis, meningitis, and otitis media (21a). Pneumococci are common inhabitants of the nasopharynx of healthy children and adults. Pneumococci can cause serious disease when they leave the nasopharynx and successfully invade the middle ear, lungs, blood, or brain (21a). Although specific antibody can mediate protection against pneumococcal infection (14, 53), there is also evidence that antibody is not necessary for immunity to colonization and that innate immunity plays a significant role (30,50,58). Moreover, the molecular mechanisms involved in the interaction of S. pneumoniae with the innate immune system are incompletely understood.Known innate immune system components that protect against bacterial infection in the upper airways include mucus flow, antibacterial proteins, such as lysozyme and lactoferrin, and antibacterial peptides, such as ␣-defensins and ␤-defensins (57). Human lactoferrin (hLf) has been reported to play a number of different roles in mammals, including immunoregulation, iron sequestration, promotion of calcium uptake, and bactericidal activity (52). The observation that pneumococcal surface protein A (PspA) specifically inhibits in vitro killing of pneumococci by apolactoferrin (apo-hLf), the iron-free for...

Tuf of Streptococcus pneumoniae is a surface displayed human complement regulator binding protein

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Multivalent Pneumococcal Protein Vaccines Comprising Pneumolysoid with Epitopes/Fragments of CbpA and/or PspA Elicit Strong and Broad Protection

“…This SM1 peptide is located just upstream of the proline-rich region of PspA in strain Rx1. Human lactoferrin is known to kill many species of bacteria, including pneumococci, and PspA has been observed to specifically inhibit in vitro killing of pneumococci by apolactoferrin (the iron-free form of lactoferrin); this action is believed to be due to the SM1 peptide that blocks the binding of lactoferrin to PspA (66). Thus, the addition of the SM1 peptide in H70 is likely to have provided enhanced protection over CD2.…”

Retention of structure, antigenicity, and biological function of pneumococcal surface protein A (PspA) released from polyanhydride nanoparticles

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