S. Kamath et al., Cellular function of elastase in Pseudomonas aeruginosa: role in the cleavage of nucleoside diphosphate kinase and in alginate synthesis, MOL MICROB, 30(5), 1998, pp. 933-941
Elastase is a major virulence factor in Pseudomonas aeruginosa that is beli
eved to cause extensive tissue damage during infection in the human host, E
lastase is secreted in non-mucoid P. aeruginosa. It is known that secretion
of most virulence factors such as elastase, lipase, exotoxin A, etc., in P
. aeruginosa is greatly reduced in alginate-secreting mucoid cells isolated
from the lungs of cystic fibrosis (CF) patients. We have previously report
ed that in mucoid P. aeruginosa, an intracellular protease cleaves the 16 k
Da form of nucleoside diphosphate kinase (Ndk) to a truncated 12 kDa form,
This smaller form is membrane associated and has been observed to form comp
lexes with specific proteins to predominantly generate GTP, an important mo
lecule in alginate synthesis, The main aim of this study was to purify and
characterize this protease. The protease was purified by hydrophobic intera
ction chromatography of the crude extract of mucoid P. aeruginosa 8821, a C
F isolate. Further analysis using a gelatin containing SDS-polyacrylamide g
el detected the presence of a 103 kDa protease, which when boiled, migrated
as a 33 kDa protein on a SDS-polyacrylamide gel. The first 10 amino acids
from the N-terminus of the 33 kDa protease showed 100% identity to the matu
re form of elastase, An elastase-negative lasB::Cm knock-out mutant in the
mucoid 8821 background was constructed, and it showed a non-mucoid phenotyp
e, This mutant showed the presence of only the 16 kDa form of Ndk both in t
he cytoplasm and membrane fractions, We present evidence for the retention
of active elastase in the periplasm of mucoid P, aeruginosa and its role in
the generation of the 12 kDa form of Ndk. Finally, we demonstrate that ela
stase, when overproduced in both mucoid and non-mucoid cells, stimulates al
ginate synthesis. This suggests that the genetic rearrangements that trigge
r mucoidy in P. aeruginosa also allow retention of elastase in the periplas
m in an active oligomeric form that facilitates cleavage of 16 kDa Ndk to i
ts 12 kDa form for the generation of GTP, required for alginate synthesis.