Journal of Microbiology

Research Article

LasB activation in Pseudomonas aeruginosa: Quorum sensing-mediated release of an auto-activation inhibitor: Cheol Seung Lee, Xi-Hui Li, Chae-Ran Jeon, Joon-Hee Lee; J. Microbiol. 2025;63(2):e2411005. Published online February 27, 2025; DOI: https://doi.org/10.71150/jm.2411005

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Abstract PDF: Pseudomonas aeruginosa secretes three major proteases: elastase B (LasB), protease IV (PIV), and elastase A (LasA), which play crucial roles in infection and pathogenesis. These proteases are activated sequentially from LasB in a proteolytic cascade, and LasB was previously thought to undergo auto-activation. However, our previous study suggested that LasB cannot auto-activate independently but requires additional quorum sensing (QS)-dependent factors for activation, as LasB remained inactive in QS-deficient P. aeruginosa (QS-) even under artificial overexpression. In this study, we provide evidence for the existence of a LasB inhibitor in QS- mutants: inactive LasB overexpressed in QS- strains was in its processed form and could be reactivated upon purification; when full-length LasB was overexpressed in Escherichia coli, a heterologous bacterium lacking both LasB activators and inhibitors, the protein underwent normal processing and activation; and purified active LasB was significantly inhibited by culture supernatant (CS) from QS- strains but not by CS from QS+ strains. These findings demonstrate that a LasB inhibitor exists in QS- strains, and in its absence, LasB can undergo auto-activation without requiring an activator. Based on these results, we propose an updated hypothesis: the QS-dependent LasB activator functions by removing the LasB inhibitor rather than acting directly on LasB itself, thus preventing premature LasB activation until QS response is initiated.

Journal Article

Mutational analysis on stable expression and LasB inhibition of LasB propeptide in Pseudomonas aeruginosa: Youngsun Shin , Xi-Hui Li , Cheol Seung Lee , Joon-Hee Lee; J. Microbiol. 2022;60(7):727-734. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-1671-5

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Abstract: Three major proteases, elastase B (LasB), protease IV (PIV), and elastase A (LasA) expressed in Pseudomonas aeruginosa play important roles in infections and pathogeneses. These are activated by a proteolytic cascade initiated by the activation of LasB. In this study, we investigated whether LasB could be inhibited using its propeptide (LasBpp). Although LasA and PIV were inhibited by their propeptides, LasB was not inhibited by purified LasBpp because LasB degraded LasBpp. To address this problem, mutant LasBpp variants were constructed to obtain a mutant LasBpp resistant to LasB degradation. A C-terminal deletion series of LasBpp was tested in vivo, and two positive candidates, T2 and T2-1, were selected. However, both caused growth retardation and were unstably expressed in vivo. Since deleting the C-terminal end of LasBpp significantly affected its stable expression, substitution mutations were introduced at the two amino acids near the truncation site of T2-1. The resulting mutants, LasBppE172D, LasBppG173A, and LasBppE172DG173A, significantly diminished LasB activity when overexpressed in vivo and were stably expressed in MW1, a quorum sensing mutant that does not produce LasB. In vitro analysis showed that purified LasBppE172DG173A inhibited LasB activity to a small extent. Summarizing, Cterminal modification of LasBpp profoundly affected the stable expression of LasBpp, and little enhanced the ability of LasBpp to resist degradation by LasB.

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