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

doi:10.71150/jm.2411005

Articles

Page Path: HOME > J. Microbiol > Volume 63(2); 2025 > Article

Full article LasB activation in Pseudomonas aeruginosa: Quorum sensing-mediated release of an auto-activation inhibitor: Cheol Seung Lee¹, Xi-Hui Li^1,2, Chae-Ran Jeon¹, Joon-Hee Lee^1,2,*; Journal of Microbiology 2025;63(2):e2411005.
DOI: https://doi.org/10.71150/jm.2411005
Published online: February 27, 2025

¹Department of Pharmacy, College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea

²Research Institute for Drug Development, Pusan National University, Busan 46241, Republic of Korea

*Correpondence Joon-Hee Lee Email: 'joonhee@pusan.ac.kr'

• Received: November 5, 2024 • Revised: November 20, 2024 • Accepted: December 19, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

168 Views
4 Download

Full Article

Download PDF

ABSTRACT
Introduction
Materials and Methods
Results
Discussion
Notes
References

ABSTRACT

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.
Keywords: Pseudomonas aeruginosa, elastase, LasB, protease, protease inhibition, quorum sensing

Introduction

Pseudomonas aeruginosa is a notorious Gram-negative pathogen that causes serious infections in people with burn wounds, cystic fibrosis, immunodeficiency, and chronic pulmonary disorders (Qin et al., 2022), and known to have multiple antibiotic resistance mechanisms, produce numerous virulence factors, and form robust biofilm, a very protective life mode (Horcajada et al., 2019). Among the virulence factors produced by P. aeruginosa, three extracellular proteases, elastase B (LasB), lysine-specific endopeptidase (PIV) and staphylolysin (LasA) are crucially implicated in the pathogenicity by damaging host tissues and immune components (Kessler et al., 1998; Li and Lee, 2019).

The expression and activation of these 3 proteases are elaborately regulated by multiple complex mechanisms. First, the transcription of these protease genes is strictly controlled by quorum sensing (QS) that is a cell-to-cell communication mechanism to regulate gene expression in a cell density-dependent manner (Miller and Bassler, 2001; Moradali et al., 2017). In P. aeruginosa, two principal QS signal synthases, LasI and RhlI (encoded by lasI and rhI, respectively) continuously synthesize N-3-oxododecanoyl homoserine lactone and N-butyryl-homoserine lactone, respectively as the QS signals (Miranda et al., 2022; Williams et al., 2004). When these QS signals reach a critical concentration, they enter cells to bind to their cognate receptors and activate the transcription of many QS regulon genes, including lasB (encoding LasB), lasA (encoding LasA), and piv (encoding PIV) (Albus et al., 1997; Ding et al., 2018; Kostylev et al., 2019).

The second regulation occurs at extracellular space during secretion. Since PIV, LasB, and LasA all have a common domain organization (a signal peptide (SP) at the N-terminus, a propeptide (PP) domain in the middle, and a mature protease domain at the C-terminus), they are initially expressed in a full length containing all domains (prepro-forms) that is inactive. But during secretion, these prepro-forms are processed by proteolytic cleavage to produce mature active forms. For example, mature LasB is a 33 kDa zinc-metalloprotease but LasB is initially synthesized as a prepro-form of 53.6 kDa. The 2.4 kDa SP is eliminated upon passage through the inner membrane into the periplasm to make pro-LasB, and the 18 kDa PP is subsequently cleaved off by proteolysis in periplasm to make mature LasB (Kessler et al., 1998; McIver et al., 1995). Before cleavage, the PP plays an essential role for the proper folding of LasB in the periplasm like some proteases mentioned above, and this proper folding allows for further autoproteolytic processing of the pro-LasB to mature LasB (McIver et al., 1991, 1995). Therefore, the cleaved PP functions as an inhibitor for LasB and intramolecular chaperone for LasB activity at the same time (Kessler and Safrin, 1988; McIver et al., 1991, 1995). PIV and LasA undergo similar processes: They are activated through propeptide cleavage during secretion in a sequential proteolytic cascade initiated by LasB. In this cascade, LasB activates pro-PIV to form active PIV, and subsequently, both LasB and PIV contribute to the activation of pro-LasA to form active LasA (Li and Lee, 2019).

So far, LasB itself has been suggested to be auto-activated and considered an initial triggering activator in this cascade activation (McIver et al., 1991, 1993). In this LasB activation model, LasB was considered to be the topmost factor that initiates activation cascade first, and was thought to not require other factors for its activation. However, our previous study has demonstrated that LasB is not activated in the QS-deficient strain of P. aeruginosa (QS^-), even with artificial overexpression (Li and Lee, 2019). Interestingly, this inactive LasB in QS^- strain was fully activated by the addition of the culture supernatant of QS⁺ strain (Li and Lee, 2019), suggesting the existence of QS-dependent activator. Therefore, the current model, which assumes LasB auto-activation, needed to be revised. In this study, we re-examined the activation mechanism of LasB and found that the extracellular activation of LasB involves not only a QS-dependent activator but also a QS-independent inhibitor. This indicates that LasB is not the primary initiator in the regulatory mechanism, but rather there exists an additional upstream regulatory system consisting of both inhibitory and activating factors. Based on these findings, we propose a new model for the initial activation mechanism of LasB.

Materials and Methods

Bacterial strains, plasmids and culture conditions

Bacterial strains and plasmids utilized in this study are listed in Table 1. Throughout this study, PAO1 or ΔlasB mutant was used for the QS⁺ strain, and MW1 (lasI^-, rhlI^-) was used for the QS^- strain. Bacterial cells in general were cultured with vigorous shaking at 37°C under 180 rpm. Luria-Bertani (LB) medium (0.5% yeast extract, 0.5% NaCl, and bacto-tryptone 1%) was used to grow the bacteria.1.5% (w/v) agar was added to solidify LB media. The bacterial growth was measured by optical density at 600 nm (OD₆₀₀) using a spectrophotometer (OPTIZEN). Gentamicin and tetracycline were added at 10 µg/ml for E. coli and 50 µg/ml for P. aeruginosa, respectively, and ampicillin, 100 µg/ml, and carbenicillin, 150 µg/ml. In order to induce protein expression, 0.5 mM Isopropyl-1-thio- β-D-galactopyranoside (IPTG) was added at OD₆₀₀ = 0.5.

Culture supernatant (CS) preparation

Bacterial cells were cultivated in 5 ml of LB with vigorous shaking at 37ºC overnight and diluted at 1:100 ratio into 5 ml fresh LB broth with 0.4% arabinose for the main culture. After further overnight culture with vigorous shaking at 37ºC, cells were removed by centrifugation at 13,000 rpm for 2 min at 4ºC and the CSs were filtered through 0.22 μm syringe filter (Satorious) and 10 times concentrated by 10 kDa cut-off centricon (Vivaspin^®, Satorious).

Overexpression and purification of LasB in BL21

LasB were overexpressed with the C-terminal histidine tag from the plasmid pQF21c-LasB (Table 1). The pQF21c-LasB plasmid was introduced to E. coli BL21 by transformation. The transformant was inoculated into 100 ml of LB with 100 μg/ml ampicillin (150 μg/ml carbenicillin) and cultured at 37ºC with vigorous shaking up to OD₆₀₀ = 0.5. Then, IPTG was added at 0.5 mM to induce the protein expression and the cells were further cultivated at 37ºC with shaking. After 16-h cultivation, cells were harvested by centrifugation at 14,000 rpm for 20 min at 4ºC and washed with 20 mM Tris-HCl (pH 8.0). The cells were resuspended in a binding buffer (20 mM Tris-HCl, 5% glycerol, pH 8.0) and disrupted by sonication. The soluble fraction was separated from insoluble fraction by centrifugation at 14,000 rpm for 20 min at 4ºC. The soluble fraction was loaded on a Ni-NTA column (Invitrogen) and washed with low concentration of imidazole (20 mM Tris-HCl, 20 mM imidazole, 5% glycerol, pH 8.0). The tightly bound proteins were eluted with increasing concentrations of imidazole (20 mM Tris-HCl, 50–500 mM imidazole, 5% glycerol, pH 8.0). The fractions containing pure LasB were collected, dialyzed in storage buffer (20 mM Tris-HCl, 5% glycerol, pH 8.0) and stored at -80ºC.

Overexpression and purification of LasB in P. aeruginosa

For the LasB purification, LasB-overexpressing plasmid (pSP201, Table 1) was introduced into the PAO1 or MW1, and the transformed cells were cultivated in 300 ml of LB medium at 37°C with vigorous shaking under 180 rpm. Arabinose was added at 0.8% to induce LasB for 16 h. Cells were then removed by centrifugation and the culture supernatant was taken. LasB was precipitated by slow addition of ammonium sulfate ((NH₄)₂SO₂) and pelleted by centrifugation. The pellet was dissolved in 20 mM Tris-HCl (pH 8.0) and the remained salt was completely removed by dialysis. This LasB-containing solution was applied to DEAE sepharose column chromatography and eluted by salt gradient (0–1 M NaCl) by using FPLC (Fast Protein Liquid Chromatography). The fractions containing pure LasB were collected and dialyzed in the storage buffer (20 mM Tris-HCl and 5% glycerol, pH 8.0). The purified LasB was aliquoted and stored at -80°C.

LasB activity assay

The elastolytic activity of LasB was measured using the Elastin-Congo red method, as previously described (Li and Lee, 2019; Shin et al., 2022). The CSs prepared from the indicated strains or purified proteins were incubated with 10 mg/ml elastin-Congo red (EPC, USA) at 37ºC for 16 h in 0.3 ml of 10 mM Tris-HCl (pH 8.0). Any precipitation was removed by centrifugation at 14,000 × g for 10 min and absorbance at 490 nm (A₄₉₀) was measured using an iMarkTM microplate reader (Bio-Rad). LasB activity was calculated by following equation: LasB activity (%) = [(A₄₉₅ of the elastolytic reaction with sample - A₄₉₅ of the elastolytic reaction with LB) / (A₄₉₅ of elastolytic reaction with PAO1 - A₄₉₅ of the elastolytic reaction with LB)] × 100. HCl, 5% glycerol, pH 8.0) and stored at -80ºC.

Statistical analysis

The significance of difference was determined by using student’s t-test (two sample assuming equal variances) in MS Office Excel (Microsoft). When p-values < 0.05, it was considered significant.

Results

The LasB inhibitor is present in QS-deficient strains, where LasB is not auto-activated even after auto-processing

Our previous study demonstrated that LasB remains inactive in the QS-deficient strain of P. aeruginosa (QS^-; MW1, Table 1), even under artificial overexpression conditions (Li and Lee, 2019). However, the addition of culture supernatant (CS) from the QS⁺ strain fully restored LasB activity in the QS^- strain (Li and Lee, 2019), suggesting the existence of QS-dependent activator. Interestingly, examination of LasB status in the CS of the LasB-overexpressing QS^- strain (CS_MW1-LasB) revealed that LasB existed in its mature form, with both SP and PP already cleaved (Fig. 1A). This finding demonstrated that despite proper processing, LasB remained in an inactive state, strongly suggesting that LasB activation requires mechanisms beyond mere proteolytic processing of pro-LasB. Therefore, we realized that we needed to distinguish between auto-processing, which refers to self-cleavage of PP, and auto-activation, which refers to self-activation.

More intriguingly, the inactive LasB regained its activity upon purification from the QS^- strain. As demonstrated in Fig. 1B, LasB purified from CS_MW1-LasB exhibited minimal activity during intermediate purification steps; however, once fully purified, it displayed activity comparable to that of LasB purified from the CS of the LasB-overexpressing wild-type (CS_WT-LasB). These findings suggested that LasB was under suppression by an inhibitory factor unique to the QS^- strain, and that removal of this factor through purification was sufficient for LasB activation without additional activating factors. Based on these observations, we hypothesized that in QS⁺ strains, QS-dependent expression of LasB-activating factors eliminates the inhibitory factor, enabling immediate activation of LasB following processing. Conversely, in QS^- strains, the persistent presence of the inhibitory factor maintains LasB in an inactive state even after proper processing.

LasB produced in the heterologous host E. coli, which lacks inhibitor, was auto-processed and auto-activated

To verify the existence of LasB inhibitor, LasB was overexpressed in E. coli rather than P. aeruginosa. Since E. coli is a heterologous host presumed to lack LasB inhibitor, the LasB expressed in E. coli was expected to maintain its activity once properly processed. When we overexpressed LasB in E. coli, and separate the whole cytoplasmic proteins of the LasB-overexpressing E. coli cells into soluble and insoluble fractions, LasB highly overexpressed by IPTG addition, existed mainly as unprocessed prepro-form (53 kDa) in the insoluble fraction, whereas without IPTG induction, the slightly overexpressed LasB was predominantly found as the processed mature form (33 kDa) in the soluble fraction (Fig. 2A). When measuring LasB activity in the soluble fraction, the slightly overexpressed LasB without IPTG showed high levels of activity, whereas the highly overexpressed LasB with IPTG exhibited very low activity (Fig. 2B). All insoluble fractions showed no LasB activity (data not shown). When the mature form of LasB was purified from the soluble fraction, it also showed high activity (Fig. 2B). This result clearly demonstrates that in E. coli, which lacks P. aeruginosa factors including inhibitors, LasB undergoes auto-processing and the auto-processed form retain activity.

The LasB that was expressed and processed in E. coli showed no differences in size compared to the LasB processed in wild type P. aeruginosa or the QS^- strain (Fig. 3A). Furthermore, LasB processed in E. coli and wild type P. aeruginosa showed no differences in activity, and its activity was also identical to that of LasB activated after purification from the QS^- strain (Fig. 3B & 1B). Therefore, despite all conditions being identical, the fact that LasB expressed and processed in the QS^- strain shows low activity before purification clearly indicates the presence of an inhibitor in the QS^- strain.

LasB is suppressed by a QS-independent inhibitor and becomes activated when the inhibitor is removed by a QS-dependent activator

In our previous study, we demonstrated the existence of a QS-dependent LasB activator (Li and Lee, 2019). In this study, we additionally discovered the presence of a LasB inhibitor that exists only in QS^- strains. Therefore, we investigated how these two factors are involved in the activation of LasB. We hypothesized that while P. aeruginosa produces a QS-independent inhibitor to suppress LasB, the QS-dependent activator removes this inhibitor, thereby releasing LasB from inhibition. In order to prove this hypothesis, we treated purified active LasB with CS prepared from the QS^- strain (CS_MW1). As a control, we similarly treated CS from the ΔlasB mutant strain (CS_ΔlasB). ΔlasB mutant is a QS⁺ strain, but lacks LasB activity. As shown in Fig. 4, only CS_MW1 could inhibit LasB activity in a dose-dependent manner, whereas there was no inhibition with the CS_ΔlasB treatment. These results clearly demonstrate that the LasB inhibitor exists only in the QS^- strain and not in the QS⁺ strain.

In our previous study, we presented evidence for the existence of a QS-dependent LasB activator (Li and Lee, 2019). This factor present in QS⁺ strain could activate the inactive LasB present in LasB-overexpressing MW1 (Li and Lee, 2019). Nonetheless, the above results in this study suggest that LasB undergoes normal auto-processing and auto-activation in the absence of inhibitors, without requiring an activator. An explanation for both of these seemingly contradictory statements is to assume that the LasB activator serves to remove the inhibitor rather than affecting LasB itself. To prove this hypothesis, the CS_MW1 that contain the LasB inhibitor was incubated with CS_ΔlasB that contains the LasB activator. By doing this, it was expected that the LasB inhibitor would be eliminated by the LasB activator. After incubation, when the two purified active LasBs, LasB_EC, and LasB_WT were treated with the mixture of CS_MW1 and CS_ΔlasB, the inhibition on LasB activity by CS_MW1 was gradually decreased according to the increasing amount of CS_ΔlasB (Fig. 5). This result clearly demonstrates that the inhibitor in the QS^- strain is removed by the QS-dependent activator. Collectively, our findings suggest that the LasB activator is a QS-dependent factor that removes the LasB inhibitor, rather than directly processing LasB itself. Furthermore, our data indicate that the activator functions after LasB processing has occurred, since LasB processing occurred normally even in the QS^- strain.

Discussion

P. aeruginosa is an opportunistic pathogen, and LasB serves as one of its crucial virulence factors. The expression of LasB is regulated by the quorum sensing (QS) system in P. aeruginosa (Yang et al., 2021). Although it has been suggested that the full length prepro-LasB is auto-processed and auto-activated (McIver et al., 1991), our previous study has demonstrated that LasB is inactive when expressed in the QS^- mutant, and suggested that LasB cannot auto-activate alone and requires a QS-dependent factor for activation (Li and Lee, 2019). Based on this previous study, we proposed a new model for the QS-dependent extracellular activation of major proteases, LasB, PIV, and LasA, as follows (Fig. 6): In the cytoplasm, the prepro-forms of the proteases are expressed under transcriptional control of QS, and their SPs are removed during translocation over the cytoplasmic membrane. In the extracellular space, the pro-form of LasB is first activated by unknown QS-dependent activating factors. The activated LasB, in turn, activates pro-PIV to PIV, and the activated PIV activates pro-LasA into active LasA. LasB also plays an auxiliary role in activation of LasA.

However, our model suggesting the existence of QS-dependent activating factors conflicted with the previously established model of LasB auto-processing and auto-activation (McIver et al., 1991, 1993). To address this inconsistency, we verified the presence of an inhibitor in QS^- strain and subsequently proposed an updated model that incorporates the cooperative involvement of both inhibitor and activator (Fig. 6). A crucial aspect of this updated model is the distinction between LasB auto-processing and auto-activation. Although LasB can undergo auto-processing independently without other factors, our model demonstrates that even after processing, LasB remains in an inactive state in QS^- strains due to the presence of an inhibitor. In contrast to this, in the QS⁺ wild type, LasB becomes activated because the inhibitor is quickly removed by the QS-dependent activator (Fig. 6).

A key evidence for this updated model is that the inactive LasB overexpressed in QS^- strain was already in its processed form and reactivated when purified (Fig. 1A & 1B). Another key evidence is that when full length LasB was overexpressed in E. coli, a heterologous bacterium that has neither activator nor inhibitor for LasB, LasB was normally processed and activated, as proposed in the previous model (Fig. 2A & 2B). These evidences clearly establish the existence of a LasB inhibitor, even though it may not be readily observable in QS⁺ wild type strains. This is because the inhibitor is rapidly eliminated by activators expressed in QS⁺ strains. In this study, we confirmed this mechanism by utilizing QS^- strains, thereby providing an updated and more precise understanding of the LasB activation mechanism.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (RS-2024-00353202).

Conflict of Interest

We declare that we have no conflicts of interest with the contents of this article.

Fig. 1.

LasB is processed but remains inactive in the QS-deficient strain, and becomes activated upon purification. The CSs were prepared from PAO1 (CS_WT), MW1 (CS_MW1), MW1 harboring a LasB-overexprsessing plasmid, pSP101 (CS_MW1-LasB), and LasB was purified from CS_MW1-LasB and CS_WT through (NH₄)₂SO₄ precipitation and Ni-NTA column purification. (A) LasB in each CS was observed in 15% SDS-PAGE before purification. (B) LasB activity was measured and compared among the CSs, LasB purified from CS_MW1-LasB (LasB_MW1-LasB), LasB purified from CS_WT (LasB_WT), and partially purified LasB_MW1-LasB obtained from (NH₄)₂SO₄ precipitation. CSs containing 2 µg of total protein and 100 ng of purified protein were used. Heat inactivation was done at 100°C for 10 min. ^***, P < 0.005; ns, no significance.

Fig. 2.

When overexpressed in E. coli, LasB undergoes both processing and activation. LasB was overexpressed by using pQF21c-LasB in E. coli BL21. (A) crude extract was separated into soluble and insoluble fractions and subjected to 15% SDS-PAGE. Mature LasB and unprocessed prepro-LasB are indicated by black and red arrows, respectively. E. coli BL21 transformed with pQF21c as a vector control and was analyzed in parallel. (B) LasB activity was measured in the soluble fraction and LasB_WT that is LasB purified from the uninduced soluble fractions. ^***, P < 0.005.

Fig. 3.

The size and activity of LasB were identical whether purified from E. coli or P. aeruginosa. (A) SDS-PAGE analysis were performed to compare LasB proteins purified from different sources: P. aeruginosa MW1 (LasB_MW1-LasB, see Fig. 1), P. aeruginosa PAO1 (LasB_WT, see Fig. 1), or E. coli BL21 (LasB_EC, see Fig. 2). (B) the LasB activity was measured and compared using 100 ng of purified LasB_WT and LasB_EC. For comparison with LasB_MW1-LasB, refer to Fig. 1B. ns, no significance.

Fig. 4.

The LasB inhibitor exists only in the QS^- strain and not in the QS⁺ strain. The purified LasB from E. coli (LasB_EC) was mixed with CS prepared from ΔlasB mutant (CS_ΔlasB) or CS prepared from MW1 (CS_MW1), incubated for 30 min, and measured for LasB activity. The amount of CS was presented as the total amount of protein contained in CS. ^*, P < 0.05; ^**, P < 0.01; ns, no significance.

Fig. 5.

Elimination of LasB inhibitor by CS of QS⁺ strain. CS_MW1 was incubated for 30 min with increasing amount of CS_ΔlasB and added to 100 ng of LasB_EC (A) or LasB_WT (B). After 4-h incubation, LasB activity was measured. The amount of CS was presented as the total amount of protein contained in CS. ^*, P < 0.05; ^***, P < 0.005.

Fig. 6.

Updated model for the extracellular activation of LasB in P. aeruginosa. The previously proposed model is shown in black and white (Li & Lee, 2019), while the content added by this study is shown in color. The key addition is that in the extracellular space, LasB activity is inhibited by a QS-independent inhibitor, but when a QS-dependent activator is expressed, it removes the inhibitor, thereby activating LasB.

Table 1.

Bacterial strains and plasmids used in this study

Name	Description	References
Pseudomonas aeruginosa
PAO1	Wild type P. aeruginosa, QS⁺	(Pearson and Pesci et al., 1997)
MW1	lasI^-, rhlI^- double mutant of PAO1, Tc^R, QS^-	(Whiteley et al., 1999)
ΔlasB	lasB^- mutant of PAO1, Tc^R, QS⁺	(Li and Lee, 2019)
Escherichia coli
DH5α	supE44ΔlacU169(80lacZΔM15) hsdR17 recA1 gyrA96thi-1 relA1	Lab. collection
BL21(DE3)	F^-ompT hsdS_B (r_B^- m_B^-) dcm gal λ (DE3)	Lab. collection
Plasmids
pJN105	araC-pBAD promoter fusion plasmid, Gm^R	(Newman and Fuqua, 1999)
pSP201	PA3724 (lasB) in pJN105, Gm^R	(Park et al., 2014)
pQF21c	Overexpression plasmid for C-terminal histidine-tagged protein (a modified pET21c with a broad-host-range replication origin, Ori₁₆₀₀), replicable in P. aeruginosa, Cb^R (Ap^R for E. coli)	(Park et al., 2014)
pQF21c-LasB	lasB in pQF21c, Cb^R (Ap^R for E. coli)	(Li and Lee, 2019)

Tc, Tetracycline; Gm, Gentamicin; Cb, Carbenicillin; Ap, Ampicillin; Km, Kanamycin

References

Albus AM, Pesci EC, Runyen-Janecky LJ, West SE, Iglewski BH. 1997. Vfr controls quorum sensing in Pseudomonas aeruginosa. J Bacteriol. 179(12): 3928–3935. Article PubMed PMC PDF
Ding F, Oinuma KI, Smalley NE, Schaefer AL, Hamwy O, et al. 2018. The Pseudomonas aeruginosa orphan quorum sensing signal receptor QscR regulates global quorum sensing gene expression by activating a single linked operon. MBio. 9(4): e01274–18. Article PubMed PMC PDF
Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, et al. 2019. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev. 32(4): e00031–19. Article PubMed PMC PDF
Kessler E, Safrin M. 1988. Partial purification and characterization of an inactive precursor of Pseudomonas aeruginosa elastase. J Bacteriol. 170(3): 1215–1219. Article PubMed PMC PDF
Kessler E, Safrin M, Gustin JK, Ohman DE. 1998. Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem. 273(46): 30225–30231. Article PubMed
Kostylev M, Kim DY, Smalley NE, Salukhe I, Greenberg EP, et al. 2019. Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy. Proc Natl Acad Sci USA. 116(14): 7027–7032. Article PubMed PMC
Li XH, Lee JH. 2019. Quorum sensing-dependent post-secretional activation of extracellular proteases in Pseudomonas aeruginosa. J Biol Chem. 294(51): 19635–19644. Article PubMed PMC
McIver K, Kessler E, Ohman DE. 1991. Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol. 173(24): 7781–7789. Article PubMed PMC PDF
McIver KS, Olson JC, Ohman DE. 1993. Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion. J. Bacteriol. 175(13): 4008–4015. Article PubMed PMC PDF
McIver KS, Kessler E, Olson JC, Ohman DE. 1995. The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol. 18(5): 877–889. Article PubMed
Miller MB, Bassler BL. 2001. Quorum sensing in bacteria. Annu Rev Microbiol. 55(1): 165–199. Article PubMed
Miranda SW, Asfahl KL, Dandekar AA, Greenberg EP. 2022. Pseudomonas aeruginosa quorum sensing. Adv Exp Med Biol. 1386: 95–115. Article PubMed PMC
Moradali MF, Ghods S, Rehm BH. 2017. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol. 7: 39.Article PubMed PMC
Newman JR, Fuqua C. 1999. Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene. 227(2): 197–203. Article PubMed
Park SJ, Kim SK, So YI, Park HY, Li XH, et al. 2014. Protease IV, a quorum sensing‐dependent protease of Pseudomonas aeruginosa modulates insect innate immunity. Mol Microbiol. 94(6): 1298–1314. Article PubMed
Pearson JP, Pesci EC, Iglewski BH. 1997. Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacteriol. 179(18): 5756–5767. Article PubMed PMC PDF
Qin S, Xiao W, Zhou C, Pu Q, Deng X, et al. 2022. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther. 7(1): 1–27. Article PubMed PMC PDF
Shin Y, Li XH, Lee CS, Lee JH. 2022. Mutational analysis on stable expression and LasB inhibition of LasB propeptide in Pseudomonas aeruginosa. J Microbiol. 60(7): 727–734.Article PubMed PDF
Whiteley M, Lee KM, Greenberg EP. 1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 96(24): 13904–13909. Article PubMed PMC
Williams SC, Patterson EK, Carty NL, Griswold JA, Hamood AN, et al. 2004. Pseudomonas aeruginosa autoinducer enters and functions in mammalian cells. J Bacteriol. 186(8): 2281–2287. Article PubMed PMC PDF
Yang D, Hao S, Zhao L, Shi F, Ye G, et al. 2021. Paeonol attenuates quorum-sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa. Front Microbiol. 12: 692474.Article PubMed PMC

Figure & Data

References

Citations

Citations to this article as recorded by

ePub Link

Cite this Article

Cite this Article: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

LasB activation in Pseudomonas aeruginosa: Quorum sensing-mediated release of an auto-activation inhibitor

J. Microbiol. 2025;63(2):e2411005 Published online February 27, 2025

DOI: https://doi.org/10.71150/jm.2411005

XML Download

Figure

LasB activation in Pseudomonas aeruginosa: Quorum sensing-mediated release of an auto-activation inhibitor

Fig. 1. LasB is processed but remains inactive in the QS-deficient strain, and becomes activated upon purification. The CSs were prepared from PAO1 (CSWT), MW1 (CSMW1), MW1 harboring a LasB-overexprsessing plasmid, pSP101 (CSMW1-LasB), and LasB was purified from CSMW1-LasB and CSWT through (NH4)2SO4 precipitation and Ni-NTA column purification. (A) LasB in each CS was observed in 15% SDS-PAGE before purification. (B) LasB activity was measured and compared among the CSs, LasB purified from CSMW1-LasB (LasBMW1-LasB), LasB purified from CSWT (LasBWT), and partially purified LasBMW1-LasB obtained from (NH4)2SO4 precipitation. CSs containing 2 µg of total protein and 100 ng of purified protein were used. Heat inactivation was done at 100°C for 10 min. ***, P < 0.005; ns, no significance.

Fig. 2. When overexpressed in E. coli, LasB undergoes both processing and activation. LasB was overexpressed by using pQF21c-LasB in E. coli BL21. (A) crude extract was separated into soluble and insoluble fractions and subjected to 15% SDS-PAGE. Mature LasB and unprocessed prepro-LasB are indicated by black and red arrows, respectively. E. coli BL21 transformed with pQF21c as a vector control and was analyzed in parallel. (B) LasB activity was measured in the soluble fraction and LasBWT that is LasB purified from the uninduced soluble fractions. ***, P < 0.005.

Fig. 3. The size and activity of LasB were identical whether purified from E. coli or P. aeruginosa. (A) SDS-PAGE analysis were performed to compare LasB proteins purified from different sources: P. aeruginosa MW1 (LasBMW1-LasB, see Fig. 1), P. aeruginosa PAO1 (LasBWT, see Fig. 1), or E. coli BL21 (LasBEC, see Fig. 2). (B) the LasB activity was measured and compared using 100 ng of purified LasBWT and LasBEC. For comparison with LasBMW1-LasB, refer to Fig. 1B. ns, no significance.

Fig. 4. The LasB inhibitor exists only in the QS- strain and not in the QS+ strain. The purified LasB from E. coli (LasBEC) was mixed with CS prepared from ΔlasB mutant (CSΔlasB) or CS prepared from MW1 (CSMW1), incubated for 30 min, and measured for LasB activity. The amount of CS was presented as the total amount of protein contained in CS. *, P < 0.05; **, P < 0.01; ns, no significance.

Fig. 5. Elimination of LasB inhibitor by CS of QS+ strain. CSMW1 was incubated for 30 min with increasing amount of CSΔlasB and added to 100 ng of LasBEC (A) or LasBWT (B). After 4-h incubation, LasB activity was measured. The amount of CS was presented as the total amount of protein contained in CS. *, P < 0.05; ***, P < 0.005.

Fig. 6. Updated model for the extracellular activation of LasB in P. aeruginosa. The previously proposed model is shown in black and white (Li & Lee, 2019), while the content added by this study is shown in color. The key addition is that in the extracellular space, LasB activity is inhibited by a QS-independent inhibitor, but when a QS-dependent activator is expressed, it removes the inhibitor, thereby activating LasB.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

LasB activation in Pseudomonas aeruginosa: Quorum sensing-mediated release of an auto-activation inhibitor

Name	Description	References
Pseudomonas aeruginosa
PAO1	Wild type P. aeruginosa, QS⁺	(Pearson and Pesci et al., 1997)
MW1	lasI^-, rhlI^- double mutant of PAO1, Tc^R, QS^-	(Whiteley et al., 1999)
ΔlasB	lasB^- mutant of PAO1, Tc^R, QS⁺	(Li and Lee, 2019)
Escherichia coli
DH5α	supE44ΔlacU169(80lacZΔM15) hsdR17 recA1 gyrA96thi-1 relA1	Lab. collection
BL21(DE3)	F^-ompT hsdS_B (r_B^- m_B^-) dcm gal λ (DE3)	Lab. collection
Plasmids
pJN105	araC-pBAD promoter fusion plasmid, Gm^R	(Newman and Fuqua, 1999)
pSP201	PA3724 (lasB) in pJN105, Gm^R	(Park et al., 2014)
pQF21c	Overexpression plasmid for C-terminal histidine-tagged protein (a modified pET21c with a broad-host-range replication origin, Ori₁₆₀₀), replicable in P. aeruginosa, Cb^R (Ap^R for E. coli)	(Park et al., 2014)
pQF21c-LasB	lasB in pQF21c, Cb^R (Ap^R for E. coli)	(Li and Lee, 2019)

Table 1. Bacterial strains and plasmids used in this study

Tc, Tetracycline; Gm, Gentamicin; Cb, Carbenicillin; Ap, Ampicillin; Km, Kanamycin