A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

Abstract

The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmSL181P, BfmSL181P/E376Q, and BfmSR393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs.

The Crc protein participates in down‐regulation of the Lon gene to promote rhamnolipid production and rhl quorum sensing in Pseudomonas aeruginosa

“…To identify genetic factors that govern rhamnolipid production in P. aeruginosa , we performed a screen for rhamnolipid defective mutants using a library of PAO1:: rhlA ‐ lacZ (Supporting Information http://onlinelibrary.wiley.com/doi/10.1111/mmi.12954/suppinfo) transposon mutants and a methylene blue/cetyltrimethylammonium bromide (CTAB) agar plate method (Kohler et al ., ; Gupta et al ., ; Cao et al ., ). Approximately 50 000 transposon insertion mutants were visually analyzed by scoring the severe decrease in the size of the blue halo surrounding the bacterial colonies on the CTAB agar plates, which indicates the production of rhamnolipid (Kohler et al ., ; Gupta et al ., ).…”

“…Given that the expression of rhlAB is controlled by the rhl QS system (Brint and Ohman, ; Ochsner and Reiser, ), we next sought to determine whether the crc deletion alters the level of the rhl QS signal C4‐HSL content. We measured the content of the RhlI‐dependent autoinducer C4‐HSL in a wild‐type PAO1 strain, a Δ crc strain and a complementary strain (Δ crc /p‐ crc ) when bacteria were grown in M8 minimal medium, using the pDO100 (pKD‐ rhlA ) system (Supporting Information http://onlinelibrary.wiley.com/doi/10.1111/mmi.12954/suppinfo) that carries a lux reporter fused with an rhlA promoter (Liang et al ., ; Cao et al ., ). We observed that the supernatants prepared from either the wild‐type PAO1 strain or the complemented strain (Δ crc /p‐ crc ), but not the Δ crc strain, markedly promoted luminescence values (Fig.…”

“…Next, we asked whether decreased rhlA promoter activity causes the reduced abundance of rhlAB mRNA. In order to assess this possibility, we measured the activities of an rhlA promoter‐ lux fusion ( rhlA ‐ lux ) (Cao et al ., ) in a wild‐type PAO1 strain, a crc deletion strain (Δ crc ) and in strain PAO1Δ crc (p‐ crc ) complemented with a plasmid‐borne copy of crc respectively. The expression of the rhlA‐lux fusion in Δ crc was significantly lower than that of the other strains when bacteria were grown in M8 minimal medium (Fig.…”

“…The las QS and rhl QS systems are indicated by dotted and closed lines respectively. This model is based on the results of the current study and some previous studies ( W hiteley et al ., ; S onnleitner et al ., 2003; 2006; S chuster and G reenberg, ; S orger‐ D omenigg et al ., ; C roda‐ G arcia et al ., ; R utherford and B assler, ; C ao et al ., ). Details are seen in the main text.…”

Biofilm: An Important Bacterial Feature Still to Deal With

“…The RHL system acts through C4-HSL (N-butanoylhomoserine lactone), responsible for the production ramnolipids and elastase. The LAS and RHL systems are still modulated by a quinolone, 2-heptyl-3-hydroxy-4-quinolone, called PQS, which increases the complexity of QS network [87,88].…”