The Ax21 protein is a cell-cell signal that regulates virulence in the nosocomial pathogen Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia encodes proteins related to the Rax proteins of Xanthomonas oryzae, which are required for the synthesis and secretion of the Ax21 protein. Here we show that Ax21 acts as a cell-cell signal to regulate a diverse range of functions, including virulence, in this nosocomial pathogen.

Fig. 1.

Mutation of ax21, raxH, or raxR has pleiotropic effects in S. maltophilia K279a. The ax21, raxH, and the raxR mutants show reduced motility in 0.6% Eiken agar (A), reduced tolerance to the aminoglycoside tobramycin at 100 μg/ml as revealed by a killing curve (B), reduced biofilm formation on glass as quantified by crystal violet staining (C), and reduced virulence in the Galleria mellonella larva infection model (D). These mutant phenotypes could be restored to wild-type levels in all cases through complementation by in trans expression of a wild-type copy of their respective gene. All methods have been described previously (3).

Fig. 2.

Ax21 and the nonsulfatable Y22A variant form of Ax21 (here designated AX21Y) are active in regulation and as extracellular signals in S. maltophilia. (A) Exogenous addition of synthetic Ax21 protein (AX21) to the medium restored motility to an ax21 mutant but not to raxH (sensor kinase) or raxR (regulator) mutants. (B) Exogenous addition of synthetic Ax21 protein to an ax21 mutant restored the levels of expression of smlt1112, smlt1390, smlt2175, and smlt3949 toward those of the wild type as measured by qRT-PCR. This effect was not seen in the raxH (sensor kinase) mutant. (C) Addition of AX21Y to the medium or in trans expression of the AX21Y variant from p0387Y22A restored motility to an ax21 mutant. (D) Addition of AX21Y to an ax21 mutant restored the levels of expression of smlt1112, smlt1390, smlt2175, and smlt3949 toward wild-type levels as measured by qRT-PCR. This effect was not seen in the raxH (sensor kinase) mutant.

Fig. 3.

Ax21 secretion is dependent on the RaxB homolog Smlt2001, a putative ABC transporter ATP binding protein. The level of Ax21 activity in culture supernatants from selected strains grown in NYGB medium to an OD at 600 nm of 2.0 was assessed by a bioassay in which the restoration of motility to an ax21 (smlt0387) mutant was measured.