Download Characterization of the receptors for the soluble pyocins S1, S2, and

Characterization of the receptors for the
soluble pyocins S1, S2, and S3 of
Pseudomonas aeruginosa
Sarah Denayer
ABSTRACT
Pseudomonas aeruginosa is an opportunistic pathogen and is associated with lung
infections in cystic fibrosis (CF) and immunocompromised patients (AIDS, cancer), with
severe burns, and with eye infections. The large and complex genome confers this organism
its versatility and adaptability to many different environments, and also the ability to produce
a wide range of virulence factors which contribute to the virulence of P. aeruginosa.
More than 90% of the P. aeruginosa strains are able to produce one or more pyocin
(Michel-Briand & Baysse, 2000). According to their structure and solubility, pyocins are
divided in three distinct groups: R-type (insoluble, contractile), F-type (insoluble, flexible)
and S-type (soluble). Currently, seven S-type pyocins have been described (S1-S5, AP41, Sa)
(Parret & De Mot, 2002). These consist of three domains: (i) an N-terminal domain involved
in receptor binding, (ii) a central translocation domain and (iii), a C-terminal domain that
exerts its activity (Michel-Briand & Baysse, 2000). Pyocins kill closely related species using a
DNAse activity (S1-S3, AP41), a tRNAse activity (S4) or a pore-forming activity (S5). For
some S-type pyocins (S2, S3, and Sa), the activity is increased by iron limitation (Smith et al.,
1992). This has been demonstrated for pyocin S3 which uses the type II pyoverdine receptor,
FpvAII (De Chial et al., 2003).
Using a site directed mutagenesis of some aromatic residues in loops 1 to 5 on FpvAII
[loop 2 (I323→A, D324→A), loop 3 (Δ365-368, Δ379-382, Δ390-393) and loop 5 (Y512→A)],
the importance of these residues in the binding of both pyoverdine and pyocin S3 was
demonstrated. Both substrates probably use common binding sites. Mainly the third loop is
important in generating an aromatic binding pocket for utilization of pyoverdine and even
more for pyocin S3 binding.
The receptor for pyocin Sa, which is produced by P. aeruginosa J1003, was also
predicted to be a siderophore receptor but nor the pyocin, nor its receptor were characterized
(Smith et al., 1992). We constructed a genomic library of the Sa producing strain J1003 and
screened for S-type pyocin producing clones. Subcloning and sequence analysis revealed that
pyocin Sa is S2.
Using an allelic fpvAI -mutant, we could demonstrate the use of the type I ferripyoverdine receptor, FpvAI, by pyocin S2 to enter sensitive cells. This confirmed the
hypothesis obtained from a thorough screening of a large collection of clinical P. aeruginosa
strains and isolates from the Belgian Woluwe river. However, five resistant strains (eg. PA14)
bearing FpvAI and lacking the S1S2 immunity protein exist. Sequence analysis revealed a
single amino acid substitution in the receptor (V46→I46) of these resistant strains. However,
expression of both receptor types in trans rendered a type II strain (7NSK2fpvA-fpvB-)
sensitive for the pyocin and induced type II pyoverdine synthesis in this strain, suggesting that
this mutation is not likely to be the sole explanation for the resistance of some fpvAI strains to
pyocin S2. Similarly, the fpvR (coding for an anti-sigma) sequences have amino acid
substitutions with F80S81 or F80G81 (in the case of PA14) for the resistant strains, while
sensitive strains have L80G81. The function of these substitutions remains unclear. To unravel
the resistance mechanism of these strains, we isolated four pyocin S2 sensitive transposon
mutants from PA14. They all had insertions in genes related to cell wall biosynthesis. The
exact mechanism conferring resistance to these strains still has to be determined, but a
difference in cell wall composition (in particular LPS) has to be considered.
Pyocin S1 does not seem to enter cells via a pyoverdine receptor. Through the analysis
of less sensitive transposon mutants we could demonstrate the importance of
lipopolysaccharides in pyocin susceptibility. All mutants with a decreased sensitivity were
affected in the sugar metabolism. Interestingly, one mutant (12H8) was in a UDP-glucose
dehydrogenase (PA3559) and is involved in lipid A modification, which also leads to
resistance to polymyxin. This predicts the involvement of lipopolysaccharides in the uptake of
pyocin S1.
The rapid rise and spread of multi-resistant strains, due to the extensive use of
antibiotics, has forced the search for of alternative methods to combat infections. Our results
support the potential of pyocins as anti-P. aeruginosa drug.