The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS

Abstract

Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.

Figure 1. PA14 is a ladS mutant.

(A) Alignment of the ladS open reading frame of PAO1 and PA14 starting at +1021 bp. The bold underlined sequence in PA14 shows the 49 bp duplication, which alters the PA14 reading frame compared to PAO1, illustrated by vertical lines. PCR primers are indicated by arrows. (B) PCR products of the genomic region indicated in (A), separated by agarose gel electrophoresis. (C) Predicted domain organization of LadS in PA14 and PAO1. Nonsense protein sequence in PA14 is shown with a dotted line.

Figure 2. Biofilm formation.

Biofilm formation of PA14 and ladS _R in M9 medium. Graph: Biofilm formation in 24-well polystyrene plates at 37°C in shaking conditions. Inset: Crystal violet stained biofilms in glass tubes after 8 h incubation at 30°C. Data is representative of at least two independent experiments.

Figure 3. qRT-PCR analysis of target genes in ladS _R compared to PA14.

Positive values indicate that mRNA levels are higher in ladS _R. All mRNA levels have been normalized to 16S rRNA.

Figure 4. Immunoblots of PA14 and ladS _R cell lysates using antibodies directed against structural components of the T3SS and T6SS (PcrV and Hcp1 respectively).

The calcium chelator EGTA was added to the growth medium to induce the T3SS as indicated.

Figure 5. Lactate dehydrogenase (LDH) release of HeLa cells infected with the indicated bacterial strains for 1 hour.

The LDH release of PA14 has been set to 100%. Data are representative of three independent experiments. The difference between PA14 and ladS _R is significant with p = 0.018.