Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection

Abstract

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.

Figure 1. Implementation of the Pos-STM screening.

A) First round of screening of a P. aeruginosa PAO1293 STM mini-Tn5 library, consisting of 6912 mutants, arranged into 96 arrays of 72 mutants each . The 72 P. aeruginosa mutants of each array were pooled, processed for agar beads preparation and injected into C57BL/6NCrlBR mice (input pool). After 14 days from challenge, mice were sacrificed, and bacteria recovered from lungs by plating homogenates. Bacterial cfu per lung and the percentage of infected mice were evaluated. B) In the first round of screening, clones that infected at least one of the two mice were identified as positive and selected for the following rounds of screening. For the second screening, we constructed 21 positive input pools with variable numbers of STM-mutants per pool, ranging from 72 to 9. Similarly, 15 positive input pools with variable numbers of STM-mutants per pool, ranging from 28 to 3 were constructed for the third screening. Mice were infected in second and third rounds of screening increasing to five the number of mice and selecting for more than 50% chronic infection. A total of 34 clones which passed the third round were tested singularly in the fourth round increasing the stringency for positive scoring and using 10–15 mice per group. STM mutants were identified by multiplex PCR. 16 mutants which increased significantly chronic infection (>70%) when compared to PAO1293 (12.5%) were considered positive Pos-STM mutants and analyzed further.

Figure 2. Virulence of P. aeruginosa Pos-STM mutants in a murine model of airways infection.

C57Bl/6NCrlBR mice were infected with Pos-STM P. aeruginosa mutants embedded in agar beads. Mortality induced by bacteremia (red) and survival (grey) were evaluated on challenged mice. Clearance (white) and capacity to establish chronic airways infection (green) after 14 days from challenge were determined on surviving mice. The data show the percentage of mice infected with P. aeruginosa pooling together two to three independent experiments (PAO1293 wt: n = 10; STM mutants n = 10–15). Statistical significance by Chi square is indicated: *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Murine lung histology and localization of P. aeruginosa Pos-STM mutants after 14 days.

Mice were infected with 2×10⁶ cfu/lung of P. aeruginosa Pos-STM strains embedded in agar beads. In this panel, PA5053- hslV (A and B), PA2998-nqrB (D and H), PA4554-pilY1 (E and I) and PA0499 (F and J) are taken as representative of the whole set of 16 Pos-STM mutants. Control mice were infected with PAO1293 wt (C, G and K). The lungs were stained with H&E (A–G) or with specific antibody against P. aeruginosa strains (red) (H–K). Counterstaining was performed with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (blue) (H–K). Bronchi and pulmonary parenchyma are characterized by chronic lesion (*) infiltrated by macrophages, lymphocytes and neutrophils. Severity of lesions and lung involvement is heterogeneous in different lobes of the same mice (A and B). Bronchi contain massive granulocyte infiltration of bacteria and agar beads (arrow) (D and H) or bronchial wall is disrupted and the bronchial lumen is filled by foamy macrophages and bacteria presumably internalized by inflammatory cells (F and J). Bacterial macrocolonies are visible in the pulmonary parenchyma outside the beads (E and I). PAO1293 wt infected mice resolved the infection and inflammation (C, G and K). A–C: 2.5×; D–K: 20×; insert I–J: 63×. Confocal microscopy (H–K). Bars, 100 µm.

Figure 4. Stimulation of A549 with P. aeruginosa Pos-STM mutants.

A) Fold of invasion relative to PAO1293 after 2 h of stimulation with Pos-STM mutants. B) IL-8 secretion was quantified by ELISA after stimulation with Pos-STM mutants for 2 h. Measurements were performed in triplicate. *p<0.05, **p<0.01, ***p<0.001 in the Student's t-test.