Comparative Signature-Tagged Mutagenesis Identifies Pseudomonas Factors Conferring Resistance to the Pulmonary Collectin SP-A

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A(+/+)) and SP-A-deficient (SP-A(−/−)) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A

The <i>pch</i> and <i>ptsP</i> Mutants Are Preferentially Permeabilized and Killed by SP-A

<div><p>(A) Fluorescence of a cleavage-activated phosphatase substrate ELF97 was examined in the presence or absence of 50 μg hSP-A for a period of 60–90 min. Human SP-A (hSP-A) preferentially permeabilized LPS mutant <i>wbpL,</i> but not the parental wild-type PA01. The <i>wbpL</i> strain was used as positive control for membrane permeability. The difference in membrane permeabilization between <i>wbpL</i> mutant and PA01 was significant from the 28^th min onward. *<i>p</i> < 0.05.</p><p>(B) A mutant strain <i>STMG2A7</i> that is virulent in both SP-A^+/+ and SP-A^−/− mice was as resistant to SP-A-mediated membrane permeabilization as the parental wild-type PA01.</p><p>(C–D) Membrane permeability of the STM mutant <i>pch</i> and <i>PA06331</i>, a <i>P. aeruginosa</i> strain which is deleted in all of the structural genes required for pyochelin biosynthesis (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010031#ppat-0010031-t001" target="_blank">Table 1</a>.). The difference in membrane permeabilization between the mutants against wild-type PA01 was significant from the 35^th and 18^th min onward for mutant strains <i>pch</i> and <i>PA06331</i>, respectively. *<i>p</i> < 0.05.</p><p>(E–F) Membrane permeability of the STM mutant <i>ptsP</i> and the Δ<i>ptsP,</i> a <i>P. aeruginosa</i> strain with a nonpolar, inframe deletion of the <i>ptsP</i> gene. The difference in membrane permeabilization between the mutants against wild-type PA01 was significant from the 29^th and 28^th min onward, for mutant strains <i>ptsP</i> and Δ<i>ptsP</i>, respectively. *<i>p</i> < 0.05.</p></div

SP-A-Mediated Membrane Permeabilization Directly Kills the <i>pch</i> and <i>ptsP</i> Mutants

<div><p>(A) Live/Dead staining was performed on <i>E. coli</i> K12, PA01, <i>pch,</i> and <i>ptsP</i> cells, following 1 h membrane permeabilization with 100 μg/ml SP-A. Green-stained cells are alive whereas red-stained cells are dead.</p><p>(B) Enumeration of live or dead bacteria following a 1 h exposure to hSP-A. At least 600–1,000 bacterial cells were counted under a fluorescence microscope. The mean of two experiments is shown.</p></div

SP-A Deficient Mice Are More Susceptible to Infection by <i>P. aeruginosa</i>

<div><p>(A) SP-A^+/+ and SP-A^−/− mice (group of six) were infected intranasally with 1 × 10⁷<i>P. aeruginosa</i> PA01. Bacteria were recovered from homogenized lung tissue 16 h after inoculation. <i>p</i> < 0.01.</p><p>(B) Representative lung histology sections (stained with hematoxylin and eosin) from SP-A^+/+ and SP-A^−/− mice 16 h post intranasal instillation of PA01. Original magnification: 10 ×.</p></div

PCR-Based Signature-Tagged Mutagenesis

<div><p>(A) Schematic drawing of PCR-based STM. Pools of 72 uniquely tagged mutants were intranasally inoculated into SP-A^+/+ and SP-A^−/− mice; 16 h later, lungs were harvested, homogenized, and plated. Approximately 10,000 colonies were harvested from the plates for genomic DNA extraction. PCR-amplification of tags was performed on the genomic DNA to screen for the presence or absence of DNA tags of each of the 72 mutants. Mutants whose DNA tags were present in the input pool and in the SP-A^−/− pool, but absent in the SP-A^+/+ pool (see white arrows) were further screened for susceptibility to SP-A.</p><p>(B) Agarose gel of PCR-based STM, identifying the <i>pch</i> mutant (left panel). Attenuation in the first SP-A^+/+ mouse (right panel, m1) was confirmed in two additional SP-A^+/+ mice (right panel, m2 and m3).</p><p>(C) Genetic loci of <i>P. aeruginosa,</i> when mutated, conferred increased sensitivity to in vivo killing by SP-A. DNA regions flanking pUTmini-Tn<i>5</i> transposon insertions were cloned and sequenced. Similarity BLAST searches were performed against <i>P. aeruginosa</i> PA01 genomic sequence on NCBI and on <a href="http://www.pseudomonas.com" target="_blank">http://www.pseudomonas.com</a>. Black arrows indicate the approximate insertion site within the mutated ORFS.</p><p>(D) TLC analyses indicate that the <i>pch</i> STM mutant is unable to synthesize pyochelin (see arrows). Wild-type PA01 grown in LB and the <i>pch</i> mutant grown in LB supplemented with 1 mM salicylate produced green color pyochelin (see arrows). In contrast, no observable pyochelin was produced by the <i>pch</i> and <i>PA06331</i> strains. Pure pyochelin was used as control.</p><p>(E) Restriction fragment length polymorphism analysis between parental wild-type PA01 and STM mutants confirmed that mutations in <i>pch</i> and <i>ptsP</i> were caused by a single insertion.</p></div

Complementation Studies of SP-A-Sensitive Mutants <i>pch and ptsP</i>

<div><p>(A) Culturing <i>pch</i> bacteria in LB supplemented with sodium salicylate restored its resistance to SP-A mediated membrane permeabilization.</p><p>(B) In control experiments, we demonstrated that the enzymatic activity of bacterial phosphatases in <i>ptsP</i> bacterial cells was not inhibited by 1 mM sodium salicylate.</p><p>(C) Provision of the wild-type <i>ptsP</i> gene on the pUCP19 <i>in trans (pUCP-ptsP)</i> restores the ability of STM mutant <i>ptsP</i> to resist SP-A mediated membrane permeabilization. In contrast, <i>ptsP</i> mutant expressing GFP gene on the pUCP19 <i>(pstP-gfp)</i> is permeabilized to similar levels as <i>ptsP</i>.</p></div

Bacterial Opsonization by SP-A Does Not Result in Differential In Vivo Phagocytosis and Killing of STM Mutants

<div><p>(A) In vitro phagocytosis assays were performed using live, GFP-expressing PA01 and isogenic <i>pch and ptsP</i> bacteria, with alveolar macrophages isolated from SP-A^+/+ mice<i>.</i> Internalized bacteria were counted using a phase contrast fluorescence microscope. The means of three experiments are shown. Two hundred macrophages were counted for each mouse. <i>p</i> = 0.0058 (PA01 vs PA01 + SP-A), <i>p</i> = 0.075 (<i>pch</i> vs <i>pch +</i> SP-A), and <i>p</i> = 0.109 (<i>ptsP</i> vs <i>ptsP +</i> SP-A).</p><p>(B) In vitro phagocytosis of PA01, <i>pch,</i> and <i>ptsP</i> by human neutrophils isolated from healthy volunteers. Phagocytosis experiments were performed as described for (A). <i>p</i> = 0.037 (PA01 vs PA01 + SP-A), <i>p</i> = 0.577 (<i>pch</i> vs <i>pch +</i> SP-A), and <i>p</i> = 0.919 (<i>ptsP</i> vs <i>ptsP +</i> SP-A).</p><p>(C–E) SP-A did not affect uptake or killing of STM mutants. PA01 (C), but not the STM mutants <i>pch</i> (D) and <i>ptsP</i> (E) were killed in significant amounts by neutrophils, independent of SP-A. The mean of three separate experiments is shown. <i>p</i> < 0.001 when comparing PA01 versus PA01 + neutrophils, and when comparing PA01-SP-A versus PA01-SP-A + neutrophils.</p></div