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. 2015 Mar 17;83(4):1339–1346. doi: 10.1128/IAI.02874-14

Type IV Pilus Glycosylation Mediates Resistance of Pseudomonas aeruginosa to Opsonic Activities of the Pulmonary Surfactant Protein A

Rommel M Tan 1, Zhizhou Kuang 1, Yonghua Hao 1, Francis Lee 1, Timothy Lee 1, Ryan J Lee 1, Gee W Lau 1,✉
Editor: B A McCormick
PMCID: PMC4363409  PMID: 25605768

Abstract

Pseudomonas aeruginosa is a major bacterial pathogen commonly associated with chronic lung infections in cystic fibrosis (CF). Previously, we have demonstrated that the type IV pilus (Tfp) of P. aeruginosa mediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A). Interestingly, P. aeruginosa strains with group I pilins are O-glycosylated through the TfpO glycosyltransferase with a single subunit of O-antigen (O-ag). Importantly, TfpO-mediated O-glycosylation is important for virulence in mouse lungs, exemplified by more frequent lung infection in CF with TfpO-expressing P. aeruginosa strains. However, the mechanism underlying the importance of Tfp glycosylation in P. aeruginosa pathogenesis is not fully understood. Here, we demonstrated one mechanism of increased fitness mediated by O-glycosylation of group 1 pilins on Tfp in the P. aeruginosa clinical isolate 1244. Using an acute pneumonia model in SP-A+/+ versus SP-A−/− mice, the O-glycosylation-deficient ΔtfpO mutant was found to be attenuated in lung infection. Both 1244 and ΔtfpO strains showed equal levels of susceptibility to SP-A-mediated membrane permeability. In contrast, the ΔtfpO mutant was more susceptible to opsonization by SP-A and by other pulmonary and circulating opsonins, SP-D and mannose binding lectin 2, respectively. Importantly, the increased susceptibility to phagocytosis was abrogated in the absence of opsonins. These results indicate that O-glycosylation of Tfp with O-ag specifically confers resistance to opsonization during host-mediated phagocytosis.

INTRODUCTION

Pseudomonas aeruginosa is one of the most common causes of nosocomial infections in humans, especially chronic infection in patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (1–3). It is also a primary cause of death and sepsis in immunocompromised individuals (4, 5). Antibiotic-resistant P. aeruginosa is a serious clinical problem that can lead to denial for lung transplant, infection, and death (6). Thus, there is an urgent need to explore alternative strategies, including the possibility of augmenting the expression of pulmonary innate immunity proteins to manage P. aeruginosa-mediated infections.

Bacterial type IV pilus (Tfp) is important for multiple cellular functions, including surface motility, microcolony and biofilm formation, host-cell adhesion, cell signaling, DNA uptake by natural transformation, and phage attachment (7). Tfp is also an important virulence factor of P. aeruginosa (8). P. aeruginosa can be separated into five groups based on the presence or absence of varying downstream accessory genes flanking the pilin gene, pilA (9, 10). The PilA pilins of these five P. aeruginosa groups have different amino acid sequences, lengths, and presence of posttranslational modifications (9–13). The downstream accessory genes function in either pilin posttranslational glycosylation (group I and group IV) or modulation of pilus assembly (group III and group V) (9, 10, 14, 15).

Group I alleles (e.g., strain 1244), which contain a glycosyltransferase (PilO/TfpO) that glycosylates each pilin of Tfp with one subunit of O-antigen (O-ag), are frequently associated with CF and environmental isolates (10). The O-ag repeating subunit was noted to be the trisaccharide pseudaminic acid (5NβOHC47NFmPse)-(2→4)-xylose-(1→3)-N-acetylfucosamine (FucNAc), bound to serine residue 148 at the carboxyl terminal of the pilin (16, 17). The O-ag glycan decorating PilA is the product of the same O-ag biosynthetic pathway for the lipopolysaccharide (LPS) O-ag of the same strain (18). Group II alleles, which do not contain an accessory flanking gene, include common laboratory strains PAO1 and PAK (10). Group III alleles contain the accessory gene tfpY and include the human clinical isolate PA14 (10). PA14 was shown to produce a lower 50% lethal dose (LD50) and a higher mortality rate than PAO1 (19). This difference in virulence was partially attributed to ybtQ, a gene present in PA14 but not PAO1 (19). Also, PA14 contains the pathogenicity islands PAPI-1 and PAPI-2, which have been shown to contribute to virulence (20, 21). Group IV alleles contain two accessory genes, tfpW and tfpX (10). The PilA pilins of group IV alleles are glycosylated with a homo-oligomer of α-1,5-linked d-arabinofuranose, which is similar to the lipoarabinomannan polymer found in the cell wall of Mycobacterium spp. (12). Group V alleles contain the accessory TfpZ gene (10). TfpY of group III alleles and TfpZ of group V alleles have been shown to be important for the surface expression of PilA (14).

Most recently, we have demonstrated that Tfp of P. aeruginosa mediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A), an important innate immunity protein that induces opsonization and membrane permeability in microbes (22). Despite reports showing frequent association of P. aeruginosa strains in the TfpO-expressing group I alleles with CF and the importance of TfpO-mediated glycosylation as a virulence factor in mouse lung infection (10, 11), the molecular basis behind the importance of Tfp glycosylation in P. aeruginosa pathogenesis, especially in the context of alveolar epithelium, is not fully understood. In the present study, we examined the mechanisms by which Tfp glycosylation with O-ag contributes to lung infection.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The clinical P. aeruginosa strain 1244, the TfpO-deficient mutant strain 1244G7 (ΔtfpO strain), and the genetically complemented strain (ΔtfpO-comp strain) were generously provided by Peter Castric (Duquesne University) (11) (Table 1). Laboratory wild-type strains PAO1 and PAK and their ΔrfbC mutants (Table 1) were generous gifts from Reuben Ramphal (University of Florida). The ΔrfbC mutant was constructed in strain 1244 by gene replacement with a gentamicin resistance gene cassette as described previously (23) (Table 1). Strain 1244 was grown in plain Luria-Bertani (LB) broth (Fisher Scientific); the ΔtfpO and ΔrfbC strains were grown in LB broth containing 75 μg of gentamicin (Life Technologies)/ml, and the ΔtfpO-comp strain was grown in LB medium containing 200 μg of carbenicillin (Lab Scientific)/ml at 37°C overnight. The strains were then stored at −80°C in 25% glycerol (Sigma-Aldrich). Before each experiment, bacteria were streaked from frozen stock onto LB agar with or without antibiotic for 18 h at 37°C. One colony from this streak was then cultured in 5 ml of LB broth to stationary phase to an optical density at 600 nm of ∼3.0 by using a Genesys 10 UV spectrophotometer (Thermo Scientific).

TABLE 1.

Bacterial strains and plasmids used in this study

P. aeruginosa strain Description Source or reference
1244 Wild type 11
1244G7 (ΔtfpO) Nonpolar gene replacement of the tfpO gene in the wild-type strain 1244 with a gentamicin cassette 11
1244G7-comp (ΔtfpO-comp) ΔtfpO mutant complemented with a copy of wild-type gene in trans 11
1244ΔfgtA Nonpolar replacement of the fgtA gene in the wild-type strain 1244 with a gentamicin cassette This study
PAO1 Wild type 23, 44
PAO1ΔfgtA Nonpolar replacement of the fgtA gene in the wild-type strain PAO1 with a gentamicin cassette 23, 44
PAK Wild type 23, 44
PAKΔfgtA Nonpolar replacement of the fgtA gene in the wild-type strain PAK with a gentamicin cassette 23, 44
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Mouse infection assays.

Wild-type C3H/HeN (SP-A+/+) mice were purchased from Harlan Laboratories (Indianapolis, IN). Isogenic SP-A−/− mice were gifts from Francis McCormack (University of Cincinnati College of Medicine). Animal studies were carried out in strict accordance with the protocol approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana-Champaign. SP-A+/+ and SP-A−/− mice (groups of 10) were given a single intranasal inoculation of 3 × 106 CFU of the parental clinical strain 1244 or the ΔtfpO mutant. After 18 h, mouse lungs were harvested for histology (n = 3) or bacterial enumeration (n = 7) as previously described (22, 24). For mortality studies, animals (n = 10) were monitored for up to 48 h. Moribund animals that displayed rough hair coat, hunched posture, distended abdomen, lethargy, or inability to eat or drink were euthanized and counted as dead.

In vivo phagocytosis assays.

The phagocytosis rates between different P. aeruginosa strains were compared using a modified gentamicin exclusion assay as previously described (22, 24). Briefly, C3H/HeN and C3H SP-A−/− mice (n = 3) were given a single intranasal inoculation of 107 CFU of 1244 or ΔtfpO strain. After 90 min, mouse lungs were lavaged to collect the alveolar macrophages and neutrophils. The white blood cells were then incubated in phosphate-buffered saline (PBS) with 100 μg of piperacillin (Sigma-Aldrich)/ml to kill the remaining extracellular bacteria (25). Piperacillin was used instead of gentamicin because the ΔtfpO mutant contains a gentamicin resistance cassette (11). The macrophages were lysed with 1% Triton X-100 solution (Fisher Scientific) and serially diluted for P. aeruginosa CFU enumeration. The ratio of CFU counts between C3H/HeN and C3H SP-A−/− mice was computed for the fold increase of phagocytosis mediated by SP-A. The changes in phagocytosis were then normalized to 1244, which was set to the baseline value of 1.

Purification of hSP-A.

Discarded lung washings from anonymous alveolar proteinosis patients were generously provided by Francis McCormack (University of Cincinnati College of Medicine). Pulmonary alveolar proteinosis is a rare lung disease with abnormally high accumulation of surfactant proteins that occurs within the alveoli (26). Human SP-A (hSP-A) was purified as previously described (27). Briefly, raw lung washings, equilibrated with 1 mM CaCl2 (Sigma-Aldrich), were passed through a Sepharose 6B column (GE Healthcare) laden with mannose (Sigma-Aldrich). The captured SP-A was then eluted using elution buffer (2 mM EDTA, 5 mM Tris-HCl [pH 7.4]). The eluted fractions were dialyzed using the dialysis buffer (150 mM NaCl, 5 mM Tris-HCl [pH 7.4]) to remove the EDTA. The purity of hSP-A preparations was confirmed by Coomassie blue analysis.

Murine macrophage culture and in vitro phagocytosis assays.

Murine RAW 264.7 macrophages (ATCC TIB-71) were maintained in Dulbecco modified Eagle medium (DMEM; Corning) supplemented with 10% fetal bovine serum (Phoenix Research Products) at 5% CO2 and 37°C (28). The phagocytosis rates between different P. aeruginosa strains were compared using the modified gentamicin exclusion assay. Briefly, 106 RAW 264.7 macrophages/ml were plated in six-well cell culture plates overnight at 37°C and 5% CO2. For dose-dependent SP-A-mediated phagocytosis, P. aeruginosa strains were preincubated with 12.5 or 25 μg of hSP-A/ml in the presence of 2 mM CaCl2 for 1 h in a rotating incubator at 37°C. For time-dependent SP-A-mediated phagocytosis, P. aeruginosa strains were preincubated with 25 μg of hSP-A/ml in the presence of 2 mM CaCl2 for 1, 6, or 12 h in a rotating incubator at 37°C. The resulting mixture was then incubated with the RAW 264.7 cells at a ratio of 10 bacteria to 1 macrophage for 90 min. The macrophages were then washed and incubated with DMEM with 100 μg of piperacillin/ml to kill the remaining extracellular bacteria. The macrophages were lysed with 1% Triton X-100 solution, and internalized bacteria were serially diluted for enumeration. The ratio of CFU between treated and untreated bacteria was computed for the fold increase of phagocytosis mediated by hSP-A. The changes in phagocytosis were then normalized to strain 1244, which was set to the baseline value of 1.

Opsonic phagocytosis assays with pulmonary opsonin SP-D (Novoprotein, catalog no. C541) and circulatory opsonin mannose-binding lectin 2 (MBL-2; Novoprotein, catalog no. C488) were performed by preincubating P. aeruginosa strains with the opsonins in a rotating incubator at 37°C for 1 h before exposure to RAW 264.7 macrophages. Nonopsonic phagocytosis studies were performed with the same volume of sterile PBS as a substitute for opsonins.

Membrane permeabilization assays.

Membrane permeabilization effects of hSP-A on P. aeruginosa were observed using the thiol-specific fluorophore ThioGlo (Calbiochem), as previously described (28, 29). Stationary-phase P. aeruginosa bacteria were washed and incubated with 50 μg of hSP-A/ml for 15 min at 37°C. For the ThioGlo assay, bacterium/SP-A mixture was sedimented, and the bacterium-free supernatant was incubated with 10 μM ThioGlo reagent. Fluorescence was measured using an excitation wavelength of 405 nm and an emission wavelength of 535 nm. The fluorescence measurement was read using a SpectraMax Gemini EM spectrophotometer (Molecular Devices).

Statistical analysis.

Quantitative data were expressed as the means ± the standard errors. Statistical significance comparisons for samples with equal variances were determined by using a parametric Student t test for two unpaired samples. To compare the means of groups of three or more, data were analyzed for statistical significance by the one-way analysis of variance (ANOVA), followed by Tukey's tests for comparison between the means. Comparison of mouse mortality was performed by using the Fisher exact test. A significant difference was considered to be P < 0.05.

RESULTS

Glycosylation of Tfp with O-ag is important for resistance to SP-A-mediated lung clearance.

To determine the contribution of O-ag on Tfp in resistance to SP-A-mediated clearance, we compared the lung infection by the wild-type P. aeruginosa 1244 and the isogenic glycosyltransferase-deficient ΔtfpO mutant in a mouse model of acute pneumonia. At 18 h after intranasal inoculation with 1244, SP-A+/+ mice showed no mortality, while SP-A−/− mice showed a 7/10 mortality. In contrast, the ΔtfpO mutant caused no mortality in SP-A+/+ mice but only a 1/10 mortality in the SP-A−/− mice (Fig. 1A). The number of viable ΔtfpO bacteria in SP-A−/− mice was significantly higher (2.39 logs) than in SP-A+/+ mice. In contrast, the burden of 1244 bacteria was only 1 log higher in the SP-A−/− mice than in the SP-A+/+ mice (Fig. 1B). The 1244 and ΔtfpO mutant strains showed comparable growth rates, indicating that attenuation in the lungs of mice infected with the latter was not due to potential growth defects caused by the gene deletion (Fig. 1C). These data suggest that the presence of O-glycosylation of Tfp with O-ag allows for increased resistance to SP-A-mediated lung clearance.

FIG 1.

FIG 1

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O-glycosylation of Tfp allows for increased resistance to SP-A during acute pneumonia. (A) Mortality rate of SP-A+/+ and SP-A−/− mice (n = 10) infected with either 1244 or ΔtfpO. *, P < 0.01 when comparing the mortality rate of SP-A+/+ versus SP-A−/− mice infected by strain 1244 and when comparing the mortality rate of SP-A−/− mice infected by 1244 versus the ΔtfpO mutant. Statistical analyses were performed by using the Fisher exact test. (B) Respiratory tract infections with wild-type strain 1244 versus the ΔtfpO mutant were performed by intranasal inoculation of anesthetized SP-A+/+ or SP-A−/− mice. Mouse lungs were harvested 18 h postinfection for bacterial enumeration. The data are the mean CFU ± the standard errors (SE; n = 7 per group). Statistical significance comparisons among various groups were determined by using one-way ANOVA (P < 0.01). *, P < 0.01 when comparing the bacterial loads between the 1244 strain versus the ΔtfpO mutant infecting SP-A+/+ mice, between the 1244 strain versus the ΔtfpO mutant infecting SP-A−/− mice versus SP-A+/+ mice, and between SP-A+/+ mice versus SP-A−/− mice infected by the ΔtfpO mutant determined using Tukey's test. (C) The growth kinetics of 1244 and ΔtfpO bacteria were determined by measuring optical density at 600 nm. The experiments were performed three times independently in triplicates. The representative growth curve from one of three independent experiments is shown.

The aforementioned virulence studies were further supported by histopathological analysis of infected lungs (Fig. 2). The parental clinical strain 1244 caused moderate bronchopneumonia (Fig. 2A), whereas the ΔtfpO mutant caused mild bronchopneumonia (Fig. 2B) in SP-A+/+ mice. In contrast, 1244 caused severe bronchopneumonia (Fig. 2C), whereas the ΔtfpO mutant caused only moderate bronchopneumonia in the lungs of SP-A−/− mice (Fig. 2D). These results indicate that O-glycosylation of Tfp increases the resistance of P. aeruginosa to SP-A-mediated lung clearance.

FIG 2.

FIG 2

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Histopathology of P. aeruginosa-infected lungs. SP-A+/+ and SP-A−/− mice were infected with the 1244 strain or the ΔtfpO mutant as described in Fig. 1. Representative hematoxylin-eosin-stained lung sections from SP-A+/+ and SP-A−/− mice (n = 3) at 18 h after intranasal instillation of 1244 (A and C) and ΔtfpO (B and D) bacteria.

O-glycosylation of Tfp does not alter resistance of P. aeruginosa to SP-A-mediated membrane permeabilization.

As discussed above, SP-A mediates its antimicrobial effects by enhancing microbial clearance through membrane permeabilization and opsonization (22, 24, 28–32). To further examine whether O-glycosylation of P. aeruginosa Tfp with O-ag increases the resistance to SP-A-mediated membrane permeability, we measured the amounts of leaked thiol containing proteins from 1244 versus ΔtfpO mutant after exposure to hSP-A. The three 1244, ΔtfpO, and ΔtfpO-comp strains were equally susceptible to SP-A-mediated permeability (Fig. 3). These results indicate that O-glycosylation of Tfp does not modulate susceptibility or resistance to membrane permeabilization function of SP-A. Thus, SP-A-mediated membrane permeabilization is unlikely to contribute to preferential clearance of the ΔtfpO mutant strain from SP-A+/+ lungs.

FIG 3.

FIG 3

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O-glycosylation of Tfp does not confer increased resistance to SP-A-mediated membrane permeabilization. An in vitro ThioGlo assay was performed. 1244, ΔtfpO, and 1244G7-comp strains were preincubated with 50 μg of hSP-A/ml for 15 min. The bacterium-free supernatants were then mixed with ThioGlo. The absorbance was measured at an excitation wavelength of 405 nm and an emission wavelength of 535 nm. *, P < 0.05 when comparing the relative fluorescence units of SP-A-treated bacteria versus untreated bacteria using one-way ANOVA.

O-glycosylation of Tfp increases the resistance of P. aeruginosa to SP-A-mediated phagocytosis.

Next, we examined whether the ΔtfpO bacteria were more susceptible to SP-A-mediated opsonization. In the presence of 12.5 and 25 μg of SP-A/ml, ΔtfpO bacteria were phagocytosed 2.5- to 3.4-fold more efficiently than the parental strain 1244 (Fig. 4A). Also, we examined the phagocytosis of ΔtfpO bacteria in a time-dependent manner. We found that ΔtfpO bacteria were more susceptible to SP-A-mediated opsonization than 1244 at 1 h after exposure to RAW 264.7 macrophages (Fig. 4B). Prolonged exposure abolished the difference of phagocytic efficiency between 1244 and ΔtfpO strains, most probably due to the production of exoproteases that degraded SP-A, as we had previously reported (28, 32). These observations were confirmed by in vivo phagocytosis assays, which showed that ΔtfpO bacteria were 4.5-fold more susceptible to SP-A-mediated phagocytosis than was strain 1244 (Fig. 4C). The in vivo phagocytosis assay measured the total phagocytosis activity involving both alveolar macrophages and neutrophils. Collectively, these results indicate that O-glycosylation of P. aeruginosa Tfp promotes resistance to opsonization mediated by host opsonins, including the pulmonary SP-A.

FIG 4.

FIG 4

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The ΔtfpO mutant is more susceptible to SP-A-mediated opsonization. RAW 264.7 macrophages were infected with either 1244 or the ΔtfpO mutant in the presence or absence of hSP-A. The ratio of ingested bacteria between those exposed to hSP-A and those unexposed was expressed as the fold increase in phagocytosis. The changes in phagocytosis were then normalized to 1244, which was set to the baseline value of 1. (A) Phagocytosis of the 1244 and ΔtfpO strains in the presence of different concentrations of hSP-A. Bacteria were preincubated with the indicated concentrations of hSP-A for 1 h prior to phagocytosis. (B) Time-dependent phagocytosis of 1244 versus the ΔtfpO mutant in the presence of 25 μg of hSP-A/ml. (C) In vivo phagocytosis of 1244 versus the ΔtfpO mutant. The fold changes in phagocytosis were determined in SP-A+/+ and SP-A−/− mice (n = 3) and normalized against the phagocytosis in SP-A−/− mice. The changes in phagocytosis were then normalized to strain 1244, which was set to the baseline value of 1. All phagocytosis experiments were independently performed three times. The means ± the standard errors from a representative experiment are shown. *, P < 0.05 when comparing the numbers of phagocytosed ΔtfpO mutant versus 1244 by using one-way ANOVA (A and B) and a parametric Student t test (C).

O-glycosylation of Tfp specifically confers resistance to opsonization by both pulmonary and circulatory opsonins.

We postulated that O-glycosylation of Tfp with O-ag might be specifically required for resistance of P. aeruginosa to opsonization mediated by pulmonary and/or circulatory opsonins (30, 31, 33–36). To test this hypothesis, we examined the phagocytosis of 1244 versus ΔtfpO bacteria in the absence of SP-A. In contrast to data presented in Fig. 4, the uptake of 1244 bacteria was indistinguishable from that of ΔtfpO bacteria, suggesting that O-glycosylation of Tfp was specifically required for conferring resistance to opsonization by SP-A (Fig. 5A).

FIG 5.

FIG 5

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O-glycosylation of TFP with O-ag confers resistance to pulmonary and circulatory opsonins. (A) Phagocytosis of 1244 and the ΔtfpO mutant in the absence of opsonin. The changes in phagocytosis were normalized to strain 1244, which was set to the baseline value of 1. All phagocytosis experiments were independently performed three times in triplicates. The means ± the standard errors from a representative experiment are shown. (B and C) Phagocytosis of strain 1244 and the ΔtfpO mutant in the presence or absence of hSP-D and MBL-2. The ratio of ingested bacteria between animals exposed to hSP-D or MBL-2 versus those unexposed is expressed as the fold increase in phagocytosis. The changes in phagocytosis were then normalized to strain 1244, which was set to the baseline value of 1. All phagocytosis experiments were independently performed three times in triplicates. The means ± the standard errors from a representative experiment are shown. *, P < 0.05 when comparing the number of the phagocytosed ΔtfpO mutant against strain 1244 by using a parametric Student t test.

To confirm the findings in Fig. 4 and 5A, we examined the susceptibility of ΔtfpO bacteria to other opsonins. Significantly, when the 1244 and ΔtfpO strains were preopsonized by pulmonary surfactant protein D (SP-D) and the circulatory opsonin MBL-2, the phagocytosis of the ΔtfpO mutant by RAW 264.6 macrophages increased by 55 and 49%, respectively (Fig. 5B and C). Collectively, these results indicate that O-ag glycosylation of Tfp specifically confers resistance to opsonization by both pulmonary and circulatory opsonins.

Glycosylation on flagella of P. aeruginosa is not important for resistance to SP-A-mediated opsonization.

We have previously shown that flagellum, another prominent appendage of P. aeruginosa, is important for resistance to SP-A-mediated opsonization and membrane permeabilization (32, 37). Similar to Tfp, the flagella of P. aeruginosa can be classified into two groups (a-type and b-type) based on the posttranslational O-glycosylation of flagellins with deoxyhexose (23, 38–44). P. aeruginosa strain PAK harbors the a-type O-glycosylated flagellins, whereas strains PAO1 and 1244 have the b-type O-glycosylated flagellins (41, 44). Originally named rfbC (23), the gene encoding the flagellar glycosyltransferase was later renamed fgtA (PA1091) (44). Flagellin glycosylation has been reported to be important for virulence in a burn infection (42). In addition, flagellar glycosylation plays a role in colonization of the gut by Campylobacter jejuni 81-176 (45). Furthermore, glycosylation of flagellum in the plant-pathogenic Pseudomonas syringae pv. glycinea has been reported to determine the recognition of virulence by host plants, resulting in the hypersensitivity reaction to flagellin (46). Given our data showing the importance of Tfp glycosylation against opsonization, we examined the role of flagellin glycosylation in the resistance to opsonization by SP-A. Interestingly, no significant differences were noted in SP-A-mediated phagocytosis when comparing 1244, PAO1, and PAK against their ΔfgtA mutants deficient in flagellin glycosylation (Fig. 6).

FIG 6.

FIG 6

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Glycosylation of flagella does not modulate resistance to SP-A-mediated opsonization. (A to C) RAW 264.7 macrophages were infected with 1244, PAO1, and PAK and their respective glycosylation-deficient ΔrfbC mutants in the presence or absence of hSP-A. The ratio of ingested bacteria between those exposed to hSP-A and those unexposed is expressed as the fold increase in phagocytosis. The changes in phagocytosis were then normalized to parental wild-type strains 1244, PAO1, and PAK, respectively, which were set to a baseline value of 1. All phagocytosis experiments were independently performed three times in triplicates. The means ± the standard errors from a representative experiment are shown. P = 0.56, 0.56, and 0.67 when comparing the number of phagocytosed 1244, PAO1, and PAK organisms to their respective ΔtfpO mutants by using a parametric Student t test.

DISCUSSION

The pilin subunits of the group 1 P. aeruginosa strains, including strain 1244, are glycosylated with O-ag containing pseudaminic acid by the TfpO glycosyltransferase. TfpO of group I P. aeruginosa strains has been shown to be important for the overall fitness of 1244 in the host. In a mixed infection within mouse lungs, the ΔtfpO mutant is less competitive against its parental strain 1244 (11). However, the mechanism of attenuation is not clear. We proposed that one of the mechanisms of higher fitness in 1244 was due to the increased resistance to SP-A-mediated effects, afforded by the O-glycosylation of O-ag subunits to each pilin monomer on Tfp. Using a mouse model of acute pneumonia infection, we showed that 1244 caused mortality when inoculated into SP-A−/− mice but no mortality when inoculated in SP-A+/+ mice. This demonstrates the importance of SP-A in innate immune resistance against 1244. Importantly, the cognate glycosyltransferase ΔtfpO mutant was attenuated during the infection of SP-A+/+ mice, suggesting that O-ag contributes to the virulence of P. aeruginosa strain 1244 against wild-type mouse lung expressing SP-A. This conclusion is supported by the finding that the ΔtfpO mutant burden is significantly higher in the SP-A−/− mice than in the SP-A+/+ mice. Furthermore, the ΔtfpO mutant was able to cause moderate bronchopneumonia in the SP-A−/− mice but was only able to cause mild bronchopneumonia in the SP-A+/+ mice. These results suggest that O-glycosylation of Tfp with O-ag confers resistance to antimicrobial properties of SP-A.

To further decipher the mechanism of SP-A resistance by 1244, we examined whether the O-ag on the pilin subunits of Tfp confers resistance to SP-A-mediated opsonization or membrane permeability. The presence of O-ag on pilin subunits of Tfp does not increase the resistance to SP-A-mediated membrane permeability. Instead, we show that O-glycosylation of Tfp with O-ag confers resistance to SP-A-mediated opsonization and clearance through phagocytosis. Under both in vitro and in vivo experimental conditions, the O-glycosylation-deficient ΔtfpO mutant was more susceptible to opsonization by hSP-A. However, the increase in the phagocytosis of the ΔtfpO mutant is most prominent in vivo. This is not surprising because the ΔtfpO mutant is also susceptible to opsonization by additional pulmonary and circulatory opsonins present within the alveolar surfactant layer, including SP-D and MBL-2.

Previous studies in other microbes have implicated the importance of O-ag for resistance to phagocytosis, including Vibrio anguillarum (47), Burkholderia cenocepacia (48), Salmonella enterica (49), Escherichia coli (50, 51), Haemophilus ducreyi (52), and Neisseria gonorrhoeae (53). In E. coli, S. enterica, H. ducreyi, and N. gonorrhoeae, it has been noted that O-ag confers resistance to phagocytosis by masking the N-acetylglucosamine residues of LPS core polysaccharide, which is the ligand for the dendritic cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN)/CD209 (51, 52). However, the role of Tfp glycosylation with O-ag has not been studied with regard to resistance to SP-A or other opsonins. The results of the present study indicate that O-glycosylation of Tfp confers not only resistance to phagocytosis by pulmonary opsonins SP-A and SP-D but also resistance to circulatory opsonin MBL-2, which presents in lesser abundance in the lung.

Macrophage mannose receptor, a pattern recognition receptor for various microorganisms, has been shown to be important for the phagocytosis of microbes (53). In V. anguillarum, it is speculated that O-ag masks putative mannose residues that interact with the mannose receptors of skin epithelial cells (47). SP-A has been shown to upregulate the expression of mannose receptor (54). Upregulation of mannose receptor by SP-A and SP-D is important in the phagocytosis of Mycobacterium avium by monocyte-derived macrophages (55). Our preliminary analyses suggest that SP-A-upregulated mannose receptor plays a minimal role in enhancing the phagocytosis of the ΔtfpO mutant. This is because mannose at concentrations ≥1 mM or ∼25 times higher than physiological levels (56) only minimally attenuates phagocytosis of ΔtfpO bacteria in the RAW 264.7 murine macrophages (R. M. Tan and G. W. Lau, unpublished data). However, we cannot rule out the possibility that this discrepancy may be due to different macrophages used in the studies. We are currently examining the interactions between Tfp glycosylation, SP-A, and mannose receptor using monocyte-derived macrophages.

In summary, we have shown that the glycosylation of O-ag subunit to Tfp enhances the virulence of P. aeruginosa during acute pneumonia infection. The increase in virulence is associated with the resistance to both pulmonary opsonins SP-A and SP-D, as well as the circulatory opsonin MBL-2, which is found at low concentrations in the lung. Given that antibiotic-resistant P. aeruginosa is a serious clinical issue, there is an added urgency to explore the use of novel, non-antibiotic-based antibacterial peptides to combat life-threatening infections. Unraveling the mechanisms governing resistance or susceptibility to SP-A may potentially lead to new treatment strategies for life-threatening lung infections. Future efforts will investigate the feasibility of using drugs to disrupt Tfp glycosylation, which may augment the clearance of P. aeruginosa in an opsonin-dependent manner, without increasing the burden of antibiotic resistance.

ACKNOWLEDGMENTS

We thank Jeff Whitsett and Tom Korfhagen (Cincinnati Children Hospital) for the gift of the Swiss Black SP-A−/− mice and Frank McCormack (University of Cincinnati College of Medicine) for the C3H SP-A−/− mice and alveolar proteinosis lavage. Also, we want to acknowledge the generosity of Peter Castric (Duquesne University) for the provision of the P. aeruginosa 1244, ΔtfpO, and 1244G7-comp strains and Reuben Ramphal (University of Florida) for the provision of P. aeruginosa PAO1, PAK, and their ΔrfbC mutant strains. Finally, we thank Shawn Choe for critical editing of the manuscript and Jingjun Lin for help with statistical analysis.

This study was supported by the National Institutes of Health (HL090699) and American Lung Association DeSouza Research Award (DS-192835-N) grants to G.W.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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