Pseudomonas aeruginosa is a gram-negative, biofilm-forming, aerobic rod that is commonly found throughout the environment in soil, surface water, sewage, plants, and foods. It is also an opportunistic pathogen capable of causing disease in multiple tissues including eye, skin, urinary tract, upper and lower respiratory tracts, and blood. Immunocompromised patients, including those on mechanical ventilation, are at risk for serious infections by P. aeruginosa (Sadikot et al., 2005). Hospital acquired infections are common, and patients with chronic obstructive pulmonary disease (COPD) are at risk for chronic pulmonary infection (Murphy, 2009). In patients with cystic fibrosis (CF), chronic P. aeruginosa infection causes significant inflammation and tissue damage and thus is a major cause of morbidity and mortality (Sadikot et al., 2005).

CF is a major respiratory disease that affects 70,000 people worldwide with 1,000 people being diagnosed every year. It is an autosomal recessive disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride ion channel (Folkesson et al., 2012). The loss of this channel results in defective chloride ion transport, leading to the production of a highly adhesive mucus, which inhibits bacterial clearance and promotes respiratory infections (Rubin, 2007). One study found that 97% of children with CF were colonized with P. aeruginosa by the age of 3 years; among all CF patients, over 58% are chronically infected with P. aeruginosa (Sadikot et al., 2005). P. aeruginosa infection in CF is associated with increased morbidity and mortality (Sadikot et al., 2005). Due to the widespread presence of P. aeruginosa in the environment and the high incidence of hospital acquired infections, it is important to understand the survival mechanisms responsible for its persistence within the CF lung (Sadikot et al., 2005).

P. aeruginosa strains isolated from patients with CF and COPD are often phenotypically different from those isolated from the environment, most prominent of which is their mucoid colony morphology. During colonization of the airway, P. aeruginosa undergoes mucoid conversion whereby a normally nonmucoid strain changes to a highly mucoid phenotype, which is indicative of the over production of a capsular exopolysaccharide called alginate (Govan and Deretic, 1996). Alginate is high molecular weight polymer of D-mannuronic acid and L-guluronic acid, where the D-mannuronate residues are also acetylated. Alginate is synthesized and secreted via a 12 gene cluster known as the algD operon. High levels of alginate are found in the sputum of patients with CF who are chronically infected with P. aeruginosa (Folkesson et al., 2012). Also, CF strains often lack flagellin and pilin, and produce ‘rough’ lipopolysaccharides (LPS) that do not have the long O-side chains characteristic of ‘smooth’ LPS (Sadikot et al., 2005). These adaptations promote persistence in the lung apparently by allowing P. aeruginosa to evade the host immune system. P. aeruginosa is also known for its ability to form biofilms, which are associated with the production of exopolysaccharides.

Alginate production may alter the innate immune response to the infecting organisms. Although alginate production may not be the initial event allowing for persistence or evasion of the immune response, it is likely to be a primary reason for maintaining a chronic infection by inhibiting its clearance by alveolar macrophages. It has been reported that mucoid P. aeruginosa is resistant to opsonic and non-opsonic phagocytosis, and that alginate production confers protection against IFN-γ mediated opsonic killing of a P. aeruginosa biofilm (Ruhen et al., 1980; Cabral et al., 1987; Krieg et al., 1988; Pier et al., 2001; Leid et al., 2005). Phagocytosis is an actin-dependent process that involves the use of multiple cell surface receptors to bind pathogens (Flannagan et al., 2012; Underhill and Goodridge, 2012). Subsequent internalization requires a complicated series of membrane remodeling, cytoskeletal rearrangements and intracellular signaling events, which also result in the release of inflammatory cytokines.

The mechanism responsible for alginate inhibition of phagocytosis and its effects on subsequent production of inflammatory mediators is not understood. To address how alginate interferes with phagocytosis and its potential effects on subsequent production of inflammatory mediators, we use cell-based host-pathogen interaction assays to compare a mucoid CF-adapted isolate of P. aeruginosa to its isogenic nonmucoid mutant, defective in expression of the algD operon for alginate biosynthesis. These studies identified macrophage surface receptors that are blocked from ligand binding by alginate expression. Interestingly, inhibition of receptor-ligand interactions and activation of signaling did not affect production of the chemokines MIP-2 and IL-8. Taken together these findings provide additional insight into the mechanisms by which mucoid conversion in P. aeruginosa promotes chronic pulmonary disease.

The goal of this research was to determine the mechanism by which alginate, an exopolysaccharide secreted by mucoid P. aeruginosa, inhibits their phagocytosis and destruction by macrophages as occurs in a biofilm. The mucoid phenotype is common to strains isolated from patients with CF and COPD that have chronic pulmonary disease with P. aeruginosa. The mucoid phenotype has been shown to enhance the survival of P. aeruginosa in the lung and significantly increase the morbidity and mortality of CF patients (Govan and Deretic, 1996; Sadikot et al., 2005). Mucoid P. aeruginosa is protected against non-opsonic phagocytosis in both the planktonic and biofilm modes of growth (Ruhen et al., 1980; Cabral et al., 1987; Krieg et al., 1988; Pier et al., 2001; Leid et al., 2005). Therefore, a better understanding of how alginate protects P. aeruginosa from phagocytosis could lead to the development of novel therapeutics for chronic pseudomonal infections. In this study we sought to quantitatively measure the effect of alginate production on the inhibition of non-opsonic phagocytosis of planktonic P. aeruginosa.

In initial experiments, the human monocytic cell line THP-1 was used as a model for phagocytosis. However, since alveolar macrophages are known to have characteristics that distinguish them from peripheral macrophages, we primarily used the murine alveolar macrophage cell line MH-S which is more similar to those present in the lung infection environment (Monick et al., 2004; Gwinn and Vallyathan, 2006). Nevertheless, comparable results were obtained with both macrophage cell lines. P. aeruginosa strain FRD1, a genetically manipulable mucoid CF clinical isolate (Ohman and Chakrabarty, 1981), was used to address the effect of alginate production (Alg⁺) on phagocytosis. FRD1131 is a non-mucoid isogenic algD::Tn501-33 mutant (Chitnis and Ohman, 1993), and the site of the transposon was precisely mapped here. The Alg⁺ phenotype was readily restored to FRD1131 by genetic complementation. Since the only difference between these bacterial strains was the production of alginate, we could specifically examine the effect of alginate expression on phagocytosis. Both strains had the mucA mutation, which is typical in mucoid conversion (Martin et al., 1993), so both have the complex σ²² stress-response system activated (Wood et al., 2006; Wood and Ohman, 2009). Phagocytosis was assessed using two assays that measured different aspects of phagocytosis. The total number of bacteria that were phagocytized was determined using the gentamicin protection assay where macrophages were lysed and CFUs counted. The fraction of macrophages engaged in phagocytosis was determined by using GFP-expressing bacteria and flow cytometry. Initial experiments confirmed that Alg⁺ FRD1 was indeed significantly more resistant to opsonic and non-opsonic phagocytosis than its Alg^- mutant derivative, FRD1131.

Since flagella-mediated motility plays a major role in the effectiveness of phagocytic uptake of P. aeruginosa (Amiel et al., 2010; Lovewell et al., 2011), it was important that we also consider the role of the flagellum. Alginate production and flagellum expression are inversely expressed in P. aeruginosa when mucA mutation activates σ²² (Tart et al., 2005; Tart et al., 2006). Consistent with this, both FRD1 and FRD1131, which have a mucA mutation, appeared to be non-motile under laboratory conditions. Interestingly, flagellar motility and not just the flagellum itself appears to be responsible for the higher rate of phagocytosis of P. aeruginosa (Amiel et al., 2010; Lovewell et al., 2011). Flagellar motility is also critical for internalization, but not binding to phagocytic cells (Mahenthiralingam et al., 1994; Mahenthiralingam and Speert, 1995). Interestingly, we observed a 10-fold reduction in phagocytosis when flagellum expression in FRD1 was removed by mutation. However, the same flagella mutation in mucA+ PAO1 resulted in a 100-fold reduction in the rate of phagocytosis. These results suggests that flagellar motility was at least transiently expressed by FRD strains under the conditions examined here despite the down regulation of flagellum expression that occurs when σ²² is activated due to mucA mutation and the apparent non-motility observed. In that both Alg⁺ FRD1 and Alg^- FRD1131 appear to be equivalent with respect to flagellar motility, then it was reasonable to assume their differences in phagocytic susceptibility is due to alginate and not motility. It is also worth noting that differences in the macrophages and P. aeruginosa strains utilized could affect the analysis of motility and phagocytosis. In this study we used human and murine macrophage cell lines, whereas previous studies used human peripheral blood monocyte-derived macrophages that may express different amounts of TLR5, which is a receptor for flagellum.

Previous studies suggest that exogenous alginate may protect planktonic and biofilm P. aeruginosa from phagocytic killing (Ruhen et al., 1980; Leid et al., 2005), raising the possibility that alginate activates a signaling pathway that inhibits phagocytosis. However, when cultured together, alginate produced by FRD1 was unable to inhibit the phagocytosis of Alg^- FRD1131. This implied that alginate did not induce a negative signal within the macrophages because the presence of alginate in the media would have inhibited phagocytosis if that were the case. Also, alginate added exogenously even at high levels (1 mg ml^-1) was unable to protect nonmucoid P. aeruginosa from phagocytosis. Again, this indicated that alginate was not negatively regulating macrophages or else this would have inhibited the phagocytosis of Alg^- FRD1131. This suggested that expression of alginate on the bacterial surface interfered with a receptor-ligand interaction that is necessary for promoting phagocytosis.

The initial interaction between phagocytes and bacteria involves multiple receptors and the activation of signaling cascades. Previous studies have implicated both the PI3K and MAPK pathways in phagocytosis (Kannan et al., 2008). Furthermore, CD11b (i.e., CR3 in mice) and CD14, which are implicated in uptake of P. aeruginosa, can contribute to PI3K signaling (Thieblemont et al., 1995; Medvedev et al., 1998; Heale et al., 2001; Sun et al., 2010), which is one of the multiple signaling pathways implicated in phagocytosis (Flannagan et al., 2012). Thus, to explore the mechanism underlying alginate induced resistance to phagocytosis, the effect of FRD1 and FRD1131 infection on the down-stream phosphorylation of AKT and ERK in macrophages was compared. The results showed that soon after infection, Alg^- FRD1131 induced p-AKT and p-ERK. However, by comparison, Alg⁺ FRD1 showed reduced activation and it was delayed. The addition of AKT inhibitors significantly inhibited the phagocytosis of P. aeruginosa, confirming that this pathway is indeed important for the phagocytosis of P. aeruginosa by alveolar macrophages. Overall, these findings suggest that alginate production by P. aeruginosa blocks phagocyte-bacterial interaction, which depresses PI3K signaling and leads to inhibition of phagocytosis.

The first step in phagocytosis is the binding of bacterial ligands to receptors on the macrophage surface, which tethers the bacteria to the phagocyte. Since exogenous alginate or alginate produced in a mixed culture were unable to inhibit phagocytosis, it was possible that alginate on the bacterial surface could interfere with binding to the macrophages and thus the activation of signaling pathways that are required for phagocytosis. To determine if alginate inhibits binding to macrophages, cytochalasin D was used to inhibit actin polymerization and therefore prevent phagocytosis, but the drug would not affect cell surface binding. Alg⁺ strains FRD1 and FRD1131C’ were shown to bind significantly less than the Alg^- FRD1131, suggesting that alginate production inhibits binding to macrophages. To identify receptors that may be blocked by alginate, we investigated the contribution of CD11b and CD14, which are cell surface receptors that have been reported to facilitate the phagocytosis of P. aeruginosa (Heale et al., 2001). CR3(CD11b) has been shown to bind to a number of ligands associated with P. aeruginosa including LPS, APG, and β-glucan (Brown, 2006; Zhou et al., 2013). Blocking either CD11b or CD14 with specific antibodies led to a significant decrease in phagocytosis of non-mucoid P. aeruginosa by murine macrophages, indicating that these receptors play a role in the uptake of strain FRD. However, this inhibitory effect was not significant with mucoid P. aeruginosa, suggesting that both of these receptors were being blocked by alginate and that further blockade with the antibodies was ineffective. CD11b and CD14 can activate PI3K and MEK1/2. CD14/TLR4 signaling through MyD88, IRAK1/4, and TRAF6 activate PI3K and MEK1/2 (Schepetkin and Quinn, 2006). CR3 can also lead to PI3K and MEK activation through PLC and protein kinase C (PKC) (Schepetkin and Quinn, 2006; Hajishengallis, 2010; Underhill and Goodridge, 2012). AKT and ERK phosphorylation were used to measure PI3K and MEK1/2 activation. Both ERK and AKT activation were decreased by alginate production. While PI3K activation was found to be critical for phagocytosis of P. aeruginosa, loss of MEK activation had no significant effect. However, reduced ERK activation by alginate may lead to altered inflammatory signaling despite not being necessary for phagocytosis.

Interestingly, we found no significant difference in the amount of IL-8 or MIP-2 induced by Alg⁺ and Alg^- P. aeruginosa. This could be because MIP-2 induction was found to be at least partially contact-independent, which is often the case in cytokine induction (Underhill and Goodridge, 2012). It is possible that while IL-8 and MIP-2 production are NF-κB dependent, NF-κB may be sufficiently activated despite reduced AKT signaling. It is also possible that NF-κB activity is stimulated through a PI3K-independent pathway, which could be accomplished through other scavenger and pattern recognition receptor signaling.

In summary, this study demonstrated for the first time that alginate on the bacterial surface blocks binding of the bacteria to macrophages via CD14 and CR3 causing decreased intracellular signaling and reduced phagocytosis. Interestingly, the decreased binding did not appear to inhibit chemokine production. Inhibition of phagocytosis while maintaining cytokine production may exacerbate lung inflammation and subsequent tissue destruction by recruiting more neutrophils to the lung environment without clearing the infection. These findings argue that therapies specifically designed to either interfere with alginate production or enhance phagocytosis could prove useful in the treatment of chronic lung infections by mucoid P. aeruginosa.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

WR conducted all the experiments described as part of his PhD dissertation. DL and DO were co-advisors in the project. All authors contributed to the article and approved the submitted version.