Previous studies have reported isolation of P. aeruginosa RSCVs, and there is evidence that these variants play a role in disease pathogenesis. In this study, we report isolation and characterization of clonally related RSCV and wild-type isolates from a CF sputum sample. We demonstrated that our clinical RSCV displayed many of the same characteristics as laboratory biofilm RSCV isolates. One such characteristic is the contribution of the Pel and Psl EPS to the phenotype. Another similarity is the elevated c-di-GMP levels in the clinical RSCV compared to the clinical wild-type strain. Overexpression of a phosphodiesterase that degrades intracellular c-di-GMP eliminated the autoaggregation and hyper-biofilm-formation phenotypes in all the RSCV strains tested. This suggests that c-di-GMP plays a central role in modulating the transition between wild-type and RSCV phenotypes.
Our transcriptome and Biolog profiling data suggest that the clinical and laboratory-derived RSCVs that we examined are physiologically similar. The autoaggregation of RSCV cells in liquid culture affects the expression of a large subset of genes, including some genes that impact metabolism. For example, RSCV strains were characterized by increased expression of denitrification genes. The expression of these genes likely occurred in response to anaerobic pockets that developed in aggregates of cells as they consumed the available oxygen. Biolog phenotyping data suggest that autoaggregation has other effects on metabolism. RSCV cells were deficient in growth on several carbon sources (Fig. ). This also appeared to be a consequence of autoaggregation, since the wspF pelA pslBDCD
triple mutant (which exhibited no autoaggregation but had elevated c-di-GMP levels) had wild-type carbon utilization patterns. This suggests that in laboratory biofilms and CF airways, the carbon sources that RSCVs can utilize may be limited. Interestingly, we found that the type VI secretion locus HSI-I was upregulated in the clinical RSCV. Antibodies to Hcp, the secreted effector, have been used to detect Hcp in sputum from a CF patient infected for a long time; thus, type VI secretion may be another hallmark of chronic infections (28
The differential expression of a second, smaller subset of genes in RSCVs is a consequence of elevated cyclic-di-GMP levels. It appears that motility and EPS production are major functions that are transcriptionally controlled by c-di-GMP. Some of the differentially expressed genes are known to be controlled by FleQ, a cyclic-di-GMP-responsive transcriptional repressor (16
). However, some genes whose expression is impacted by elevated cyclic-di-GMP levels (e.g., PA0169 to PA0172) are not regulated by FleQ. This suggests that there may be other cyclic-di-GMP-sensitive transcriptional regulators.
Data presented here and elsewhere show that the pel
EPS biosynthetic loci are key contributors to the RSCV phenotype. Single mutations in either of these gene clusters have only a partial effect on RSCV phenotypes. Only pel psl
double mutations fully convert RSCVs to wild-type strains. It is not clear whether these EPS types are functionally redundant or if they have unique roles in RSCVs. Interestingly, in contrast to previous reports, the cupA
operon did not appear to contribute to the RSCV phenotype (26
). Insertion-deletion mutations in cupA3
appeared to suppress the RSCV phenotype; however, excision of the antibiotic cassette insertion resulted in reversion back to the RSCV phenotype. One potential explanation for this is that the antibiotic resistance cassette induces expression of the last gene in the operon, PA2133 encoding the c-di-GMP-degrading phophodiesterase. Our analysis of intracellular c-di-GMP levels supports this; the c-di-GMP levels were depleted in the insertion mutant, while excision of the antibiotic resistance cassette restored c-di-GMP levels (see Fig. S4 in the supplemental material). Meissner et al. reported upregulation of CupA fimbriae in RSCV backgrounds. We have evidence that liquid culture autoaggregation creates anaerobic pockets in the aggregates. Since microaerobic conditions induce cupA
expression, we think that reduced oxygen tension in RSCVs might have contributed to the cupA
expression in the study of Meissner et al. (2
The complementation analysis suggests that there are two classes of variants based on wspF
complementation. Even though these two classes appear to have many phenotypic similarities, we do not know whether there are any important phenotypic differences. Although the levels of intracellular c-di-GMP are high for both classes, the quantities may differ. This may affect Pel and Psl expression levels, which in turn have the potential to impact the degree of autoaggregation. RSCVs characterized by higher cyclic-di-GMP levels may have differences in key phenotypes, such as antimicrobial resistance. A challenge for the future is to identify the mutations that confer the RSCV phenotype for class B strains. Once this has been achieved, functional comparisons can be made among isogenic RSCV strains. von Gotz et al. reported that an RSCV-like strain isolated from a CF patient is highly motile and extremely cytotoxic and expresses high levels of type III secretion genes (41
). In our transcriptional analysis, we found that the RSCVs did not express elevated levels of type III secretion genes. The differences between the results illustrate the fact that not all RSCV strains are exactly alike (15
What are the selective forces in the CF lung that are responsible for selecting for RSCV formation? Are these selective forces the same as those that produce RSCVs in laboratory-cultured biofilms? Since chronic CF infections involve biofilms, some selective pressure present in both laboratory biofilms and CF biofilms might amplify the RSCV phenotype (35
). Previous studies have shown that RSCVs exhibit elevated tolerance to hydrogen peroxide (5
). Recent work by Boles and Singh also identified oxidative stress as a selective pressure for diversification in laboratory biofilms (4
). One stimulus for the generation of mucoid variants in the lung is thought to be reactive oxygen species (24
). Perhaps oxidative stress present in both lab and CF biofilms is a key selective pressure for RSCVs as well.
RSCV strains appear to grow relatively poorly on certain carbon and sulfur sources, including amino acids. Amino acids are suggested to be a major constituent of airway secretions, and they presumably support the growth of P. aeruginosa
and other colonizing species (29
). RSCVs may not be able to compete with wild-type strains (or other bacterial species) for amino acids, which may explain why they are never the predominant organisms in P. aeruginosa
strains isolated from a given patient. The heightened resistance of RSCVs to antimicrobials may allow them to successfully compete for growth substrates in microniches subjected to elevated antimicrobial stress, where wild-type P. aeruginosa
strains and other bacterial species would be impaired. Alternatively, RSCVs may be better adapted to use other growth substrates with which they do not have such a severe growth handicap, such as fatty acid and hydrophobic substrates. For example, RSCVs grew as well as wild-type strains on Tween substrates. The CF airways contain host-derived fatty acids and lipids. In addition, hydrophobic substrates are also present in older laboratory biofilms as cells begin to lyse. Consideration of all these points leads to the hypothesis that RSCVs may occupy a nutritional niche in these biofilm environments.
The CF host mounts a tremendous immune response to chronic infection (13
). This response probably exhibits temporal and spatial variability in the airways. Given their highly autoaggregative nature, RSCVs probably exist in the airways as distinct aggregates. The local host immune response surrounding these aggregates may be dampened due to reduced expression of flagella and an increase in expression of RSCV-induced immunomodulatory functions. Thus, RSCVs may represent a nidus of persistence that can contribute to reseeding of the airways after a course of antibiotic treatment. Precisely how the RSCV strains, of both clinical and biofilm origin, elicit a reduced inflammatory response is unknown. One observation from the array data is that flagellar genes are downregulated in RSCV strains. Since flagellar expression is known to cause Toll-like receptor 5-mediated inflammation in the host, this might be one factor contributing to the response in our assays (31
). Collectively, the hyper-biofilm-formation, increased antibiotic tolerance, and reduced inflammation traits of RSCVs suggest they are a subpopulation geared toward persistence in biofilms and the CF airway environment.