Mucoid switch in Burkholderia cepacia complex bacteria: Triggers, molecular mechanisms and implications in pathogenesis

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Mucoid switch in Burkholderia cepacia complex bacteria: Triggers, molecular mechanisms and implications in pathogenesis Mirela R. Ferreiraa,†, Sara C. Gomesa,†, Leonilde M. Moreiraa,b,*

a IBB-Institute for Bioengineering and Biosciences, Instituto Superior T
ecnico, Universidade de Lisboa, Lisboa, Portugal b Department of Bioengineering, Instituto Superior T
ecnico, Universidade de Lisboa, Lisboa, Portugal *Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. In vivo and in vitro Burkholderia cepacia complex mucoid-to-nonmucoid transition 3. Mucoid phenotype due to cepacian biosynthesis 3.1 Genetic organization and phylogenetic distribution of the bce loci 3.2 Molecular mechanism of cepacian biosynthesis 3.3 Genes regulating cepacian biosynthesis 3.4 Cepacian structural and regulatory genes as evolutionary targets of selection 4. Implications of regulator-mediated mucoid switch in virulence phenotypes 5. Biological functions of Burkholderia cepacia complex exopolysaccharide 6. Concluding remarks Acknowledgments References

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Abstract Bacteria produce a vast range of exopolysaccharides (EPSs) to thrive in diverse environmental niches and often display a mucoid phenotype in solid media. One such exopolysaccharide, cepacian, is produced by bacteria of the genus Burkholderia and is of interest due to its role in pathogenesis associated with lung infections in cystic fibrosis (CF) patients. Cepacian is a repeat-unit polymer that has been implicated in biofilm formation, immune system evasion, interaction with host cells, resistance against antimicrobials, and virulence. Its biosynthesis proceeds through the Wzy-dependent †

These authors contributed equally to this work.

Advances in Applied Microbiology ISSN 0065-2164 https://doi.org/10.1016/bs.aambs.2019.03.001

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2019 Elsevier Inc. All rights reserved.

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polymerization and secretion mechanism, which requires a multienzymatic complex. Key aspects of its structure, genetic organization, and the regulatory network involved in mucoid switch and regulation of cepacian biosynthesis at transcriptional and posttranscriptional levels are reviewed. It is also evaluated the importance of cepacian biosynthesis/regulation key players as evolutionary targets of selection and highlighted the complexity of the regulatory network, which allows cells to coordinate the expression of metabolic functions to the ones of the cell wall, in order to be successful in ever changing environments, including in the interaction with host cells.

1. Introduction The genus Burkholderia comprises more than 120 validly named species found in different ecological niches including water, soil, industrial settings, man-made products, symbiosis with plants, invertebrate guts, and plant and animal infections (Coenye & Vandamme, 2003; Compant, Nowak, Coenye, Cl
ement, & Ait Barka, 2008). Despite their metabolic potential for bioremediation, biocontrol and plant growth promotion, many species are important opportunistic pathogens in immunocompromised patients (Coenye & Vandamme, 2003; Depoorter et al., 2016). An interesting feature of these bacteria is their large genomes, ranging from 6 to 10 Mbp, divided by two to three chromosomes and several plasmids (Mahenthiralingam, Urban, & Goldberg, 2005). A recent phylogenetic study based on 16S ribosomal RNA gene sequence subdivided this genus into Burkholderia sensu stricto, comprising human pathogens, and novel genera such as Paraburkholderia and Caballeronia, which include the environmental isolates (Dobritsa & Samadpour, 2016; Sawana, Adeolu, & Gupta, 2014). Nevertheless, this division is not consensual. Among the Burkholderia sensu stricto species, are found the ones belonging to the Burkholderia cepacia complex (Bcc), a group of opportunistic pathogens causing infections in patients with cystic fibrosis (CF), chronic granulomatous disease and other immunocompromised patients (Drevinek & Mahenthiralingam, 2010; Leita˜o et al., 2010). Bacteria from the Burkholderia cepacia complex, which currently has over 25 different species, are transmissible among CF patients by social contact and intrinsically resistant to multiple antibiotics (Govan et al., 1993). Patient’s clinical outcome following colonization with bacteria belonging to the Bcc is variable and unpredictable. The rapid decline of lung function observed in approximately 10% of Bcc-infected CF patients, leading to a fatal necrotizing pneumonia accompanied by septicemia—the “cepacia syndrome,” is strongly feared (Isles et al., 1984).

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Bcc bacteria display a vast arsenal of virulence factors such as type I, type II, type III, type IV and type VI secretion systems, adhesins, pili, siderophores, extracellular proteases and lipases, quorum sensing molecules, and several polysaccharides (Leita˜o et al., 2010; Mahenthiralingam et al., 2005). Among these virulence factors, exopolysaccharide (EPS) production was one of the first traits to be evaluated. Sage and co-authors analyzed clinical and environmental strains (at that time cataloged as Pseudomonas cepacia) regarding the mucoid phenotype and determined the sugar composition of that acidic carbohydrate polymer (Sage, Linker, Evans, & Lessie, 1990). Since their study, many others reported the ability of Bcc clinical isolates recovered from CF respiratory infections and environmental isolates to express the mucoid phenotype due to exopolysaccharide production (Achouak, Christen, Barakat, Martel, & Heulin, 1999; Chiarini et al., 2004; Ferreira et al., 2010; Ferreira, Silva, Oliveira, Cunha, & Moreira, 2011; Richau et al., 2000; Sua´rez-Moreno et al., 2010; Zlosnik et al., 2008). Here, we review our current knowledge on the biosynthesis and regulation of exopolysaccharide in Burkholderia cepacia complex and the relevance of mucoid morphotype switch, but when information is available it will be extended to the genus Burkholderia sensu lato which also includes Paraburkholderia and Caballeronia.

2. In vivo and in vitro Burkholderia cepacia complex mucoid-to-nonmucoid transition While analyzing the mucoid morphotype of longitudinal Bcc isolates recovered from single CF patients with chronic infections, Zlosnik and co-workers found 15 morphotype transitions; 13 of them were from mucoid-to-nonmucoid and 2 from nonmucoid-to-mucoid (Zlosnik et al., 2008). This predominant mucoid-to-nonmucoid switch, observed mostly in Burkholderia multivorans isolates, can be seen in Fig. 1A, which shows an example of sequential clonal isolates recovered from the same CF patient over a 13-years period, with the first 8 isolates being mucoid producers and the remaining 10 being nonmucoid or having reduced mucoid phenotype. Similar trends were observed for other CF chronic infections where early isolates were mucoid and later isolates became nonmucoid. Nevertheless, from one CF patient with a chronic respiratory infection it was recovered simultaneously clonal mucoid and nonmucoid isolates. This is explained by the fact that during the course of the infection, several lineages might evolve from the initial colonizers, some of them being unable to

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Fig. 1 (A) Evolution of the mucoid morphotype in Burkholderia multivorans longitudinal isolates recovered from a single CF patient grown in mannitol-rich medium for 2 days. Isolates 1–8 are mucoid due to high amount of polysaccharide produced (shown are isolates 1, 3, and 5), while isolates 9–18 are mostly nonmucoid (shown are isolates 9, 11, and 18). (B) Nonmucoid variants derived from the mucoid wild-type strain of B. multivorans D2095 after prolonged stationary phase in mannitol-rich medium. (C) Structure of cepacian heptasaccharide repeat-unit. Gal, galactose; Rha, rhamnose; GlcA, glucuronic acid; Man, mannose; and Glc, glucose.

produce EPS while others retained this trait (Silva et al., 2016), highlighting the complexity of bacterial phenotypes present in these infections. The relevance of the mucoid phenotype in CF lung function performance evidenced that patients infected exclusively with nonmucoid Bcc isolates had a more rapid decline in lung function than those infected with mucoid bacteria (Zlosnik et al., 2011). To understand which environmental conditions trigger the mucoid-tononmucoid switch, experiments have been carried out in which clinical and environmental isolates of Burkholderia and Paraburkholderia were exposed to one or more stresses that mimic the lung environment and alterations in the mucoid morphotype evaluated (Silva, Tavares, Ferreira, & Moreira, 2013). Prolonged stationary phase due to 21 days of growth under static conditions or growth in the presence of 2.5  the minimal inhibitory concentration (MIC) of ciprofloxacin were identified as mucoid-to-nonmucoid triggers in the Bcc strains of B. cepacia, B. contaminans, B. multivorans, B. stabilis, B.

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dolosa, B. ambifaria, and B. anthina and the non-Bcc strains of Paraburkholderia phymatum STM815 and Paraburkholderia xenovorans LB400 with a frequency of nonmucoid variants ranging from 0.2% to 30% (Silva et al., 2013). An example of mucoid wild-type large colonies and small nonmucoid colonies is shown in Fig. 1B. The strain with the highest rate of mucoid-to-nonmucoid transition under the reported conditions, B. multivorans D2095, was exposed to other stresses such as sub-inhibitory concentration of antibiotics like ceftazidime, amikacin and kanamycin, oxidative, osmotic and nitrosative stresses, temperature below and above the optimum, and microaerophilic conditions. Except for microaerophilic conditions and nitrosative stress with less than 1% of the colonies becoming nonmucoid, all the other stresses exerted an effect in the mucoid morphotype with 1–30% of the colonies becoming nonmucoid. Additionally, the mucoid-to-nonmucoid transition was stable for most of them since the alleviation of the stress imposed did not restore the mucoid phenotype of the obtained variants (Silva et al., 2013). In another in vitro study, the antibiotics ciprofloxacin and ceftazidime have also been shown to trigger the mucoid-to-nonmucoid switch in Burkholderia cenocepacia and B. multivorans clinical isolates (Zlosnik et al., 2011). Although these environmental factors triggering mucoid-to-nonmucoid switch were identified under laboratory settings, one cannot exclude that they may act alone or together under the host environmental conditions. During CF patient’s chronic lung infections, adverse conditions such as oxygen limitation, oxidative stress, and antibiotic therapies are present (Sass et al., 2013; Yang, Jelsbak, & Molin, 2011) and are likely triggering the mechanisms of adaptation which alter this and other bacterial traits.

3. Mucoid phenotype due to cepacian biosynthesis Three decades ago Sage and co-workers described the exopolysaccharide produced by CF isolates of Burkholderia, grown on excess of glucose or mannitol as carbon source, as being composed of five different sugars (Sage et al., 1990). This polysaccharide, later named cepacian, turned out to be the main exopolysaccharide produced by many strains in the Burkholderia genus (Cescutti et al., 2000; Conway, Chu, Bylund, Altman, & Speert, 2004; Cuzzi et al., 2014; Ferreira et al., 2010; Hallack et al., 2010). Cepacian (CEP) is a branched acetylated heptasaccharide repeat-unit polymer with D-glucose, D-rhamnose, D-mannose, D-galactose, and D-glucuronic acid in

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the ratio of 1:1:1:3:1 (Fig. 1C) (Cerantola, Lemassu-Jacquier, & Montrozier, 1999; Cescutti et al., 2000; Cescutti, Foschiatti, Furlanis, Lagatolla, & Rizzo, 2010). In addition to cepacian, at least seven additional EPSs have been identified in the Burkholderia genus. These are PS-I (Cerantola, Marty, & Montrozier, 1996); dextran; levan; galactan-Kdo (GAL-KDO), a linear polysaccharide composed by three galactose residues, one acyl substituent and one 3-deoxy-D-manno-oct-ulosonic acid (Cescutti et al., 2003); BV-EP, a B. vietnamiensis EPS with a hexasaccharide repeat-unit containing fucose, glucose and glucuronic acid (Cescutti, Cuzzi, Herasimenka, & Rizzo, 2013); CO-CEP, a polysaccharide containing galactose and glucuronic acid (Cuzzi et al., 2014); and a linear exopolysaccharide containing rhamnose and mannose and named rhamnomannan (Cuzzi et al., 2014). Depending on the strains, some produce only one type of EPS while others produce a mixture. For instance, Burkholderia contaminans IST408 (formerly Burkholderia cepacia) and B. multivorans ATCC 17616 only produce cepacian (Cescutti et al., 2000; Ferreira et al., 2010), while B. cenocepacia C9343 produces simultaneously PS-I, cepacian and levan (Conway et al., 2004). In another study, Cuzzi and co-workers analyzed 10 isolates from 9 species within the Bcc, all grown in mannitol-rich medium, and identified that all strains produced mixtures of two or more EPSs (Cuzzi et al., 2014). In all strains the major fraction was composed by cepacian, but a minor fraction of CO-CEP or one of the other polysaccharides mentioned above was also present. Depending on the growth medium used, the proportion of the different polysaccharides was changing and this was particularly evident during biofilm formation (Cuzzi et al., 2014). The mucoid phenotype observed by Bcc strains grown in broth or agar medium containing high carbon to nitrogen ratio (usually mannitol or glucose) is a consequence of cepacian production (Cescutti et al., 2000; Ferreira et al., 2010).

3.1 Genetic organization and phylogenetic distribution of the bce loci Despite the sequenced genomes and the determination of the chemical structure of the several polysaccharides produced by Burkholderia, the genetic basis for their biosynthesis is mostly unknown. One of the exceptions is cepacian, known to be synthesized by a cluster of at least 20 genes named bce (Burkholderia cepacia exopolysaccharide) which can be together in the same genomic locus or fragmented in two different loci (clusters bce-I and bce-II) generally of chromosome 2, but in a few strains, of chromosome 1 (Fig. 2) (Ferreira et al., 2010; Moreira et al., 2003). These gene products are

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Fig. 2 Genetic loci implicated in cepacian biosynthesis by some members of the Burkholderiaceae family. Distribution and functional category of bce genes in the Bcc strain Burkholderia multivorans ATCC 17616, the non-Bcc Burkholderia thailandensis E264, Paraburkholderia xenovorans LB400, and Caballeronia glathei DSM 50014. The genomic distance between bce-I and bce-II clusters is indicated in kilobase pairs.

responsible for the synthesis of activated sugar-nucleotide precursors, the glycosyltransferases and acetyltransferases responsible for repeating-unit formation and decoration, and the enzymes involved in polymerization and export to the cell surface. Search of bce genes in more than 1500 Burkholderia, Paraburkholderia and Caballeronia strains with sequenced genomes showed their presence in most of them, being the sole exceptions Paraburkholderia rhizoxinica HKI 454 with both gene clusters absent and the species Burkholderia mallei having only the cluster bce-II. In addition, bce-I cluster was absent from five B. cenocepacia CF isolates (BC7, VC12821, VC14801, VC15495, and VC15495); one environmental (LMG29308) and one CF isolates (ATCC BAA-247) of B. multivorans; and three soil isolates of Burkholderia ubonensis (MSMB1138WGS, MSMB1188WGS, and MSMB2028WGS). Contrastingly, in Burkholderia contaminans MS14, the bce-I cluster was duplicated, with one copy in chromosome 1 and another in chromosome 2. The bce-II cluster was fully absent from two Burkholderia isolates (sp. PAMC 28687 and sp. E7m39), but genes bceM, bceP, and bceU were often missing. Several genomes where the bce genes were partially lost are from isolates recovered from CF patients chronic respiratory infections, characterized by suffering genome reduction during long-term colonization (Lee et al., 2017; Silva et al., 2016). Still, genome reduction was much higher in the endosymbiont P. rhizoxinica HKI 454 with a genome size of 3.8 Mbp and in the animal pathogen B. mallei with a median genome size of 5.7 Mbp, while the other

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species have genome sizes ranging from 6 to approximately 10 Mbp. Few additional genes encoding small size hypothetical proteins are present between bceK and bceM/bceN and before bceO of P. xenovorans and C. glathei clusters (Fig. 2), but it is unknown whether these are implicated in cepacian biosynthesis. Concatenation of bce gene products from different strains with fully sequenced genomes from the genus Burkholderia, Paraburkholderia and Caballeronia, followed by phylogenetic analysis is shown in Fig. 3. This neighbor-joining phylogenetic tree evidences three main branches. Branch I contains 18 different species belonging to the Burkholderia cepacia complex. The two bce clusters map in two different genomic regions separated by a minimum of 130 kbp in Burkholderia latens AU17928 to a maximum of 314 kbp in B. multivorans ATCC 17616. Branch II includes non-Bcc plant and animal pathogens in which the two bce gene clusters are distant from each other with a minimum of 147 kbp for Burkholderia thailandensis E264 and Burkholderia oklahomensis EO 147 and a maximum of 1016 kbp for the plant pathogen Burkholderia glumae BGR1. Branch III includes strains from the genus Paraburkholderia and Caballeronia and are characterized by having all bce genes in the same genomic location (Fig. 2).

3.2 Molecular mechanism of cepacian biosynthesis Cepacian is a repeating unit-containing polymer which is synthesized by the Wzy-dependent pathway, a mechanism well characterized in Escherichia coli group 1 capsular polysaccharide (Cuthbertson, Mainprize, Naismith, & Whitfield, 2009). In cepacian, five different activated sugar-nucleotide precursors are required for synthesis of the heptasaccharide repeat-unit (Fig. 4). Gene bceA encodes a bifunctional protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities required for the formation of GDP-D-mannose (Sousa et al., 2007). Burkholderia genomes have a second bceA gene homolog. Therefore, deletion of bceA gene does not prevent cepacian biosynthesis despite an observed 50% reduction (Sousa et al., 2007). Using GDP-D-mannose as substrate, the gene products of bceM and bceN supply the activities of GDP-mannose-4,6-dehydratase and GDP-6deoxy-D-lyxo-4-hexulose reductase for the synthesis of GDP-D-rhamnose (Sousa, Feliciano, Pinheiro, & Leita˜o, 2013). Although gene bceM is absent from the bce gene cluster of P. glumae, P. phymatum and P. xenovorans, a gene encoding a homolog protein is found in another location of their genome. For the synthesis of UDP-D-glucose, cells might use the product of gene

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Fig. 3 See legend on next page.

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bceT, an enzyme with putative activity of UDP-glucose pyrophosphorylase. UDP-D-glucose is then the precursor for UDP-D-glucuronic acid and UDP-D-galactose (Fig. 4). The enzyme required for the synthesis of UDP-D-glucuronic acid is encoded by the bceC gene and its product, with activity of UDP-glucose dehydrogenase, has been extensively characterized (Loutet, Bartholdson, Govan, Campopiano, & Valvano, 2009; Rocha et al., 2011). Deletion of bceC gene does not abrogate cepacian biosynthesis since these microorganisms have at least one more copy of an homolog gene. The gene encoding the enzyme UDP-glucose epimerase required for synthesis of UDP-D-galactose is absent from bce gene clusters but located elsewhere in the genome. Assembly of cepacian heptasaccharide repeat-unit catalyzed by glycosyltransferases encoded by bceB, bceG, bceH, bceJ, bceK and bceR genes occurs at the cytoplasmic side of the inner membrane (Fig. 4). The only glycosyltransferase that has been characterized so far is BceB which catalyzes the transfer of glucose to the isoprenoid lipid carrier, having a undecaprenylphosphate glycosyl-1-phosphate transferase activity (Videira, Garcia, & Sa´-Correia, 2005). From the remaining glycosyltransferases, the protein encoded by bceR gene is predicted to be a bifunctional enzyme with two glycosyltransferase domains of CAZy GT-4 family with a retaining mechanism of glycosyl residue transfer and adopting a GT-B fold. BceR has been shown to be essential for cepacian biosynthesis, as confirmed by the absence of exopolysaccharide in the growth medium of B. contaminans IST408 bceR insertion mutant (Ferreira et al., 2010). BceH, BceJ and BceK are predicted to belong to CAZy GT-1 family, which comprises retaining glycosyltransferases, and BceG is predicted to belong to the CAZy GT-2 family of enzymes that transfer glycosyl residues by an inverting mechanism (Ferreira et al., 2010; Moreira et al., 2003). BceB, BceJ and BceK possess

Fig. 3 Phylogeny of gene clusters bce-I and bce-II encoding products. Neighbor-joining phylogenetic tree of the deduced amino acid sequences of Bce predicted proteins from 30 species belonging to three different genera of Burkholderiaceae family. The comparative sequence analysis was performed using MEGA-X v.10.5.0 (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). The Bce protein sequences of each gene cluster were first concatenated and independently aligned by Clustal W (Thompson, Higgins, & Gibson, 1994) using 1000 bootstrap replicates. Then sequence alignments obtained were concatenated and a neighbor-joining phylogenetic tree constructed. The three branches obtained include the Bcc species (I) and non-Bcc species (II) of genus Burkholderia, and environmental isolates from genera Paraburkholderia and Caballeronia (III). Scale: number of substitutions per site.

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Fig. 4 See legend on next page.

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transmembrane domains, being most likely integral or membrane associated proteins while no such domains were detected in BceG, BceH and BceR (Ferreira et al., 2010; Moreira et al., 2003; Videira et al., 2005). The order of sugar addition to assemble the repeat-unit has been proposed (Cescutti et al., 2010), with a mannose substituted with galactose being added to the glucose linked to the lipid carrier. Then, glucuronic acid is added to this trisaccharide followed by the incorporation of two substituents; the first being galactose and the second, the disaccharide rhamnose and galactose (Fig. 1C). Cepacian repeat-unit acetylation occurs at the inner membrane during and after its assembly, but the number of substitutions seems to be strain dependent (Cescutti et al., 2010). The enzymes required for these modifications are putatively encoded by bceO, bceS and bceU genes and are predicted to be located in the inner membrane (Fig. 4), with BceO and BceU exhibiting nine predicted transmembrane domains, while BceS exhibits eight predicted transmembrane domains (Ferreira et al., 2010). Despite the weak conservation at the amino acid level, BceO, BceS and BceU are homologous to membrane proteins involved in the acylation of carbohydrate moieties of extracytoplasmic molecules. Genes bceO and bceS map into the bce-II cluster of all species where this cluster was found, but bceU seems to be absent from strains of the genus Paraburkholderia and Caballeronia, implying different patterns of acetylation in the final polymer. Experimental evidences for the role of these proteins are available solely for bceS gene of B. multivorans ATCC 17616 since its disruption caused reduction of the EPS

Fig. 4 Mechanisms of cepacian biosynthesis and gene expression regulation. Cepacian biosynthesis begins with the formation of activated sugar-nucleotide precursors required for repeat-unit formation which is assembled on a lipid carrier in the inner membrane in a reaction initiated by the BceB enzyme and continued by the other glycosyltransferases (GTs) and acetylated by acyltransferases (ACTs). The lipid-linked repeat-units are translocated across the inner membrane by the putative BceQ protein and polymerization occurs at the periplasmic face of the inner membrane by BceI whose activity is possibly regulated by another membrane protein, the BceF tyrosine kinase. BceD is a protein tyrosine phosphatase enzyme responsible for dephosphorylating BceF. BceE forms a channel structure for export of EPS chains to the outside. The proposed regulatory mechanisms are based on data obtained from B. multivorans, B. contaminans, B. cenocepacia, and P. xenovorans, as explained in the text. Abbreviations: GlcA, glucuronic acid; Gal, galactose; Glc, glucose; Rha, rhamnose, Man, mannose; PGI, phosphoglucose isomerase; PGM, phosphoglucomutase; UGE, UDP-glucose epimerase; PMM, phosphomannomutase; ATP, adenosine triphosphate; GDP, guanosine diphosphate; UDP, uridine diphosphate; P, phosphate; HSL, homoserine lactones; IM, inner membrane; OM, outer membrane; PL, peptidoglycan layer.

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acetylation content by approximately 20%, confirming BceS involvement in the repeat-unit decoration (Ferreira et al., 2010). After repeat-unit assembly, polymer biosynthesis proceeds via the Wzydependent pathway (Fig. 4). According to this model, the lipid carrierlinked repeat-units are exported across the inner membrane by a polysaccharide-specific transport protein (a so-called flippase), followed by repeat-units polymerization at the periplasmic face of the inner membrane by a polysaccharide polymerase. Since bceQ and bceI gene products have 12 and 10 predicted transmembrane domains, respectively, and show similarity with other Wzx flippases and Wzy polysaccharide polymerases, it is suggested that BceI is the putative polymerase and BceQ the putative flippase. Both genes are essential for cepacian biosynthesis, as demonstrated by the EPS deficient phenotype of the B. contaminans IST408 insertion mutants for bceI and bceQ genes (Ferreira et al., 2010; Moreira et al., 2003). The proteins encoded by bceD and bceF genes are also involved in EPS polymerization and export (Fig. 4). BceF belongs to the bacterial tyrosine kinase (BY-kinase) family which includes Wzc homologs predicted to act as the polysaccharide co-polymerase component (Bechet et al., 2010; Cuthbertson et al., 2009). These proteins assist polymerization, but also control polymer chain length and guide the nascent polymer through the periplasm to the outer membrane auxiliary protein from the Wza family (Cuthbertson et al., 2009). BceF possesses two transmembrane domains that flank a large periplasmic loop and a cytoplasmic located C-terminal region required for autophosphorylation (Ferreira et al., 2007). The periplasmic domain is predicted to adopt a coiled-coil structure important for interaction with other proteins such as BceI polysaccharide polymerase and the outer membrane protein BceE, in a similar way to the model proposed for E. coli capsule group 1 biosynthesis (Cuthbertson et al., 2009; Tocilj et al., 2008). Also based on the current model for E. coli capsule group 1 biosynthesis, it is likely that cepacian biosynthesis occurs by transferring the nascent chain between adjacent BceI proteins, with BceF controlling chain length by affecting assembly of the previous enzyme units. In this case, chain growth would be terminated in the absence of an adjacent BceI. Disruption of bceF gene in B. contaminans and B. multivorans abrogates cepacian biosynthesis (Ferreira et al., 2007; Moreira et al., 2003; Silva et al., 2018), but the importance of BceF tyrosine autophosphorylation activity in cepacian biosynthesis is unknown. Nevertheless, C-terminal tyrosine phosphorylation of other Wzc homologs seems to be essential for assembly of high molecular weight EPS (Cuthbertson et al., 2009). BceD was shown to be

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a phosphotyrosine phosphatase (PTP) protein that interacts with BceF, promoting this BY-kinase tyrosine dephosphorylation (Ferreira et al., 2007). A B. contaminans IST408 mutant lacking the bceD gene shows reduction of 30% of the wild-type EPS amount, but forms less viscous solutions, suggesting an alteration in chain length (Ferreira et al., 2007). Protein BceE is a Wza homolog (Fig. 4) predicted to be the outer membrane polysaccharide export protein (OPX) responsible for the final stage of polysaccharide export. OPX family of proteins are predicted to be lipoproteins that adopt an octameric configuration with a large central cavity that facilitates polysaccharide export through the periplasm and across the outer membrane (Nickerson et al., 2014). A B. contaminans IST408 bceE mutant is impaired in cepacian biosynthesis demonstrating the importance of this protein in the synthesis of this polymer (Ferreira et al., 2013). Finally, bce-II gene cluster contains the bceP gene, encoding a protein of unknown function. BceP secondary structure was predicted to be exclusively composed of β-strands, and its location can be the periplasm, the outer membrane or even the extracellular milieu. Structural homology modeling suggests that BceP resembles to a polysaccharide degrading xyloglucanase of Clostridium thermocellum. Thus, BceP may be responsible for processing the EPS before and/or after export depending on its cellular localization. Despite that, the product of bceP gene does not seem to be required for cepacian biosynthesis since this gene is absent from the bce cluster of several Burkholderia strains.

3.3 Genes regulating cepacian biosynthesis Cepacian biosynthesis is controlled by several players within a complex regulatory network. Although some have been identified, there is still a lot to understand in this process. Here, we describe the already identified mechanisms. A major regulator of cepacian biosynthesis and of mucoid-tononmucoid switch is the response regulator OmpR from a two-component signal transduction system where the associated kinase is EnvZ (Fig. 4) (Silva et al., 2018). This response regulator was identified through whole genome sequence of B. multivorans nonmucoid colonies derived from mucoid ancestors exposed to stress conditions, where the main target of mutations was the coding region of ompR gene. Several of the ompR mutants had frameshift mutations leading to very short truncated proteins, while others had nonsynonymous substitutions or short deletions mapping either in the N-terminal receiver domain or the C-terminal DNA-binding domain.

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These mutations were stable, except for one of the mutants with a leucine substituted by proline at position 53 of OmpR, possibly affecting phosphorylation of aspartate at position 57. Plating cells of this OmpRL53P mutant in mannitol-rich medium resulted in a few mucoid colonies that accumulated a second mutation in ompR gene, namely, replacing aspartate at position 13 by valine. This region of the protein is the site for Mg2+ coordination, and possibly changes protein conformation, suppressing the negative effect of the L53P substitution. Quantification of bce genes expression in several ompR mutants confirmed their downregulation (Silva et al., 2018). Nevertheless, it is unknown whether OmpR regulates bce genes expression directly or mediated through another regulator and whether this is dependent on phosphorylation events. Transcriptomic analysis of one of the ompR mutants identified more than 700 genes differentially expressed, including several other regulators, confirming that OmpR is a major regulator. The second signal transduction system implicated in the regulation of cepacian biosynthesis is the NtrB histidine kinase and the NtrC response regulator (Fig. 4). Many bacteria respond to nitrogen starvation by activating the nitrogen regulatory (Ntr) system to facilitate nitrogen scavenging from other sources. The Ntr system monitors the intracellular ratio of glutamine to α-ketoglutarate. Under nitrogen limiting conditions, the PII signal transduction proteins encoded by glnB and glnK genes are uridylylated, controlling the kinase and phosphatase activities of histidine kinase NtrB. This sensor kinase phosphorylates the response regulator NtrC, which then binds to promoter sequences and together with the alternative sigma factor σ54 (RpoN) activates transcription (Reitzer, 2003; Wyman, Rombel, North, Bustamante, & Kustu, 1997). The initiation of σ54-dependent transcription usually requires such an interaction with an enhancer binding protein (EBP) which in the case of nitrogen starvation conditions is NtrC (Bush & Dixon, 2012). The response of B. cenocepacia H111 to nitrogen starvation was shown to be largely dependent on sigma factor σ54 (Lardi et al., 2015) and a mutant in this gene displayed decreased expression of the bce-I and bce-II gene clusters, as well as reduced amount of cepacian production. Stringent search for σ54 consensus sequences failed to identify potential binding sites in the bce-I and bce-II promoter regions (Lardi et al., 2015), but a more permissive search identified potential σ54 boxes in front of bceE, bceJ, and bceR, that still need confirmation. Further studies, confirmed that a ntrC mutant has the same reduced production of polysaccharide as the sigma factor σ54 mutant and that complementation of the ntrC mutant restored EPS production to the level of the wild-type (Liu, Lardi, Pedrioli, Eberl, & Pessi, 2017). Biochemical data

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on the binding of NtrC and the sigma factor σ54 to the bce promoters are still required to confirm the direct or indirect involvement of this signal transduction system and the sigma factor in mediating bce gene expression. In line with these results, control of EPS synthesis by NtrC has been reported in several bacteria including the human pathogen Vibrio vulnificus (Kim, Park, & Lee, 2009), and the plant nitrogen fixation symbiont Sinorhizobium meliloti (Wang, Xue, Yin, Xie, & Luo, 2013), but again biochemical proof of direct binding to promoters is missing. Quorum sensing (QS) has been shown to interfere with exopolysaccharide production in different bacteria like Pantoea stewartii (von Bodman, Majerczak, & Coplin, 1998) or Sinorhizobium meliloti (Pellock, Teplitski, Boinay, Bauer, & Walker, 2002). Burkholderia employ at least three different types of QS signals, including the N-acylhomoserine lactones (AHL), 2-heptyl-4-quinolone (HHQ), and cis-2-dodecenoic acid (BDSF) (Deng, Boon, Eberl, & Zhang, 2009; Diggle et al., 2006; Schmid et al., 2012). The major AHL system is the Cep system, consisting of the acylhomoserine lactone (AHL) synthase CepI and the transcriptional regulator CepR, which becomes activated after binding to AHL. Activated CepR binds to cep boxes, which are often located upstream of the target genes, and results in activation or repression (Gotschlich et al., 2001). Burkholderia strains have 2–3 of these systems. The BDSF system relies on the biosynthesis of BDSF signals by the bifunctional crotonase RpfF and the BDSF receptor protein RpfR (Deng et al., 2012). Although this diffusible signal factor has been implicated in production of extracellular polysaccharide in Xanthomonas campestris (He et al., 2006), no information about Burkholderia is available. Regarding the Cep system, a study in P. xenovorans and P. unamae has shown that the BraI/BraR QS system (a Cep homolog) which produces several acyl homoserine lactones (3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL) positively regulates EPS production in mannitol-rich medium (Fig. 4) (Sua´rez-Moreno et al., 2010). Investigation on the role of QS in cepacian biosynthesis by B. contaminans IST408 showed that the expression of a lactonase enzyme able to degrade the homoserine lactone ring abrogated EPS production (our unpublished data). Additionally, under those conditions, both bce-I and bce-II gene clusters showed decreased expression when comparing to the control not expressing lactonase. Nevertheless, the B. contaminans IST408 cepR insertion mutant still produced EPS, suggesting that another QS system present in this organism must be the responsible for cepacian biosynthesis regulation.

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Although posttranscriptional regulation by small RNA is an important mechanism in the regulation of alginate biosynthesis by P. aeruginosa (Hay, Wang, Moradali, Rehman, & Rehm, 2014), not much is known in Burkholderia. The only evidences of a possible involvement of sRNAs in cepacian biosynthesis comes from the observation that a mutant in the RNA chaperone Hfq produces 50% less EPS than the wild-type strain (Fig. 4) (Sousa, Ramos, Moreira, & Leita˜o, 2010). In this type of mechanism, sRNAs exert their regulatory action by base pairing with specific mRNAs (the mRNA targets) with the help of the RNA chaperone Hfq. The sRNA-mRNA double stranded complex often hinders the ribosome binding site (RBS) of the mRNA, leading to repression of translation or, if the double-stranded complex exposes the RBS of the mRNA, resulting in increased translation of the encoded protein (Feliciano, Grilo, Guerreiro, Sousa, & Leita˜o, 2016). The amount of cepacian being produced also depends on whether cells grow as single entities or as planktonic cellular aggregates. A B. multivorans ATCC 17616 double mutant in the LysR-type transcriptional regulator LdhR and the D-lactate dehydrogenase LdhA grown in mannitol-rich medium displayed higher EPS production than the wild-type strain. Besides this, it was noticed that in the tested growth conditions, B. multivorans ATCC 17616 forms macroscopic aggregates while the ldhRA mutant grows mainly as free cells and microscopic aggregates (Silva et al., 2017). Therefore, the negative effect in EPS biosynthesis by LdhR and LdhA might be a consequence of planktonic cellular aggregate formation. Most cells of the ldhRA mutant contribute to EPS production while wild-type larger aggregates have smaller contributions to EPS biosynthesis, explaining the lower yield in the presence of several carbon sources.

3.4 Cepacian structural and regulatory genes as evolutionary targets of selection To identify mutations and their influence in the phenotype displayed by bacterial cells, sequencing of genomes from longitudinal isolates of Bcc recovered from CF patients have been conducted (Lee et al., 2017; Lieberman et al., 2014, 2011; Silva et al., 2016). This general strategy involves comparison of genome sequences of isolates recovered from a single patient to the first isolate genome and identification of single nucleotide polymorphysm (SNPs) and indels. Such analysis conducted in B. dolosa isolates recovered from 14 different patients (Lieberman et al., 2011), 1 patient chronically colonized by B. multivorans (Silva et al., 2016) and 16 patients

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from B. cenocepacia (Lee et al., 2017) showed mutations in some of the bce genes as well as in cepacian biosynthesis regulators. A common mutation found in many B. cenocepacia isolates unable to produce EPS is a frameshift mutation in bceB gene, impairing the synthesis of the priming glycosyltransferase. Whether this is the sole cause for the nonmucoid phenotype of these isolates remains to be determined. In B. dolosa it was found a nonsynonymous mutation in bceA gene in isolates from three patients while in B. multivorans, several isolates accumulated nonsynonymous mutations in bceH (G103R), bceJ (G378S), bceR (Y156C), and bceU (L72H and a frameshift mutation) (Silva et al., 2016). Evaluation of the mucoid morphotype in these B. multivorans isolates containing mutations in bce-I or bce-II genes did not allow to establish a correlation between mutations and the nonmucoid morphotype. Also, complementation of B. multivorans nonmucoid isolates with the wild-type copy of mutated bce gene did not restore the mucoid phenotype, implying that other mutations might be responsible for the observed phenotype. This suggests that the observed decrease of mucoidy during chronic colonization (Zlosnik et al., 2008) might be mainly caused by changes at bce gene expression level possibly caused by mutations in regulatory genes. The signal transduction systems EnvZ/OmpR and NtrB/NtrC, as well as the chaperone Hfq and the quorum sensing regulator CepR accumulated a few mutations in B. multivorans isolates and are likely important in adaptation to the lung environment. Nevertheless, these mutations alone could not explain loss of mucoid phenotype in some of the isolates (Silva et al., 2016).

4. Implications of regulator-mediated mucoid switch in virulence phenotypes The regulators here described as implicated in cepacian biosynthesis are not exclusively dedicated to this function as they regulate many other traits. This implies that in order to adapt to changing environments, cells need to simultaneously adapt the expression of many properties and that is possible by the coordination of major regulators. Taking as an example the EnvZ/OmpR two-component regulatory system, it has been demonstrated that mutations in OmpR block cepacian biosynthesis (Silva et al., 2018). But this is just one phenotype among the several affected by mutations in ompR gene. As expected, the mucoid wild-type and the ompR mutants have different ability to grow under high-salt concentration, with the mutants being more adapted to high osmotic conditions as shown by

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their reduced duplication time and high biomass in stationary phase when compared to the wild-type strain. This difference is illustrated by the differential expression of several genes encoding porins as well as the protein profile of the outer membrane fractions of wild-type and mutants. These differences at the cell envelope have consequences in other cell properties. For example, ompR mutants are more susceptible to antibiotics such as, aztreonam and piperacillin, and show higher swimming and swarming motilities. Concerning biofilm formation under normal osmotic conditions, the ompR mutants form less biofilm biomass. But, under high-salt concentration, the opposite effect is seen with the mutants producing 3–5  more biofilm biomass. Similar situation is seen for adhesion to CFBE44o- lung epithelial cells in which the wild-type strain adheres more efficiently under normal salt concentration, but the mutants have higher adhesion under high-salt concentration. Also, virulence using Galleria mellonella as infection model showed some virulence attenuation for the ompR mutants (Silva et al., 2018). Altogether, this two-component regulatory system might sense differences in osmolarity and triggers a cell response which involves changes in the expression of many genes related to metabolism adjustment and cell wall structure and composition, that will help cells to adapt under these new conditions. What is certain is that cepacian biosynthesis is co-regulated with many other cellular phenotypic traits.

5. Biological functions of Burkholderia cepacia complex exopolysaccharide Most of the studies assessing the importance of EPS production by Bcc bacteria are related to virulence phenotypes. Being extracellular components, EPSs have privileged contact with the surrounding environment, as, for example, the immune system cells. Effectively, in vitro studies confirmed the ability of Bcc EPS to interfere with the innate immune system by neutralizing reactive oxygen species, inhibiting neutrophil chemotaxis and interfering with neutrophil phagocytosis, most likely due to the masking of bacterial surface antigens recognized by immune cells (Bylund, Burgess, Cescutti, Ernst, & Speert, 2006; Conway et al., 2004). Several in vivo studies, namely, by using the wax moth G. mellonella and mice, also confirmed the importance of EPS in Bcc virulence. For example, B. contaminans IST408 mutants in the bceF and bceI genes were found attenuated in G. mellonella and gp91phox / mouse (Ferreira et al., 2013; Sousa et al., 2007). In another study, infection of a BALB/c mouse pulmonary infection

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model with two sequential clinical isolates of B. cenocepacia displaying different morphotype (mucoid vs. nonmucoid) showed that the mucoid clinical isolate had increased persistence in the lungs (Conway et al., 2004). Similarly, another pair of mucoid/nonmucoid clinical isolates from B. multivorans has shown that the nonmucoid isolate displayed acute virulence attenuation in G. mellonella (Silva et al., 2011). This decreased ability of the nonmucoid isolates to establish acute infections in G. mellonella was also observed for several of the B. multivorans-derived nonmucoid variants obtained under several stress conditions (Silva et al., 2013). Overall, these evidences suggest that EPS production seems to favor acute virulence and can be considered a virulence determinant. However, when clinical isolates were analyzed, there was a negative correlation between EPS production ability and CF patients lung function deterioration (Zlosnik et al., 2011). Another study comparing cepacia syndrome blood isolates and 2 months earlier sputum isolates confirmed the lower activity of bce genes in the sputum isolates (Kalferstova, Kolar, Fila, Vavrova, & Drevinek, 2015). Observations from these last studies seem to be against the role of EPS as virulence determinant described above. One possible explanation is that when analyzing in vivo data, we are looking at long-term chronic infections that may last years while in the tested animal infection models we are looking at acute infections that last a few days. It is possible that mucoid strains have an easier escape from the G. mellonella immune system than the nonmucoid ones, justifying the increased virulence of the former. It is also possible that when colonizing the CF lungs, mucoid bacteria have an initial advantage against the immune system and antibiotic therapies than nonmucoid ones, and here EPS would contribute for persistence. But it is also possible that with the progressive lung function deterioration bacteria adopt other strategies to resist antimicrobials and the immune system, such as through formation of microcolonies where polysaccharides such as cepacian might not play a major role. Many polysaccharides have key roles as matrix components within biofilms. In Bcc bacteria this matter is particularly complex, since they can produce several polysaccharides, depending on the environmental signals. Mutants in genes required for cepacian biosynthesis (bceF, bceI), or that affect its molecular mass (bceA, bceC, bceD) or acetylation pattern (bceS) showed lower ability to form biofilms than the wild-type strains (Cunha et al., 2004; Ferreira et al., 2010, 2007), suggesting that wild-type cepacian is not required for biofilm formation, but it has a role in optimal biofilm maturation. In another study that analyzed EPS production of several Bcc

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isolates grown in mannitol-rich medium in biofilm and non-biofilm mode of growth, concluded that biosynthesis of cepacian was stimulated in both conditions (Cuzzi et al., 2014). However, when biofilm was formed on cellulose membranes deposited on MH agar plates, polysaccharides other than cepacian were prevalent, underlying that the type of EPSs synthesized not only varies with the medium used, but it is also influenced by the presence of the solid support where it develops. In addition to the environmental conditions, the genetic background is also very important for EPS production. When sequential isolates from CF patients were analyzed, the ones with increased biofilm formation were low or non-EPS producers. This is the case of a mucoid/nonmucoid pairs of B. multivorans D2095/D2214 and B. cenocepacia C9343/C8963 in which the nonmucoid isolates form more biofilm (Conway et al., 2004; Silva et al., 2011). This increase of biofilm formation observed in nonmucoid clinical isolates can be the result of compensation from other traits subjected to the same regulatory mechanisms. Low levels of cepacian are detrimental to biofilm formation (Cunha et al., 2004; Ferreira et al., 2007), but if this effect is suppressed by the overproduction of other structures with relevance to biofilm initiation, such as pili and adhesins, it may result in increased biofilm formation. By conducting experimental evolution, Traverse and colleagues have shown that adaptation of B. cenocepacia to a biofilm life style is accompanied by numerous mutations in genes that control important features like cyclic-di-GMP-based signaling, cell wall modifications, namely, in LPS and EPS, and metabolic adjustment (Traverse, Mayo-Smith, Poltak, & Cooper, 2013). Since EPS can neutralize ROS and render neutrophils unable to kill Bcc bacteria by oxidative means, the antimicrobial action of those cells will be entirely dependent on the non-oxidative cationic antimicrobial peptides. However, Bcc bacteria are also resistant to non-oxidative antimicrobial peptides and this ability was attributed to the presence of EPS (Herasimenka et al., 2005). In fact, due to the negative charge given by acetyl substituents, cepacian was shown to interact with positively charged antimicrobial peptides forming complexes that lower the antimicrobial peptides biological activity. In line with that, the exposure of Bcc bacteria to the human antimicrobial peptides cathelicidin LL-37 and β-defensin hBD-3 and other mammal peptides has shown that EPS-producing bacteria were more resistant to these antimicrobials (Benincasa et al., 2009). The idea that EPS limits antibiotic penetration within biofilms was evaluated in B. pseudomallei mutants in the genes bpsI (encoding an autoinducer synthase), the alternative

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sigma factor RpoS encoding gene and in ppk, encoding polyphosphate kinase, all of them relevant for biofilm formation (Mongkolrob, Taweechaisupapong, & Tunpradabkul, 2015). It was observed similar reduction in exopolysaccharide production and antibiotic resistance, including different ratios of monosaccharide types in the EPS of the biofilm by the mutants, suggesting that effects on exopolysaccharide amount/composition might lead to the ineffective function of the biofilm matrix as an antibiotic barrier. Protective functions against desiccation and toxic metal ions concentration have also been attributed to EPS and these roles are of major importance concerning the adaptation of bacteria to different environments. Given its hygroscopic properties, EPS may decrease the rate of water loss from the cells and provide bacterial cells with means to survive drying and desiccation (Potts, 1994). Indeed, the external addition of EPS to P. xenovorans LB400 and B. multivorans ATCC 17616 isolates prior desiccation enhanced their tolerance (Ferreira et al., 2010). The protective effect of cepacian on Burkholderia isolates exposed to metal ion stress was also shown. The metal-binding properties of EPS might be due to the carbonyl, carboxyl and hydroxyl groups within the EPS matrix that attach cations and scavenge metals (Potts, 1994). The ability of Burkholderia to withstand desiccation and metal ion stress in the presence of cepacian suggest that this EPS plays a role in their survival, thus representing an advantage for bacteria to thrive in adverse environments.

6. Concluding remarks Bacteria from the Burkholderia genus produce many virulence factors including several polysaccharides. Among them, cepacian has been confirmed as a virulence determinant in several animal models of infection and surveys to assess the ability of clinical isolates recovered from CF patients’ airways to produce EPS confirmed its high prevalence, at least in early infection periods. Although a lot has been learnt over the past few years about the biosynthetic enzymes and their activities, further experiments to elucidate interactions between the polymerization/secretion apparatus still need to be done. Also, of great importance are the molecular mechanisms involved in regulation of cepacian biosynthesis with several key questions persisting. What the specific environmental cues inducing cepacian production in the various species are and the mechanisms behind how these signals are detected remain unclear. Furthermore, how the regulatory network is

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controlled with so much cross-talk between regulators remains an open question. This is particularly complex if we consider that regulators such as OmpR, NtrC, RpoN, and quorum sensing signaling are all heavily involved in other regulatory networks. Whatever the exact mechanisms are, it is evident that the co-regulation of cepacian biosynthesis with other phenotypic traits such as biofilm formation, motility, antimicrobial resistance, among others, leaves Bcc bacteria with increased ability to adapt to different environments and perhaps to persist longer within the CF host. A better understanding of these regulatory mechanisms and the complex networks behind EPS production will allow future identification of potential therapeutic targets.

Acknowledgments This work was supported by Fundac¸a˜o para a Ci^encia e a Tecnologia, Portugal (project UID/ BIO/04565/2013) and by Programa Operacional Regional de Lisboa 2020 (LISBOA-010145-FEDER-007317).

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