Unique Biofilm Signature,Drug Susceptibility and Decreased Virulence in Drosophila through the Pseudomonas aeruginosa Two-Component System PprAB

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.     

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria.

Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.     

FppA, a novel Pseudomonas aeruginosa prepilin peptidase involved in assembly of type IVb pili

Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.     

Assembly of fimbrial structures in Pseudomonas aeruginosa: functionality and specificity of chaperone-usher machineries

Fimbrial or nonfimbrial adhesins assembled by the bacterial chaperone-usher pathway have been demonstrated to play a key role in pathogenesis. Such an assembly mechanism has been exemplified in uropathogenic Escherichia coli strains with the Pap and the Fim systems. In Pseudomonas aeruginosa, three gene clusters (cupA, cupB, and cupC) encoding chaperone-usher pathway components have been identified in the genome sequence of the PAO1 strain. The Cup systems differ from the Pap or Fim systems, since they obviously lack numbers of genes encoding fimbrial subunits. Nevertheless, the CupA system has been demonstrated to be involved in biofilm formation on solid surfaces, whereas the role of the CupB and CupC systems in biofilm formation could not be clearly elucidated. Moreover, these gene clusters were described as poorly expressed under standard laboratory conditions. The cupB and cupC clusters are directly under the control of a two-component regulatory system designated RocA1/S1/R. In this study, we revealed that Roc1-dependent induction of the cupB and cupC genes resulted in a high level of biofilm formation, with CupB and CupC acting with synergy in clustering bacteria for microcolony formation. Very importantly, this phenotype was associated with the assembly of cell surface fimbriae visualized by electron microscopy. Finally, we observed that the CupB and CupC systems are specialized in the assembly of their own fimbrial subunits and are not exchangeable.     

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.     

Environmental switching during biofilm development in a cold seep system and functional determinants of species sorting

The functional basis for species sorting theory remains elusive, especially for microbial community assembly in deep‐sea environments. Using artificial surface‐based biofilm models, our recent work revealed taxonomic succession during biofilm development in a newly defined cold seep system, the Thuwal cold seeps II, which comprises a brine pool and the adjacent normal bottom water (NBW) to form a metacommunity via the potential immigration of organisms from one patch to another. Here, we designed an experiment to investigate the effects of environmental switching between the brine pool and the NBW on biofilm assembly, which could reflect environmental filtering effects during bacterial immigration to new environments. Analyses of 16S rRNA genes of 71 biofilm samples suggested that the microbial composition of biofilms established in new environments was determined by both the source community and the incubation conditions. Moreover, a comparison of 18 metagenomes provided evidence for biofilm community assembly that was based primarily on functional features rather than taxonomic identities; metal ion resistance and amino acid metabolism were the major species sorting determinants for the succession of biofilm communities. Genome binning and pathway reconstruction of two bacterial species (Marinobacter sp. and Oleispira sp.) further demonstrated metal ion resistance and amino acid metabolism as functional traits conferring the survival of habitat generalists in both the brine pool and NBW. The results of this study shed new light on microbial community assembly in special habitats and bridge a gap in species sorting theory.     

MorA defines a new class of regulators affecting flagellar development and biofilm formation in diverse Pseudomonas species

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in Pseudomonas putida. MorA is conserved among diverse Pseudomonas species, and homologues are present in all Pseudomonas genomes sequenced thus far. In P. putida, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In Pseudomonas aeruginosa, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of P. putida, the motility of the P. aeruginosa mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of Pseudomonas species.     

The tad locus: postcards from the widespread colonization island

The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.     

Extracellular DNA in single- and multiple-species unsaturated biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

Impairment of the bacterial biofilm stability by triclosan

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition--isolated from sediments of the Eden Estuary (Scotland, UK)--on non-cohesive glass beads (<63 μm) and exposed to a range of triclosan concentrations (control, 2-100 μg L(-1)) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects.     

Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms

Detailed knowledge of the developmental process from single cells scattered on a surface to complex multicellular biofilm structures is essential in order to create strategies to control biofilm development. In order to study bacterial migration patterns during Pseudomonas aeruginosa biofilm development, we have performed an investigation with time-lapse confocal laser scanning microscopy of biofilms formed by various combinations of colour-coded P. aeruginosa wild type and motility mutants. We show that mushroom-shaped multicellular structures in P. aeruginosa biofilms can form in a sequential process involving a non-motile bacterial subpopulation and a migrating bacterial subpopulation. The non-motile bacteria form the mushroom stalks by growth in certain foci of the biofilm. The migrating bacteria form the mushroom caps by climbing the stalks and aggregating on the tops in a process which is driven by type-IV pili. These results lead to a new model for biofilm formation by P. aeruginosa.     

Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.     

Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces. In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage. Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P. aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA*. The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli. Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes. However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis. The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.     

Use of synthetic peptides to confirm that the Pseudomonas aeruginosa PAK pilus adhesin and the Candida albicans fimbrial adhesin possess a homologous receptor-binding domain

Pseudomonas aeruginosa PAK pili and Candida albicans fimbriae are adhesins present on the microbial cell surfaces which mediate binding to epithelial cell-surface receptors. The receptor-binding domain (adhesintope) of the PAK pilus adhesin has been shown previously to reside in the carboxy-terminal disulphide-bonded region of P. aeruginosa pilin (PAK128-144). The delineation of the C. albicans fimbrial adhesintope was investigated in these studies using synthetic peptides which correspond to the whole (PAK128-144) or part of (PAK134-140) adhesintope of the PAK pilus and their respective anti-peptide antisera and biotinylated PAK pili (Bt-PAK pili), fimbriae (Bt-fimbriae), P. aeruginosa whole cells (Bt- P. aeruginosa ) and C. albicans whole cells (Bt- C. albicans ). The results from these studies confirmed that a structurally conserved motif akin to the PAK(128-144) peptide sequence is present in C. albicans fimbrial adhesin and that the seven-amino-acid residue PAK(134-140) sequence plays an important role in forming the adhesintope for both P. aeruginosa PAK pilus and C. albicans fimbrial adhesins.     

Green and red fluorescent protein vectors for use in biofilm studies of the intrinsically resistant Burkholderia cepacia complex

Cystic fibrosis isolates of the Burkholderia cepacia complex (BCC) have demonstrated a propensity to associate intimately with Pseudomonas aeruginosa in mixed community biofilms, which may impact on their overall pathogenicity during infection of the lungs in cystic fibrosis. Here, we describe the construction and use of novel green and red fluorescent protein expression vectors suitable for labeling biofilm cells of multi-resistant clinical isolates of the BCC for microscopic analysis of both single species biofilms and mixed community associations with P. aeruginosa. Antimicrobial susceptibility testing established that tetracycline and/or trimethoprim were suitable selective agents for widespread use in BCC. The green and red fluorescent protein genes, driven by constitutively active promoters, were cloned into two mobilizable plasmids pBBR1MCS-3 and pBBR1Tp, carrying tetracycline and trimethoprim resistance cassettes, respectively. The fluorescence of transformed BCC and P. aeruginosa planktonic cells was detectable using fluorescence microscopy and/or fluorometry. The plasmids were stable in the absence of selection for at least 3 days in planktonic and biofilm cultures, and fluorescence was still visible in a 4-day glass coverslip flow cell biofilm. The plasmids functioned well to distinguish the two species in a mixed community biofilm, with no indications of plasmid transfer between species or cross-talk of the fluorescent signals. These vectors represent the first green and red fluorescent vectors to be constructed and analyzed specifically for wide spread use in BCC and P. aeruginosa single and mixed biofilm cultures.     

Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.