Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced into Aeromonas salmonicida 449 which produces a surface protein array known as the A-layer. Kanamycin-resistant exconjugants of 449 with altered ability to produce the A-layer were selected by virtue of their altered colonial morphology and color on medium containing the dye Congo red. Analysis of culture supernatants, periplasmic shock fluid, outer membranes, and whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to A-protein revealed five classes of single-insertion mutations that affected the ability of cells to produce and export A-protein and to assemble the A-layer. These studies suggest that A-protein is produced from a single chromosomal gene. The subunits subsequently pass through the periplasm and across the outer membrane. At least one gene product is required for this export. Assembly of A-layer on the cell surface then requires the presence of O polysaccharide chains on the lipopolysaccharide. In one case, insertion of Tn5 resulted in loss of ability to produce both A-protein and lipopolysaccharide with O polysaccharide chains, suggesting that synthesis of A-protein and synthesis of lipopolysaccharide may involve coordinate regulation.     

Immunoproteomic Analysis of Proteins Expressed by Two Related Pathogens,Burkholderia multivorans and Burkholderia cenocepacia,during Human Infection

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.     

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.     

Differential modulation of innate immune cell functions by the Burkholderia cepacia complex: Burkholderia cenocepacia but not Burkholderia multivorans disrupts maturation and induces necrosis in human dendritic cells

Burkholderia cepacia complex (BCC) bacteria cause pulmonary infections that can evolve into fatal overwhelming septicemia in chronic granulomatous disease or cystic fibrosis patients. Burkholderia cenocepacia and Burkholderia multivorans are responsible for the majority of BCC infections in cystic fibrosis patients, but B. cenocepacia is generally associated with a poorer prognosis than B. multivorans. The present study investigated whether these pathogens could modulate the normal functions of primary human monocyte-derived dendritic cells (DCs), important phagocytic cells that act as critical orchestrators of the immune response. Effects of the bacteria on maturation of DCs were determined using flow cytometry. DCs co-incubated for 24 h with B. cenocepacia, but not B. multivorans, had reduced expression of costimulatory molecules when compared with standard BCC lipopolysaccharide-matured DCs. B. cenocepacia, but not B. multivorans, also induced necrosis in DCs after 24 h, as determined by annexin V and propidium iodide staining. DC necrosis only occurred after phagocytosis of live B. cenocepacia; DCs exposed to heat-killed bacteria, bacterial supernatant or those pre-treated with cytochalasin D then exposed to live bacteria remained viable. The ability of B. cenocepacia to interfere with normal DC maturation and induce necrosis may contribute to its pathogenicity in susceptible hosts.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.     

The Burkholderia cenocepacia peptidoglycan‐associated lipoprotein is involved in epithelial cell attachment and elicitation of inflammation

The Burkholderia cepacia complex (Bcc) is a group of Gram‐negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti‐virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan‐associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88‐fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O‐antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X‐ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5‐fold reduced stimulation of IL‐8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o^? cells, but adhesion of Pal‐expressing recombinant E. coli to CFBE41o^? cells was enhanced compared to wild‐type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.     

Quantitative proteomics (2-D DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy

Chronic respiratory infections caused by Burkholderia cenocepacia in patients with cystic fibrosis (CF) are characterized by low responsiveness to antibiotic therapy and, in general, to a more rapid decline of lung function. To get clues into the molecular mechanisms underlying the adaptive strategies employed to deal with the stressing conditions of the CF lung including antibiotic therapy, quantitative proteomics (2-D DIGE) was used to compare the expression programs of two clonal isolates retrieved from a chronically infected CF patient. Isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after 3 years of persistent infection and intravenous therapy with ceftazidime/gentamicin. This isolate exhibits higher resistance levels towards different classes of antimicrobials. Proteins of the functional categories Energy metabolism, Translation, Nucleotide synthesis, Protein folding and stabilization are more abundant in IST4113, compared with IST439, suggesting an increased protein synthesis, DNA repair and stress resistance in IST4113. The level of proteins involved in peptidoglycan, membrane lipids and lipopolysaccharide synthesis is also altered and proteins involved in iron binding and transport are more abundant in IST4113. The quantitative comparison of the two proteomes suggests a genetic adaptation leading to increased antimicrobial resistance and bacterial persistence in the CF airways.     

Genotypic characterization of Burkholderia cenocepacia strains by rep-PCR and PCR-RFLP of the fliC gene

Thirty-five strains of Burkholderia cenocepacia from clinical and environmental sources were characterized genotypically by repetitive sequence PCR (ERIC- and BOX-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC). In cluster analysis based on the repetitive PCR profiles the strains were composed of five clusters, of which clusters 1, 2 and 3 were more closely related to each other than to clusters 4 and 5. It has been reported that the majority of Burkholderia cepacia complex strains can be separated into two types on the basis of fliC size (types I and II correspond to 1.4 and 1.0 kb, respectively). When the strains were analysed by PCR of fliC, all strains yielded amplified products of 1.0 kb except for three strains. The latter strains gave PCR products of 0.7 kb (atypical type), which belonged to repetitive PCR cluster 5. These results indicated that the majority of B. cenocepacia strains belonged to flagellin type II. In the RFLP analysis of the large fliC amplicons with HaeIII, 10 patterns were observed indicating remarkable variation. Strains grouping in repetitive PCR cluster 4 had a unique fliC RFLP pattern. The results of repetitive PCR typing and PCR-RFLP analysis of fliC showed a strong correlation. Strains belonging to the repetitive PCR clusters 4 or 5 were distinctly different from other B. cenocepacia strains as shown by PCR-RFLP analysis of the fliC gene and phenotypic assays.     

Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

A total of 38 strains of atypical Aeromonas salmonicida , three oxidase-negative but otherwise typical Aer. salmonicida , three typical Aer. salmonicida , and two reference strains, isolated from several countries and fish species were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (> 55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer. salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile.     

High-affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a ¹²⁵I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. ¹²⁵I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48 degrees C, pH optimum at pH 9 and a molecular weight of 87500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

Host cell invasion and intracellular residence by Aeromonas salmonicida: role of the S-layer

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages. Here we report that S+ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants. When reconstituted with exogenous S-layers, these S- mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S+ and S- A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S+ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.     

Cable pili and the associated 22 kDa adhesin contribute to Burkholderia cenocepacia persistence in vivo

Background

Infection by Burkholderia cenocepacia in cystic fibrosis (CF) patients is associated with poor clinical prognosis. Previously, we demonstrated that one of the highly transmissible strains, BC7, expresses cable pili and the associated 22 kDa adhesin, both of which contribute to BC7 binding to airway epithelial cells. However, the contribution of these factors to induce inflammation and bacterial persistence in vivo is not known.

Methodology/Principal Findings

Wild-type BC7 stimulated higher IL-8 responses than the BC7 cbl and BC7 adhA mutants in both CF and normal bronchial epithelial cells. To determine the role of cable pili and the associated adhesin, we characterized a mouse model of B. cenocepacia, where BC7 are suspended in Pseudomonas aeruginosa alginate. C57BL/6 mice were infected intratracheally with wild-type BC7 suspended in either alginate or PBS and were monitored for lung bacterial load and inflammation. Mice infected with BC7 suspended in PBS completely cleared the bacteria by 3 days and resolved the inflammation. In contrast, mice infected with BC7 suspended in alginate showed persistence of bacteria and moderate lung inflammation up to 5 days post-infection. Using this model, mice infected with the BC7 cbl and BC7 adhA mutants showed lower bacterial loads and mild inflammation compared to mice infected with wild-type BC7. Complementation of the BC7 cblS mutation in trans restored the capacity of this strain to persist in vivo. Immunolocalization of bacteria revealed wild-type BC7 in both airway lumen and alveoli, while the BC7 cbl and BC7 adhA mutants were found mainly in airway lumen and peribronchiolar region.

Conclusions and Significance

B. cenocepacia suspended in alginate can be used to determine the capacity of bacteria to persist and cause lung inflammation in normal mice. Both cable pili and adhesin contribute to BC7-stimulated IL-8 response in vitro, and BC7 persistence and resultant inflammation in vivo.