Twenty-eight divergent polysaccharide loci specifying within and amongst strain capsule diversity in three strains of Bacteroides fragilis

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsule polysaccharide is a bacterial decoy for antimicrobial peptides

Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil alpha-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.

The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-kappaB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Effect of short-term macrophage depletion in the development of posterior capsule opacification in rodents

Aim: To evaluate the role of macrophages in the development of posterior capsule opacification (PCO). Methods: For this purpose, an extracapsular lens extraction was performed in 18 consecutive Sprague-Dawley rats. Animals were treated with liposomal clodronate (Cl MDP-lip-treated group, n = 10) or phosphate-buffered saline (PBS) (control group, n = 8) 1 day preoperatively and on the first day postoperatively, and sacrificed 3 days postoperatively. Masked clinical, light microscopy and immunohistochemistry studies were conducted. The Fisher exact test and randomisation test were used to assess statistically differences between groups. Results: A statistically significant reduction in the number of macrophages (ED1+, ED7+, ED8+) was found in the Cl MDP-lip-treated group compared with the PBS-lip-treated group (p = 0.048, p = 0.004, p = 0.027, respectively). There were no statistically significant differences with regards to the presence/absence of central opacification (p = 0.29) and capsular wrinkling (p = 0.21) as detected clinically between groups. Similarly, a qualitative evaluation of the degree of PCO with regards to lens epithelial cell (LEC) proliferation, capsular wrinkling and Soemmerring ring formation showed no statistically significance between groups (p = 0.27, p = 0.061, p = 1.0, respectively). However, a statistically significant reduction in the number of lens epithelial cells (LEC) counted in the centre of the posterior capsule was found in the Cl MDP-lip- treated group (p = 0.009). Conclusion: Depletion of macrophages was accompanied by a reduction in LEC in the centre of the posterior capsule in rodents.

Effect of TGF-β2 and anti-TGF-β2 antibody in a new in vivo rodent model of posterior capsule opacification

PURPOSE. This study evaluated the effect of transforming growth factor (TGF)-ß2 and anti-TGF-ß2 antibody in a rodent model of posterior capsule opacification (PCO). METHODS. An extracapsular lens extraction (ECLE) was performed in 72 Sprague-Dawley rats. At the end of the procedure, 10 µL TGF-ß2 (TGF-ß2-treated group), fetal calf serum (FCS)/phosphate- buffered saline (PBS; FCS/PBS-treated control group), a human monoclonal TGF-ß2 antibody (anti-TGF-ß2-treated group), or a null control IgG4 antibody (null antibody-treated control group) was injected into the capsule. Animals were killed 3 and 14 days postoperatively. Eyes were evaluated clinically prior to euthanatization, then enucleated and processed for light microscopy and immunohistochemistry afterward. PCO was evaluated clinically and histopathologically. Student's t-test and ? were used to assess differences between groups. RESULTS. There were no statistically significant clinical or histopathological differences in degree of PCO between the TGF-ß2- and FCS/PBS-treated groups at 3 and 14 days after ECLE. Nor were there differences between the anti-TGF-ß2- and the null antibody-treated groups, with the exception of the histopathology score for capsule wrinkling 3 days after ECLE (P = 0.02). a-Smooth-muscle actin staining was observed in the lens capsular bag only in areas where there was close contact with the iris. CONCLUSIONS. No sustained effect of TGF-ß2 or anti-TGF-ß2 antibody on PCO was found in rodents at the dose and timing administered in this study. Iris cells may play a role in the process of epithelial mesenchymal transition linked to PCO. Copyright © Association for Research in Vision and Ophthalmology.

Posterior capsule opacification in mice

Objective: To present a new model of posterior capsule opacification (PCO) in mice. Methods: An extracapsular lens extraction was performed in 28 consecutive mice. Animals were humanely killed 0 and 24 hours and 3 and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. Results: In 20 animals (71%), the eye appeared well healed before death. In 8 animals (29%), postoperative complications were noted. All animals developed PCO 2 weeks after surgery. Immediately after extracapsular lens extraction, lens epithelial cells were present in the inner surface of the anterior capsule and at the lens bow. At 24 hours, lens epithelial cells started to migrate toward the center of the posterior capsule. At 3 days, multilayered lens epithelial cells throughout the lens capsule and capsular wrinkling were apparent. Lens fibers and Soemmerring ring formation were observed 14 days after surgery. CD45 and CD11b macrophages were found in greater numbers 24 hours and 3 days after surgery (CD45 , P = .04 and P <.001, respectively; and CD11b , P = .01 and P = .004, respectively). The number of CD45 cells remained statistically significantly higher (P = .04) 14 days after surgery. Conclusion: In mice, PCO occurs following extracapsular lens extraction and is associated with low-grade but significant macrophage response. Clinical Relevance: The use of genetically modified mice to evaluate the pathogenic mechanisms of PCO and search for new therapeutic modalities to prevent or treat PCO is now possible.

A new model of posterior capsule opacification in rodents

PURPOSE. To describe a new model of posterior capsule opacification (PCO) in rodents METHODS. An extracapsular lens extraction (ECLE), by continuous curvilinear capsulorrhexis and hydrodissection, was performed in 42 consecutive Brown Norway rats. Animals were killed at 0, 6, and 24 hours and 3, 7, and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. RESULTS. In 34 (81%) of the animals the operated eye appeared well healed before death, with a clear cornea and a well-formed anterior chamber. In eight (19%) there was no view of anterior segment structures because of hyphema, fibrin, or corneal opacification. PCO was clinically evident 3 days after ECLE and was present in all animals at 2 weeks. Immediately after ECLE, lens epithelial cells (LECs) were present in the inner surface of the anterior capsule and lens bow. Twenty-four hours after surgery, LECs started to migrate toward the center of the posterior capsule. At 3 days, multilayered LECs, some spindle shaped, were present throughout the lens capsule. Capsular wrinkling was apparent. Lens fibers and Soemmering's ring were observed in all animals 14 days after surgery, indicating some degree of cellular differentiation. Activated macrophages were present in greater numbers at 3 and 14 days after surgery (P <0.05), when proliferation and migration of LECs appeared to be greatest, and lens fiber differentiation was evident, respectively. CONCLUSIONS. In rodents PCO occurs after ECLE and is associated with low-grade inflammation, mostly of mononuclear macrophages. Although no intraocular lens implantation was performed, this model appears to be valuable for studying the sequence of events that leads to PCO after cataract surgery and the extracellular matrix cues that promote lens fiber differentiation.

An investigation into the structure and bioavailability of a-tocopherol dispersions in Gelucire 44/14

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, ±-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of ±-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU ±-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total ±-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.