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Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: [ugd.sub.BCAL2946], [ugd.sub.BCAM0855] and [ugd.sub.BCAM2034], all of which were individually inactivated. Only inactivation of [ugd.sub.BCAL2946] resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either [ugd.sub.BCAL2946] or [ugd.sub.BCAM0855] but not [ugd.sub.BCAM2034] was expressed from plasmids. The growth of a conditional [ugd.sub.BCAL2946] mutant, created in the [DELTA][ugd.sub.BCAM0855] background, was significantly impaired under non-permissive conditions. Growth could be rescued by either [ugd.sub.BCAL2946] or [ugd.sub.BCAM0855] expressed in trans, but not by [ugd.sub.BCAM2034]. Biochemical analysis of the purified, recombinant forms of [ugd.sub.BCAL2946] and [ugd.sub.BCAM0855] revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and [NAD.sup.+]. Purified [Ugd.sub.BCAM2034] showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of [ugd.sub.BCAL2946] was 5.4- and 135-fold greater than that of [ugd.sub.BCAM0855] and [ugd.sub.BCAM2034], respectively. Together, these data indicate that the combined activity of [Ugd.sub.BCAL2946] and [Ugd.sub.BCAM0855] is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, [ugd.sub.BCAL2946], is required for polymyxin B resistance.

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.

The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET1 2 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

Pseudomonas aeruginosa is the pathogen most commonly associated with morbidity and mortality in cystic fibrosis (CF) patients. The host-pathogen interactions responsible for progressive CF lung diseases are complex. However, there is growing interest in the role of hypermutable P. aeruginosa (that is, those strains with an increased mutation frequency due to mutations in mismatch repair and error prevention genes), in terms of both bacterial adaptation and antimicrobial resistance. The prevalence of hypermutable P. aeruginosa in chronic CF infection has been established, and at 37% is surprisingly high. To the authors' knowledge, there are no reports of prevalence during the early stages of infection, in environmental pseudomonas, which are believed to be the primary source of infection, and in epidemic strains, which have emerged as a major challenge. The aim of this study was to establish the prevalence of hypermutable P. aeruginosa in these pseudomonas populations. The hypothesis was that hypermutability would be rare in early and in environmental P. aeruginosa but in contrast would explain the relatively recent emergence of epidemic strains. It was found that 10/100 (10%) of early isolates were strong or weak mutators, suggesting that the CF lung is not the only factor influencing the existence of mutators in this group of patients. Two weak mutators (6%) were found in 32 environmental isolates. Only two of 15 (13%) epidemic P. aeruginosa strains were hypermutable, and although closer analysis revealed this issue to be complex, on the whole the data suggested that the atypical characteristics of these highly transmissible strains cannot solely be explained by this phenomenon. The higher than predicted prevalence of mutators in early infection, and in environmental isolates, reinforces the importance of early and aggressive treatment for P. aeruginosa infection in CF.

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N. doi:10.1128/JB.00153-07

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified [...]

Studies were conducted for the BCP homologue of Burkholderia cenocepacia (BcBCP) and bacterioferritin comigratory protein (BCP) of Escherichia coli to clarify the catalytic mechanism related to the active and resolving cysteine and unresolving cysteine of BCP peroxiredins. The characterization supported the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.