46 research outputs found
The changing epidemiology of Burkholderia species infection at an adult cystic fibrosis centre
Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex
The Human Cathelicidin LL-37 Preferentially Promotes Apoptosis of Infected Airway Epithelium
Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria-epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37-mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens
Garlic Revisited: Antimicrobial Activity of Allicin-Containing Garlic Extracts against Burkholderia cepacia Complex
The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics
Epistatic Roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in Coping with Reactive Oxygen Species-Induced DNA Damage
Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H2O2)-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H2O2-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed
Cathelicidin and its role in defence against bacterial infections of epithelial cells
Cathelicidins are antimicrobial peptides (AMPs) that were first discovered to have
microbicidal properties but more recently to be multifunctional immunomodulators and thus
important in influencing host defence against infectious disease. Whilst roles in various
organs have been demonstrated, their expression patterns in health and disease in other
organs are less clear and their key immunomodulatory functions remain undefined,
particularly with regard to the balance of immunomodulatory properties and microbicidal
activity in their ability to promote defence against infection.
I therefore set out to describe LL-37 expression (human cathelicidin) in the female
reproductive tract (across the menstrual cycle) and in the lung (during specific lung diseases),
to define the effects on the function of airway epithelial cells during bacterial infection and to
evaluate the key in vivo roles of endogenous cathelicidin (using a knockout mouse model) as
well as the effect of therapeutic administration of LL-37 in a pulmonary Pseudomonas
aeruginosa infection model.
I demonstrated that cathelicidin protein and transcription shows a cyclical pattern of
expression in female reproductive tissues which is maintained at high levels in decidua. LL-
37 protein was also detected in hTERT endometrial epithelial cells but despite the suggestion
that cathelicidin may be regulated by steroid hormones there was no direct effect of
progesterone on transcription. LL-37 is barely detected in healthy airways however is well
known to increase during infection or inflammation. I observed that sputum from patients
with bronchiectasis showed a correlation between the level of LL-37, TNF, MPO and chronic
colonisation of Pseudomonas aeruginosa. Patients with lung cancer expressed much less LL-
37 than the bronchiectasis patients but there was a trend towards increased production postsurgery
compared to pre-surgery.
LL-37 was previously shown by our lab to selectively promote BAX and caspase-dependant
death of infected epithelial cells. I went on to show that this appears to be a partially caspase-
1 dependent mechanism and that human bronchial epithelial (HBE) cells and A549 cell lines
both express several of the components required to form inflammasomes, a caspase-1
dependant form of inflammatory cell death.
Finally, I showed using murine models that cathelicidin enhances bacterial clearance during
pulmonary infection in vivo, a response which is defective in mice lacking endogenous
cathelicidin and that administration of exogenous, synthetic LL-37 at the time of infection
can promote an early protective neutrophil influx in the absence of endogenous cathelicidin
production
Pseudomonas, alginate biosynthesis and cystic fibrosis
This thesis documents the author's research on the pathogenesis and epidemiology of the
pseudomonads in particular the association of Pseudomonas aeruginosa and Pseudomonas
cepacia with pulmonary colonisation and progressive lung damage in patients with cystic
fibrosis.One of the key virulence determinants in pulmonary colonisation of cystic fibrosis patients by
P. aeluginosa is biosynthesis of alginate. This unusual bacterial polysaccharide confers a
strikingly mucoid colonial phenotype and in vivo is associated with the formation of microbial
biofilms. During the last two decades, my research has focused on the genetic and molecular
regulation of alginate biosynthesis. The origins of these studies derive from my PhD research
on pyocins which led to the development of an internationally recognised typing system for
P. aeruginosa. In the early 1970s, pyocin typing data suggested that mucoid P. aeruginosa arise
in vivo, by mutation or environmental stimulation following primary asymptomatic colonisation
with a nonmucoid parent strain. My genetic studies of alginate regulation were developed in
the mid 1970s during an MRC Travelling Fellowship at Monash University, Melbourne and
produced the first evidence for chromosomal genes controlling the mucoid phenotype. These
embryonic studies and the muc mutations which were identified were to play an important
part in the subsequent molecular unravelling of the sensory regulation of alginate biosynthesis
in collaboration with colleagues in San Antonio. From 1980, my research on the
microbiology of pulmonary infections in cystic fibrosis patients has included studies of
antibiotic therapy, the epidemiology of pseudomonads and the use of animal models of
chronic respiratory infection. Currently, in collaboration with colleagues at the MRC Human
Genetics Unit in Edinburgh, I am involved in microbiological studies of the newly developed
cystic fibrosis mouse.The most common microbial pathogens in cystic fibrosis patients are Staphylococcus aureus,
Haemophilus influenzae and P. aeruginosa. From the mid 1980s, however, transmissible and
potentially fatal pulmonary infection by the phytopathogen P. cepacia has caused increasing
concern to patients and those involved in patient care. A major aim of my current research
is to clarify the epidemiology of P. cepacia and to identify the host and bacterial factors
associated with transmission and pathogenesis