F. Reen

The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms

Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs). However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

Subinhibitory concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa

Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection in cystic fibrosis (CF) lungs. The effects of subinhibitory concentrations of colistin on gene expression in P. aeruginosa were investigated by transcriptome and functional genomic approaches. Analysis revealed altered expression of 30 genes representing a variety of pathways associated with virulence and bacterial colonization in chronic infection. These included response to osmotic stress, motility, and biofilm formation, as well as genes associated with LPS modification and quorum sensing (QS). Most striking was the upregulation of Pseudomonas quinolone signal (PQS) biosynthesis genes, including pqsH, pqsB and pqsE, and the phenazine biosynthesis operon. Induction of this central component of the QS network following exposure to subinhibitory concentrations of colistin may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF lung.

Emerging Concepts Promising New Horizons for Marine Biodiscovery and Synthetic Biology

The vast oceans of the world, which comprise a huge variety of unique ecosystems, are emerging as a rich and relatively untapped source of novel bioactive compounds with invaluable biotechnological and pharmaceutical potential. Evidence accumulated over the last decade has revealed that the diversity of marine microorganisms is enormous with many thousands of bacterial species detected that were previously unknown. Associated with this diversity is the production of diverse repertoires of bioactive compounds ranging from peptides and enzymes to more complex secondary metabolites that have significant bioactivity and thus the potential to be exploited for innovative biotechnology. Here we review the discovery and functional potential of marine bioactive peptides such as lantibiotics, nanoantibiotics and peptidomimetics, which have received particular attention in recent years in light of their broad spectrum of bioactivity. The significance of marine peptides in cell-to-cell communication and how this may be exploited in the discovery of novel bioactivity is also explored. Finally, with the recent advances in bioinformatics and synthetic biology, it is becoming clear that the integration of these disciplines with genetic and biochemical characterization of the novel marine peptides, offers the most potential in the development of the next generation of societal solutions.

Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY

A steep oxygen gradient within the mucus of the cystic fibrosis (CF) lung combined with the biofilm mode of bacterial growth forces respiratory pathogens to adapt to varying oxygen availability. This study presents the novel finding that the Pseudomonas aeruginosa response to limiting oxygen stress includes induction of its type III secretion system (T3SS), which subsequently contributes towards host cell cytotoxicity. In P. aeruginosa, the global anaerobic response regulator Anr perceives low oxygen and subsequently triggers gene expression of a range of target genes, including the response regulator narL. Here we demonstrate that microaerobic induction of the T3SS is dependent on Anr, and that this is mediated through direct NarL transcriptional repression of the sRNAs rsmY and rsmZ, allowing free RsmA protein to positively regulate the T3SS. This study reveals a novel interplay between the Anr-NarL and RsmAYZ regulatory circuits, and introduces RsmA as an important regulator during P. aeruginosa adaptation to a low-oxygen environment.

Detection of the Pseudomonas Quinolone Signal (PQS) by cyclic voltammetry and amperometry using a boron doped diamond electrode

2-Heptyl-3-hydroxy-4-quinolone, known as the Pseudomonas Quinolone Signal, is a key regulator of bacterial cooperative behaviour known as quorum sensing. A simple electrochemical strategy was employed for its sensitive detection using a bare boron-doped diamond electrode by cyclic voltammetry and amperometry. PQS (and potentially other quinolones) was then detected in cultures of P. aeruginosa pqsL(-) mutant strains.

Pseudomonas aeruginosa Alkyl Quinolones Repress Hypoxia-Inducible Factor 1 (HIF-1) Signaling through HIF-1α Degradation

ABSTRACT The transcription factor hypoxia-inducible factor 1 (HIF-1) has recently emerged to be a crucial regulator of the immune response following pathogen perception, including the response to the important human pathogen Pseudomonas aeruginosa. However, as mechanisms involved in HIF-1 activation by bacterial pathogens are not fully characterized, understanding how bacteria and bacterial compounds impact on HIF-1α stabilization remains a major challenge. In this context, we have focused on the effect of secreted factors of P. aeruginosa on HIF-1 regulation. Surprisingly, we found that P. aeruginosa cell-free supernatant significantly repressed HIF-1α protein levels. Further characterization revealed that HIF-1α downregulation was dependent on a subset of key secreted factors involved in P. aeruginosa pathogenesis, the 2-alkyl-4-quinolone (AQ) quorum sensing (QS) signaling molecules, and in particular the pseudomonas quinolone signal (PQS). Under hypoxic conditions, the AQ-dependent downregulation of HIF-1α was linked to the suppressed induction of the important HIF-1 target gene hexokinase II. Furthermore, we demonstrated that AQ molecules directly target HIF-1α protein degradation through the 26S-proteasome proteolytic pathway but independently of the prolyl hydroxylase domain (PHD). In conclusion, this is the first report showing that bacterial molecules can repress HIF-1α protein levels. Manipulation of HIF-1 signaling by P. aeruginosa AQs could have major consequences for the host response to infection and may facilitate the infective properties of this pathogen.

Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans

Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens.

Respiratory Pathogens Adopt a Chronic Lifestyle in Response to Bile

Chronic respiratory infections are a major cause of morbidity and mortality, most particularly in Cystic Fibrosis (CF) patients. The recent finding that gastro-esophageal reflux (GER) frequently occurs in CF patients led us to investigate the impact of bile on the behaviour of Pseudomonas aeruginosa and other CF-associated respiratory pathogens. Bile increased biofilm formation, Type Six Secretion, and quorum sensing in P. aeruginosa, all of which are associated with the switch from acute to persistent infection. Furthermore, bile negatively influenced Type Three Secretion and swarming motility in P. aeruginosa, phenotypes associated with acute infection. Bile also modulated biofilm formation in a range of other CF-associated respiratory pathogens, including Burkholderia cepacia and Staphylococcus aureus. Therefore, our results suggest that GER-derived bile may be a host determinant contributing to chronic respiratory infection.

Structure-function analysis of the C-3 position in analogues of microbial behavioural modulators HHQ and PQS.

2-Heptyl-3-hydroxy-4-quinolone (PQS) and its precursor 2-heptyl-4-quinolone (HHQ) are key signalling molecules of the important nosocomial pathogen Pseudomonas aeruginosa. We have recently reported an interkingdom dimension to these molecules, influencing key virulence traits in a broad spectrum of microbial species and in the human pathogenic yeast Candida albicans. For the first time, targeted chemical derivatisation of the C-3 position was undertaken to investigate the structural and molecular properties underpinning the biological activity of these compounds in P. aeruginosa, and using Bacillus subtilis as a suitable model system for investigating modulation of interspecies behaviour.

A novel host-responsive sensor mediates virulence and type III secretion during Pseudomonas aeruginosa-host cell interactions

Sensitive sensory mechanisms are instrumental in affording Pseudomonas aeruginosa the capacity to establish diverse yet severe human infections, which can manifest themselves in long-term untreatable disease. The ability of P. aeruginosa to tightly regulate gene expression and virulence factor production, in response to activation of these sensory components, enables the pathogen to sustain infection despite the host immune response and aggressive antibiotic treatment. Although a number of factors are recognized as playing a role in early infection, very little is known regarding the sensors involved in this process. In this study, we identified P. aeruginosa PA3191 as a novel host-responsive sensor that plays a key role during P. aeruginosa-host interactions and is required for optimum colonization and dissemination in a mouse model of infection. We demonstrated that PA3191 contributed to modulation of the type III secretion system (T3SS) in response to host cells and T3SS-inducing conditions in vitro. PA3191 (designated GtrS) acted in concert with the response regulator GltR to regulate the OprB transport system and subsequently carbon metabolism. Through this signal transduction pathway, T3SS activation was mediated via the RsmAYZ regulatory cascade and involved the global anaerobic response regulator Anr.