Matthew Thoendel

Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms

The process of detachment, through which bacteria use active mechanisms to leave biofilms and return to the planktonic (free‐living) state, is perhaps the least understood aspect of the biofilm life cycle. Like other stages of biofilm development, detachment is a dynamic, regulated process, controlled by specific genes, and induced by particular environmental cues. In previous work we discovered Pseudomonas aeruginosa variants that exhibit accelerated biofilm detachment. These hyper‐detaching variants arise spontaneously from biofilms at a high frequency, and they exhibit robust detachment under different biofilm growth conditions. Here we show that these variants detach by a mechanism requiring the biosurfactant rhamnolipid and that this detachment mechanism rapidly restores antibiotic sensitivity to separating bacteria. We also show that rhamnolipids can bring about detachment in wild‐type P. aeruginosa biofilms. These findings raise the possibility that this detachment mechanism may be useful as a treatment to disrupt established biofilms. Interestingly, the rhamnolipid‐mediated detachment mechanism involves the formation of cavities within the centre of biofilm structures. Our data suggest a model to explain detachment that occurs via this pattern.

Peptide Signaling in the Staphylococci

Gram-negative and Gram-positive bacteria have evolved elaborate machinery to biosynthesize and respond to diverse small-molecule signals. As bacteria grow, these signals accumulate in the extracellular environment until a particular concentration is reached, usually at a specific cell density or “quorum”, activating a regulatory cascade that controls some type of cellular process. This phenomenon is generally referred to as “quorum-sensing” and has been the subject of many excellent review articles1-3. The general paradigm is that Gram-negatives recognize small chemical compounds called N-acyl homoserine lactones that are membrane permeable and bind to a cytoplasmic receptor in order to exert a regulatory output. In contrast, the Gram-positives recognize peptides with diverse post-translational modifications using either a membrane-bound histidine kinase or cytoplasmic receptors. Amongst the Gram-positives, the size and structure of these peptide signals vary widely depending on function and the producing bacterium, and published examples of these regulatory mechanisms have become abundant. As notable examples, Streptococcus pneumoniae regulates competence with a 17-residue linear peptide4. Bacillus subtilis regulates sporulation and competence with a series of linear peptides5, one of which is post-translationally modified6. Bacillus cereus regulates the expression of virulence factors and Enterococcus faecalis controls plasmid-mediated conjugation with various linear peptides7-8. As this quick overview demonstrates, peptides are regulating an impressive array of cellular events, and this list continues to grow as additional systems are being discovered. One of the more intriguing classes of peptide signals are the cyclic lactones and thiolactones. The first of these cyclic peptide signals was discovered in Staphylococcus aureus and is the focus of this review article. The peptide signal controls an autoactivation circuit and hence is referred to as an autoinducing peptide or “AIP”. With the surge of studies on quorum-sensing and bacterial genome sequencing, it is now evident that the AIP scaffold and autoactivation circuitry is conserved among many Gram-positive bacteria9. Notably, all of the staphylococcal species make similar AIP structures10-11, and in recent studies, related signals have been identified in Enterococcus faecalis12-14, Lactobacillus plantarum15-16, Listeria monocytogenes17-18, Clostridium perfringens19-20 and C. botulinum21. Genome mining has revealed additional agr-like systems in other Gram-positives 9, such as the outbreak C. difficile 027 strain22 and in some species of Bacillus. In this review, we will focus on the accessory gene regulator or “agr” quorum-sensing system in S. aureus as a paradigm model. We will describe what is known about the function of each gene product in the agr locus and the mechanism of signal production. Signal sensing and output will be reviewed, along with the contribution of other regulatory inputs to agr function. We will also describe the current status of agr in biofilms and pathogenesis, and outline the latest advances in agr-targeted therapies. Finally, the similarities and differences of the agr system in other Staphylococci will be described.

Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.

agr-Dependent Interactions of Staphylococcus aureus USA300 with Human Polymorphonuclear Neutrophils

The emergence of serious infections due to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has fueled interest in the contributions of specific staphylococcal virulence factors to clinical disease. To assess the contributions of agr-dependent factors to the fate of organisms in polymorphonuclear neutrophils (PMN), we examined the consequences for organism and host cells of feeding PMN with wild-type CA-MRSA (LAC) or CA-MRSA (LAC agr KO) at different multiplicities of infection (MOIs). Phagocytosed organisms rapidly increased the transcription of RNAIII in a time- and MOI-dependent fashion; extracellular USA300 (LAC) did not increase RNAIII expression despite having the capacity to respond to autoinducing peptide-enriched culture medium. HOCl-mediated damage and intracellular survival were the same in the wild-type and USA300 (LAC agr KO). PMN lysis by ingested USA300 (LAC) was time- and MOI-dependent and, at MOIs >1, required α-hemolysin (hla) as USA300 (LAC agr KO) and USA300 (LAC hla KO) promoted PMN lysis only at high MOIs. Taken together, these data demonstrate activation of the agr operon in human PMN with the subsequent production of α-hemolysin and PMN lysis. The extent to which these events in the phagosomes of human PMN contribute to the increased morbidity and mortality of infections with USA300 (LAC) merits further study.

Fluorescent reporters for Staphylococcus aureus

With the emergence of Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the agr quorum-sensing and sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence-activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow-cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.

A role for type I signal peptidase in Staphylococcus aureus quorum sensing.

The Staphylococcus aureus Agr quorum‐sensing system modulates the expression of extracellular virulence factors. The Agr system is controlled by an autoinducing peptide (AIP) molecule that is secreted during growth. In the AIP biosynthetic pathway, two proteolytic events are required to remove the leader and tail segments of AgrD, the peptide precursor of AIP. The only protein known to be involved in this pathway is AgrB, a membrane endopeptidase that removes the AgrD carboxy‐tail. We designed a synthetic peptide substrate and developed an assay to detect peptidases that can remove the N‐terminal leader of AIP. Several peptidase activities were detected in S. aureus extracts and these activities were present in both wild‐type and agr mutant strains. Only one of these peptidases cleaved in the correct position and all properties of this enzyme were consistent with type I signal peptidase. Subsequent cloning and purification of the two known S. aureus signal peptidases, SpsA and SpsB, demonstrated that only SpsB catalysed this activity in vitro. To investigate the role of SpsB in AIP biosynthesis, SpsB peptide inhibitors were designed and characterized. The most effective inhibitor blocked SpsB activity in vitro and showed antibacterial activity against S. aureus. Importantly, the inhibitor reduced expression of an Agr‐dependent reporter and inhibited AIP production in S. aureus, indicating a role for SpsB in quorum sensing.

Biosynthesis of Peptide Signals in Gram-Positive Bacteria

Gram-positive bacteria coordinate social behavior by sensing the extracellular level of peptide signals. These signals are biosynthesized through divergent pathways and some possess unusual functional chemistry as a result of posttranslational modifications. In this chapter, the biosynthetic pathways of Bacillus intracellular signaling peptides, Enterococcus pheromones, Bacillus subtilis competence pheromones, and cyclic peptide signals from Staphylococcus and other bacteria are covered. With the increasing prevalence of the cyclic peptide signals in diverse Gram-positive bacteria, a focus on this biosynthetic mechanism and variations on the theme are discussed. Due to the importance of peptide systems in pathogenesis, there is emerging interest in quorum-quenching approaches for therapeutic intervention. The quenching strategies that have successfully blocked signal biosynthesis are also covered. As peptide signaling systems continue to be discovered, there is a growing need to understand the details of these communication mechanisms. This information will provide insight on how Gram-positives coordinate cellular events and aid strategies to target these pathways for infection treatments.

Identification of Staphylococcus aureus AgrD Residues Required for Autoinducing Peptide Biosynthesis

Staphylococcus aureus regulates the production of extracellular virulence factors using the agr quorum-sensing system. This regulatory system responds to a secreted peptide thiolactone signal called an autoinducing peptide (AIP). The biosynthesis of AIP requires AgrD, the peptide precursor of AIP, and the integral membrane endopeptidase AgrB. In this study, we performed a molecular analysis of AgrD to identify peptide regions important for processing and AIP secretion. As a lead-in to this study, we discovered that AIP type I could be generated in Escherichia coli through the heterologous expression of the agrBD genes, allowing the use of E. coli as an expression host for investigating the biosynthetic pathway. One of the most conserved regions of AgrD is the charged C-terminal tail, and through truncation analysis, the first nine residues were found to be essential for AIP production and AgrB endopeptidase activity. Within this essential region, mutation of residues glutamate 34 or leucine 41 inhibited AIP production and AgrB activity. Following cleavage, AgrB is hypothesized to form an enzyme-bound intermediate with the AgrD N-terminal region, but clear evidence of this intermediate has never been presented. By inactivating the AgrD cysteine 28 residue, an AgrD-AgrB structure was stabilized and detected in immunoblots using N-terminal His6-tagged AgrD. Formation of the structure could not be detected using the AgrB C84S mutation, indicating the cysteine residue is essential for its formation. These studies provide new insights on the requirements and mechanism of S. aureus AIP biosynthesis.

Staphylococcus aureus regulates the expression and production of the staphylococcal superantigen‐like secreted proteins in a Rot‐dependent manner

Staphylococcus aureus overproduces a subset of immunomodulatory proteins known as the staphylococcal superantigen‐like proteins (Ssls) under conditions of pore‐mediated membrane stress. In this study we demonstrate that overproduction of Ssls during membrane stress is due to the impaired activation of the two‐component module of the quorum‐sensing accessory gene regulator (Agr) system. Agr‐dependent repression of ssl expression is indirect and mediated by the transcription factor repressor of toxins (Rot). Surprisingly, we observed that Rot directly interacts with and activates the ssl promoters. The role of Agr and Rot as regulators of ssl expression was observed across several clinically relevant strains, suggesting that overproduction of immunomodulatory proteins benefits agr‐defective strains. In support of this notion, we demonstrate that Ssls contribute to the residual virulence of S. aureus lacking agr in a murine model of systemic infection. Altogether, these results suggest that S. aureus compensates for the inactivation of Agr by producing immunomodulatory exoproteins that could protect the bacterium from host‐mediated clearance.

Random mutagenesis and topology analysis of the autoinducing peptide biosynthesis proteins in Staphylococcus aureus

The Staphylococcus aureus accessory gene regulator (agr) is a peptide signalling system that regulates the production of secreted virulence factors required to cause infections. The signal controlling agr function is a 7‐9 residue thiolactone‐containing peptide called an autoinducing peptide (AIP) that is biosynthesized from the AgrD precursor by the membrane peptidase AgrB. To gain insight into AgrB and AgrD function, the agrBD genes were mutagenized and screened for deficiencies in AIP production. In total, single‐site mutations at 14 different residues in AgrD were identified and another 20 within AgrB. In AgrD, novel mutations were characterized in the N‐terminal leader and throughout the central region encoding the AIP signal. In AgrB, most mutations blocked peptidase activity, but mutations in the K129–K131 residues were defective in a later step in AIP biosynthesis, separating the peptidase function from thiolactone ring formation and AIP transport. With the identification of residues in AgrB essential for AgrD processing, we reevaluated the membrane topology and the new model predicts four transmembrane helices and a potential re‐entrant loop on the cytoplasmic face. Finally, co‐immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. These studies provide further insight into the unique structural and functional properties of AgrB.