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1.  The role of two Pseudomonas aeruginosa anthranilate synthases in tryptophan and quorum signal production 
Microbiology  2013;159(Pt 5):959-969.
Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes infections in the lungs of individuals with the genetic disease cystic fibrosis. Density-dependent production of toxic factors regulated by the Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone; PQS) have been proposed to be involved in P. aeruginosa virulence. PQS biosynthesis requires conversion of the central metabolite chorismate to anthranilate by anthranilate synthase. This reaction is also the first step in tryptophan biosynthesis. P. aeruginosa possesses two functional anthranilate synthases, TrpEG and PhnAB, and these enzymes are not functionally redundant, as trpEG mutants are tryptophan auxotrophs but produce PQS while mutants in phnAB are tryptophan prototrophs but do not produce PQS in minimal media. The goal of the work described in this paper was to determine the mechanism for this lack of functional complementation of TrpEG and PhnAB. Our results reveal that overexpression of either enzyme compensates for tryptophan auxotrophy and PQS production in the trpEG and phnAB mutants respectively, leading to the hypothesis that differential regulation of these genes is responsible for the lack of functional complementation. In support of this hypothesis, trpEG was shown to be expressed primarily during low-density growth while phnAB was expressed primarily at high density. Furthermore, dysregulation of phnAB expression eliminated tryptophan auxotrophy in the P. aeruginosa trpEG mutant. Based on these data, we propose a model for anthranilate sequestration by differential transcriptional regulation of the two P. aeruginosa anthranilate synthase enzymes.
doi:10.1099/mic.0.063065-0
PMCID: PMC3709558  PMID: 23449919
2.  Probing Bacterial Metabolism during Infection Using High-Resolution Transcriptomics 
Journal of Bacteriology  2013;195(22):4991-4998.
A fundamental aspect of most infectious diseases is the need for the invading microbe to proliferate in the host. However, little is known about the metabolic pathways required for pathogenic microbes to colonize and persist in their hosts. In this study, we used RNA sequencing (RNA-seq) to generate a high-resolution transcriptome of the opportunistic pathogen Aggregatibacter actinomycetemcomitans in vivo. We identified 691 A. actinomycetemcomitans transcriptional start sites and 210 noncoding RNAs during growth in vivo and as a biofilm in vitro. Compared to in vitro biofilm growth on a defined medium, ∼14% of the A. actinomycetemcomitans genes were differentially regulated in vivo. A disproportionate number of genes coding for proteins involved in metabolic pathways were differentially regulated in vivo, suggesting that A. actinomycetemcomitans in vivo metabolism is distinct from in vitro growth. Mutational analyses of differentially regulated genes revealed that formate dehydrogenase H and fumarate reductase are important A. actinomycetemcomitans fitness determinants in vivo. These results not only provide a high-resolution genomic analysis of a bacterial pathogen during in vivo growth but also provide new insight into metabolic pathways required for A. actinomycetemcomitans in vivo fitness.
doi:10.1128/JB.00875-13
PMCID: PMC3811578  PMID: 23974023
3.  Oxygen Limitation within a Bacterial Aggregate 
mBio  2014;5(2):e00992-14.
ABSTRACT
Cells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >108 cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤105 bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D “microtraps” that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (1012 cells ml−1) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations.
IMPORTANCE
Before developing into large, complex communities, microbes initially cluster into aggregates, and it is unclear if chemical heterogeneity exists in these ubiquitous micrometer-scale aggregates. We chose to examine oxygen availability within an aggregate since oxygen concentration impacts a number of important bacterial processes, including metabolism, social behaviors, virulence, and antibiotic resistance. By determining that oxygen availability can vary within aggregates containing ≤105 bacteria, we establish that physiological heterogeneity exists within P. aeruginosa aggregates, suggesting that such heterogeneity frequently exists in many naturally occurring small populations.
doi:10.1128/mBio.00992-14
PMCID: PMC3994514  PMID: 24736225
4.  Microbial Communication Superhighways 
Cell  2011;144(4):469-470.
Exchange of information is critical for bacterial social behaviors. Now Dubey and Ben-Yehuda (2011) provide evidence for bacterial “nanotube” conduits that allow microbes to directly exchange cytoplasmic factors. Protein and DNA transfer between distantly related species raises the prospect of a new, widely distributed mechanism of bacterial communication.
doi:10.1016/j.cell.2011.02.001
PMCID: PMC3984534  PMID: 21335231
5.  Going local: technologies for exploring bacterial microenvironments 
Nature reviews. Microbiology  2013;11(5):337-348.
Microorganisms lead social lives and use coordinated chemical and physical interactions to establish complex communities. Mechanistic insights into these interactions have revealed that there are remarkably intricate systems for coordinating microbial behaviour, but little is known about how these interactions proceed in the spatially organized communities that are found in nature. This Review describes the technologies available for spatially organizing small microbial communities and the analytical methods for characterizing the chemical environment surrounding these communities. Together, these complementary technologies have provided novel insights into the impact of spatial organization on both microbial behaviour and the development of phenotypic heterogeneity within microbial communities.
doi:10.1038/nrmicro3010
PMCID: PMC3984535  PMID: 23588251
6.  Metatranscriptomics of the Human Oral Microbiome during Health and Disease 
mBio  2014;5(2):e01012-14.
ABSTRACT
The human microbiome plays important roles in health, but when disrupted, these same indigenous microbes can cause disease. The composition of the microbiome changes during the transition from health to disease; however, these changes are often not conserved among patients. Since microbiome-associated diseases like periodontitis cause similar patient symptoms despite interpatient variability in microbial community composition, we hypothesized that human-associated microbial communities undergo conserved changes in metabolism during disease. Here, we used patient-matched healthy and diseased samples to compare gene expression of 160,000 genes in healthy and diseased periodontal communities. We show that health- and disease-associated communities exhibit defined differences in metabolism that are conserved between patients. In contrast, the metabolic gene expression of individual species was highly variable between patients. These results demonstrate that despite high interpatient variability in microbial composition, disease-associated communities display conserved metabolic profiles that are generally accomplished by a patient-specific cohort of microbes.
IMPORTANCE
The human microbiome project has shown that shifts in our microbiota are associated with many diseases, including obesity, Crohn’s disease, diabetes, and periodontitis. While changes in microbial populations are apparent during these diseases, the species associated with each disease can vary from patient to patient. Taking into account this interpatient variability, we hypothesized that specific microbiota-associated diseases would be marked by conserved microbial community behaviors. Here, we use gene expression analyses of patient-matched healthy and diseased human periodontal plaque to show that microbial communities have highly conserved metabolic gene expression profiles, whereas individual species within the community do not. Furthermore, disease-associated communities exhibit conserved changes in metabolic and virulence gene expression.
doi:10.1128/mBio.01012-14
PMCID: PMC3977359  PMID: 24692635
7.  Pseudomonas aeruginosa Enhances Production of a Non-Alginate Exopolysaccharide during Long-Term Colonization of the Cystic Fibrosis Lung 
PLoS ONE  2013;8(12):e82621.
The gram-negative opportunistic pathogen Pseudomonas aeruginosa is the primary cause of chronic respiratory infections in individuals with the heritable disease cystic fibrosis (CF). These infections can last for decades, during which time P. aeruginosa has been proposed to acquire beneficial traits via adaptive evolution. Because CF lacks an animal model that can acquire chronic P. aeruginosa infections, identifying genes important for long-term in vivo fitness remains difficult. However, since clonal, chronological samples can be obtained from chronically infected individuals, traits undergoing adaptive evolution can be identified. Recently we identified 24 P. aeruginosa gene expression traits undergoing parallel evolution in vivo in multiple individuals, suggesting they are beneficial to the bacterium. The goal of this study was to determine if these genes impact P. aeruginosa phenotypes important for survival in the CF lung. By using a gain-of-function genetic screen, we found that 4 genes and 2 operons undergoing parallel evolution in vivo promote P. aeruginosa biofilm formation. These genes/operons promote biofilm formation by increasing levels of the non-alginate exopolysaccharide Psl. One of these genes, phaF, enhances Psl production via a post-transcriptional mechanism, while the other 5 genes/operons do not act on either psl transcription or translation. Together, these data demonstrate that P. aeruginosa has evolved at least two pathways to over-produce a non-alginate exopolysaccharide during long-term colonization of the CF lung. More broadly, this approach allowed us to attribute a biological significance to genes with unknown function, demonstrating the power of using evolution as a guide for targeted genetic studies.
doi:10.1371/journal.pone.0082621
PMCID: PMC3855792  PMID: 24324811
8.  Role of Pseudomonas aeruginosa Peptidoglycan-Associated Outer Membrane Proteins in Vesicle Formation 
Journal of Bacteriology  2013;195(2):213-219.
Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multiple Enterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogen Pseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of the Pseudomonas quinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI.
doi:10.1128/JB.01253-12
PMCID: PMC3553829  PMID: 23123904
9.  An Evolutionary Link between Natural Transformation and CRISPR Adaptive Immunity 
mBio  2012;3(5):e00309-12.
ABSTRACT
Natural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history of Aggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway.
IMPORTANCE
The human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogen Aggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competent A. actinomycetemcomitans strains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.
doi:10.1128/mBio.00309-12
PMCID: PMC3484387  PMID: 23033473
10.  Indole Production Promotes Escherichia coli Mixed-Culture Growth with Pseudomonas aeruginosa by Inhibiting Quorum Signaling 
Indole production by Escherichia coli, discovered in the early 20th century, has been used as a diagnostic marker for distinguishing E. coli from other enteric bacteria. By using transcriptional profiling and competition studies with defined mutants, we show that cyclic AMP (cAMP)-regulated indole formation is a major factor that enables E. coli growth in mixed biofilm and planktonic populations with Pseudomonas aeruginosa. Mutants deficient in cAMP production (cyaA) or the cAMP receptor gene (crp), as well as indole production (tnaA), were not competitive in coculture with P. aeruginosa but could be restored to wild-type competitiveness by supplementation with a physiologically relevant indole concentration. E. coli sdiA mutants, which lacked the receptor for both indole and N-acyl-homoserine lactones (AHLs), showed no change in competitive fitness, suggesting that indole acted directly on P. aeruginosa. An E. coli tnaA mutant strain regained wild-type competiveness if grown with P. aeruginosa AHL synthase (rhlI and rhlI lasI) mutants. In contrast to the wild type, P. aeruginosa AHL synthase mutants were unable to degrade indole. Indole produced during mixed-culture growth inhibited pyocyanin production and other AHL-regulated virulence factors in P. aeruginosa. Mixed-culture growth with P. aeruginosa stimulated indole formation in E. coli cpdA, which is unable to regulate cAMP levels, suggesting the potential for mixed-culture gene activation via cAMP. These findings illustrate how indole, an early described feature of E. coli central metabolism, can play a significant role in mixed-culture survival by inhibiting quorum-regulated competition factors in P. aeruginosa.
doi:10.1128/AEM.06396-11
PMCID: PMC3255741  PMID: 22101045
11.  A Bilayer-Couple Model of Bacterial Outer Membrane Vesicle Biogenesis 
mBio  2012;3(2):e00297-11.
ABSTRACT
Gram-negative bacteria naturally produce outer membrane vesicles (OMVs) that arise through bulging and pinching off of the outer membrane. OMVs have several biological functions for bacteria, most notably as trafficking vehicles for toxins, antimicrobials, and signaling molecules. While their biological roles are now appreciated, the mechanism of OMV formation has not been fully elucidated. We recently demonstrated that the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) is required for OMV biogenesis in P. aeruginosa. We hypothesized that PQS stimulates OMV formation through direct interaction with the outer leaflet of the outer membrane. To test this hypothesis, we employed a red blood cell (RBC) model that has been used extensively to study small-molecule–membrane interactions. Our results revealed that addition of PQS to RBCs induced membrane curvature, resulting in the formation of membrane spicules (spikes), consistent with small molecules that are inserted stably into the outer leaflet of the membrane. Radiotracer experiments demonstrated that sufficient PQS was inserted into the membrane to account for this curvature and that curvature induction was specific to PQS structure. These data suggest that a low rate of interleaflet flip-flop forces PQS to accumulate in and expand the outer leaflet relative to the inner leaflet, thus inducing membrane curvature. In support of PQS-mediated outer leaflet expansion, the PQS effect was antagonized by chlorpromazine, a molecule known to be preferentially inserted into the inner leaflet. Based on these data, we propose a bilayer-couple model to describe P. aeruginosa OMV biogenesis and suggest that this is a general mechanism for bacterial OMV formation.
IMPORTANCE
Despite the ubiquity and importance of outer membrane vesicle (OMV) production in Gram-negative bacteria, the molecular details of OMV biogenesis are not fully understood. Early experiments showed that 2-heptyl-3-hydroxy-4-quinolone (PQS) induces OMV formation through physical interaction with the membrane but did not elucidate the mechanism. The present study demonstrates that PQS specifically and reversibly promotes blebbing of model membranes dependent upon the same properties that are required for OMV formation in P. aeruginosa. These results are consistent with a mechanism where expansion of the outer leaflet relative to the inner leaflet induces localized membrane curvature. This “bilayer-couple” model can account for OMV formation under all conditions and is easily generalized to other Gram-negative bacteria. The model therefore raises the possibility of a universal paradigm for vesicle production in prokaryotes with features strikingly different from what is known in eukaryotes.
doi:10.1128/mBio.00297-11
PMCID: PMC3312216  PMID: 22415005
13.  Protein abundances are more conserved than mRNA abundances across diverse taxa 
Proteomics  2010;10(23):4209-4212.
Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression datasets from seven different species: two bacteria, yeast, nematode, fly, human, and plant. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.
doi:10.1002/pmic.201000327
PMCID: PMC3113407  PMID: 21089048
14.  Oxygen Levels Rapidly Modulate Pseudomonas aeruginosa Social Behaviors via Substrate Limitation of PqsH 
Molecular microbiology  2010;77(6):1527-1538.
Summary
Many bacteria use extracellular signals to coordinate group behaviors, a process referred to as quorum sensing (QS). The bacterium Pseudomonas aeruginosa utilizes a complex QS system to control expression of over 300 genes, including many involved in host colonization and disease. The Pseudomonas Quinolone Signal (PQS) is a component of P. aeruginosa QS, and although it contributes to virulence in some models of infection, the PQS biosynthetic pathway is not fully elucidated. Here, we show that PqsH catalyzes the terminal step in PQS production, synthesizing PQS in vitro using the substrates 2-heptyl-4-quinolone (HHQ), NADH, and oxygen. Structure function studies reveal that the alkyl side chain of HHQ is critical for PqsH activity with the highest activity observed for alkyl chain lengths of 7 and 9 carbons. Due to the PqsH requirement for oxygen, PQS and PQS-controlled virulence factors are not produced by anaerobic P. aeruginosa. Interestingly, anaerobic P. aeruginosa produced PQS in the absence of de novo protein synthesis upon introduction of oxygen, indicating that oxygen is the sole limiting substrate during anaerobic growth. We propose a model in which PqsH poises anaerobic P. aeruginosa to activate PQS-controlled factors immediately upon exposure to molecular oxygen.
doi:10.1111/j.1365-2958.2010.07303.x
PMCID: PMC3098721  PMID: 20662781
PqsH; PQS; Pseudomonas; oxygen; quorum sensing
15.  Pseudomonas aeruginosa Enhances Production of an Antimicrobial in Response to N-Acetylglucosamine and Peptidoglycan▿ †  
Journal of Bacteriology  2010;193(4):909-917.
Pseudomonas aeruginosa is an opportunistic pathogen often associated with chronic lung infections in individuals with the genetic disease cystic fibrosis (CF). Previous work from our laboratory revealed that five genes predicted to be important for catabolism of N-acetylglucosamine (GlcNAc) are induced during in vitro growth in CF lung secretions (sputum). Here, we demonstrate that these genes comprise an operon (referred to as the nag operon) and that NagE, a putative component of the GlcNAc phosphotransferase system, is required for growth on and uptake of GlcNAc. Using primer extension analysis, the promoter of the nag operon was mapped and shown to be inducible by GlcNAc and regulated by the transcriptional regulator NagR. Transcriptome analysis revealed that in addition to induction of the nag operon, several P. aeruginosa genes encoding factors critical for extracellular antimicrobial production are also induced by GlcNAc. Finally, we show that the GlcNAc-containing polymer peptidoglycan induces production of the antimicrobial pyocyanin. Based on this data, we propose a model in which P. aeruginosa senses surrounding bacteria by monitoring exogenous peptidoglycan and responds to this cue through enhanced production of an antimicrobial.
doi:10.1128/JB.01175-10
PMCID: PMC3028681  PMID: 21169497
16.  4-Quinolones: Smart Phones of the Microbial World 
Chemical reviews  2010;111(1):152-159.
doi:10.1021/cr100063u
PMCID: PMC3147023  PMID: 20701272
17.  Quantifying Pseudomonas aeruginosa Quinolones and Examining Their Interactions with Lipids 
Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.g. cell cultures) is described. Separation of PQS from extracts by Thin-Layer Chromatography (TLC) is used in combination with the natural fluorescence of the molecule for quantification. A second separation technique for the PQS precursor HHQ using High-Performance Liquid Chromatography (HPLC) is also described, and this assay exploits the molecule’s characteristic absorbance for quantification. A third method for quantification of PQS from simple mixtures (e.g. enzyme assays) using fluorescence is outlined. Finally, a protocol for determining PQS interactions with membrane lipids through Fluorescence Resonance Energy Transfer (FRET) is presented. These techniques allow for quantification and characterization of PQS from diverse environments, a prerequisite to understanding the biological functions of QS molecules.
doi:10.1007/978-1-60761-971-0_15
PMCID: PMC3129850  PMID: 21031314
Pseudomonas quinolone signal; Thin-layer chromatography; High-pressure liquid chromatography; Fluorescence resonance energy transfer
18.  Revisiting the host as a growth medium 
Nature reviews. Microbiology  2008;6(9):657-666.
The ability of the human body to play host to bacterial pathogens has been studied for more than 200 years. Successful pathogenesis relies on the ability to acquire the nutrients that are necessary for growth and survival, yet relatively little is understood about the in vivo physiology and metabolism of most human pathogens. This Review discusses how in vivo carbon sources can affect disease and highlights the concept that carbon metabolic pathways provide viable targets for antibiotic development.
doi:10.1038/nrmicro1955
PMCID: PMC3115587  PMID: 18679171
19.  Characterization of a Novel Riboswitch-Regulated Lysine Transporter in Aggregatibacter actinomycetemcomitans ▿ §  
Journal of Bacteriology  2010;192(23):6240-6250.
Aggregatibacter actinomycetemcomitans is an opportunistic pathogen that resides primarily in the mammalian oral cavity. In this environment, A. actinomycetemcomitans faces numerous host- and microbe-derived stresses, including intense competition for nutrients and exposure to the host immune system. While it is clear that A. actinomycetemcomitans responds to precise cues that allow it to adapt and proliferate in the presence of these stresses, little is currently known about the regulatory mechanisms that underlie these responses. Many bacteria use noncoding regulatory RNAs (ncRNAs) to rapidly alter gene expression in response to environmental stresses. Although no ncRNAs have been reported in A. actinomycetemcomitans, we propose that they are likely important for colonization and persistence in the oral cavity. Using a bioinformatic and experimental approach, we identified three putative metabolite-sensing riboswitches and nine small regulatory RNAs (sRNAs) in A. actinomycetemcomitans during planktonic and biofilm growth. Molecular characterization of one of the riboswitches revealed that it is a lysine riboswitch and that its target gene, lysT, encodes a novel lysine-specific transporter. Finally, we demonstrated that lysT and the lysT lysine riboswitch are conserved in over 40 bacterial species, including the phylogenetically related pathogen Haemophilus influenzae.
doi:10.1128/JB.00935-10
PMCID: PMC2981213  PMID: 20889741
20.  Metabolite Cross-Feeding Enhances Virulence in a Model Polymicrobial Infection 
PLoS Pathogens  2011;7(3):e1002012.
Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.
Author Summary
Many bacterial infections are not the result of colonization and persistence of a single pathogenic microbe in an infection site but instead the result of colonization by several. Although the importance of polymicrobial interactions and pathogenesis has been noted by many prominent microbiologists including Louis Pasteur, most studies of pathogenic microbes have focused on single organism infections. One of the primary reasons for this oversight is the lack of robust model systems for studying bacterial interactions in an infection site. Here, we use a model co-culture system composed of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans and the common oral commensal Streptococcus gordonii to assess the impact of polymicrobial growth on pathogenesis. We found that the abilities of A. actinomycetemcomitans to persist and cause disease are enhanced during co-culture with S. gordonii. Remarkably, this enhanced persistence requires A. actinomycetemcomitans catabolism of L-lactate, the primary metabolite produced by S. gordonii. These data demonstrate that during co-culture growth, S. gordonii provides a carbon source for A. actinomycetemcomitans that is necessary for establishing a robust polymicrobial infection. This study also demonstrates that virulence of an opportunistic pathogen is impacted by members of the commensal flora.
doi:10.1371/journal.ppat.1002012
PMCID: PMC3069116  PMID: 21483753
21.  Revealing the dynamics of polymicrobial infections: implications for antibiotic therapy 
Trends in microbiology  2010;18(8):357-364.
As a new generation of culture-independent analytical strategies emerge, the amount of data on polymicrobial infections will increase dramatically. For these data to inform clinical thinking, and in turn to maximise benefits for patients, an appropriate framework for their interpretation is required. Here, we use cystic fibrosis (CF) lower airway infections as a model system to examine how conceptual and technological advances can address two clinical questions that are central to improved management of CF respiratory disease. Firstly, can markers of the microbial community be identified that predict a change in infection dynamics and clinical outcomes? Secondly, can these new strategies directly characterize the impact of antimicrobial therapies, allowing treatment efficacy to be both assessed and optimized?
doi:10.1016/j.tim.2010.04.005
PMCID: PMC3034215  PMID: 20554204
22.  Characterization of the Pseudomonas aeruginosa Transcriptional Response to Phenylalanine and Tyrosine▿  
Journal of Bacteriology  2010;192(11):2722-2728.
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often associated with chronic infections in the lungs of individuals with the heritable disease cystic fibrosis (CF). Previous work from our laboratory demonstrated that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance synthesis of the cell signaling molecule Pseudomonas quinolone signal (PQS). The present study investigates the role of the aromatic amino acid-responsive regulator PhhR in mediating these phenotypes. Transcriptome analysis revealed that PhhR controls four putative transcriptional units (phhA, hpd, hmgA, and dhcA) involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. The phhA, hpd, hmgA, and dhcA promoters were mapped by primer extension, and purified His6-PhhR was shown to bind the phhA, hpd, and dhcA promoters in vitro by use of electrophoretic mobility shift assays. Our work characterizes a transcriptional regulator of catabolic genes induced during P. aeruginosa growth in CF sputum.
doi:10.1128/JB.00112-10
PMCID: PMC2876504  PMID: 20304990
23.  Probing Prokaryotic Social Behaviors with Bacterial “Lobster Traps” 
mBio  2010;1(4):e00202-10.
Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 108 bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 101 to 105 cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial “lobster traps”) defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (1012 cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations.
IMPORTANCE
Prokaryotes are social organisms capable of coordinated group behaviors, including the abilities to construct antibiotic-resistant biofilms and to communicate with small signaling molecules (quorum sensing [QS]). While there has been significant effort devoted to understanding biofilm formation and QS, few studies have examined these processes in high-density/low-cell-number populations. Such studies have clinical significance, as many infections are initiated by small bacterial populations (<105) that are organized into dense clusters. Here, we describe a technology for studying such bacterial populations in picoliter-sized porous cavities (referred to as bacterial “lobster traps”) capable of capturing a single bacterium and tracking growth and behavior in real time. We provide evidence that small changes in the size of the bacterial cluster as well as flow rate of the surrounding medium modulate QS in Pseudomonas aeruginosa. We also demonstrate that as few as ~150 confined bacteria are needed to exhibit an antibiotic-resistant phenotype similar to biofilm bacteria.
doi:10.1128/mBio.00202-10
PMCID: PMC2975351  PMID: 21060734
24.  Parallel Evolution in Pseudomonas aeruginosa over 39,000 Generations In Vivo 
mBio  2010;1(4):e00199-10.
The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung, P. aeruginosa becomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits of P. aeruginosa during chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000 in vivo generations. We identified 24 genes that were commonly regulated by all 3 P. aeruginosa lineages, including several genes encoding traits previously shown to be important for in vivo growth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial for P. aeruginosa chronic colonization of the CF lung.
IMPORTANCE
Deadly diseases like AIDS, malaria, and tuberculosis are the result of long-term chronic infections. Pathogens that cause chronic infections adapt to the host environment, avoiding the immune response and resisting antimicrobial agents. Studies of pathogen adaptation are therefore important for understanding how the efficacy of current therapeutics may change upon prolonged infection. One notorious chronic pathogen is Pseudomonas aeruginosa, a bacterium that causes long-term infections in individuals with the heritable disease cystic fibrosis (CF). We used gene expression profiles to identify 24 genes that commonly changed expression over time in 3 P. aeruginosa lineages, indicating that these changes occur in parallel in the lungs of individuals with CF. Several of these genes have previously been shown to encode traits critical for in vivo-relevant processes, suggesting that they are likely beneficial adaptations important for chronic colonization of the CF lung.
doi:10.1128/mBio.00199-10
PMCID: PMC2939680  PMID: 20856824
25.  Deathly Drool: Evolutionary and Ecological Basis of Septic Bacteria in Komodo Dragon Mouths 
PLoS ONE  2010;5(6):e11097.
Komodo dragons, the world's largest lizard, dispatch their large ungulate prey by biting and tearing flesh. If a prey escapes, oral bacteria inoculated into the wound reputedly induce a sepsis that augments later prey capture by the same or other lizards. However, the ecological and evolutionary basis of sepsis in Komodo prey acquisition is controversial. Two models have been proposed. The “bacteria as venom” model postulates that the oral flora directly benefits the lizard in prey capture irrespective of any benefit to the bacteria. The “passive acquisition” model is that the oral flora of lizards reflects the bacteria found in carrion and sick prey, with no relevance to the ability to induce sepsis in subsequent prey. A third model is proposed and analyzed here, the “lizard-lizard epidemic” model. In this model, bacteria are spread indirectly from one lizard mouth to another. Prey escaping an initial attack act as vectors in infecting new lizards. This model requires specific life history characteristics and ways to refute the model based on these characteristics are proposed and tested. Dragon life histories (some details of which are reported here) prove remarkably consistent with the model, especially that multiple, unrelated lizards feed communally on large carcasses and that escaping, wounded prey are ultimately fed on by other lizards. The identities and evolutionary histories of bacteria in the oral flora may yield the most useful additional insights for further testing the epidemic model and can now be obtained with new technologies.
doi:10.1371/journal.pone.0011097
PMCID: PMC2888571  PMID: 20574514

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