The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa

Certain members of the fluorescent pseudomonads produce and secrete phenazines. These heterocyclic, redox-active compounds are toxic to competing organisms, and the cause of these antibiotic effects has been the focus of intense research efforts. It is largely unknown, however, how pseudomonads themselves respond to – and survive in the presence of – these compounds. Using Pseudomonas aeruginosa DNA microarrays and quantitative RT-PCR, we demonstrate that the phenazine pyocyanin elicits the upregulation of genes/operons that function in transport [such as the resistance-nodulation-cell division (RND) efflux pump MexGHI-OpmD] and possibly in redox control (such as PA2274, a putative flavin-dependant monooxygenase), and downregulates genes involved in ferric iron acquisition. Strikingly, mexGHI-opmD and PA2274 were previously shown to be regulated by the PA14 quorum sensing network that controls the production of virulence factors (including phenazines). Through mutational analysis, we show that pyocyanin is the physiological signal for the upregulation of these quorum sensing-controlled genes during stationary phase and that the response is mediated by the transcription factor SoxR. Our results implicate phenazines as signalling molecules in both P. aeruginosa PA14 and PAO1

Microbial Community Composition Impacts Pathogen Iron Availability during Polymicrobial Infection

Iron is an essential nutrient for bacterial pathogenesis, but in the host, iron is tightly sequestered, limiting its availability for bacterial growth. Although this is an important arm of host immunity, most studies examine how bacteria respond to iron restriction in laboratory rather than host settings, where the microbiome can potentially alter pathogen strategies for acquiring iron. One of the most important transcriptional regulators controlling bacterial iron homeostasis is Fur. Here we used a combination of RNA-seq and chromatin immunoprecipitation (ChIP)-seq to characterize the iron-restricted and Fur regulons of the biofilm-forming opportunistic pathogen Aggregatibacter actinomycetemcomitans. We discovered that iron restriction and Fur regulate 4% and 3.5% of the genome, respectively. While most genes in these regulons were related to iron uptake and metabolism, we found that Fur also directly regulates the biofilm-dispersing enzyme Dispersin B, allowing A. actinomycetemcomitans to escape from iron-scarce environments. We then leveraged these datasets to assess the availability of iron to A. actinomycetemcomitans in its primary infection sites, abscesses and the oral cavity. We found that A. actinomycetemcomitans is not restricted for iron in a murine abscess mono-infection, but becomes restricted for iron upon co-infection with the oral commensal Streptococcus gordonii. Furthermore, in the transition from health to disease in human gum infection, A. actinomycetemcomitans also becomes restricted for iron. These results suggest that host iron availability is heterogeneous and dependent on the infecting bacterial community

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans. One of this bacterium\u27s key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field\u27s lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising \u3e1,500 unique insertions and used a sequencing-based approach to define each mutant\u27s position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen

Phage inhibit pathogen dissemination by targeting bacterial migrants in a chronic infection model

The microbial communities inhabiting chronic infections are often composed of spatially organized micrometer-sized, highly dense aggregates. It has recently been hypothesized that aggregates are responsible for the high tolerance of chronic infections to host immune functions and antimicrobial therapies. Little is currently known regarding the mechanisms controlling aggregate formation and antimicrobial tolerance primarily because of the lack of robust, biologically relevant experimental systems that promote natural aggregate formation. Here, we developed an in vitro model based on chronic Pseudomonas aeruginosa infection of the cystic fibrosis (CF) lung. This model utilizes a synthetic sputum medium that readily promotes the formation of P. aeruginosa aggregates with sizes similar to those observed in human CF lung tissue. Using high-resolution imaging, we exploited this model to elucidate the life history of P. aeruginosa and the mechanisms that this bacterium utilizes to tolerate antimicrobials, specifically, bacteriophage. In the early stages of growth in synthetic sputum, planktonic cells form aggregates that increase in size over time by expansion. In later growth, migrant cells disperse from aggregates and colonize new areas, seeding new aggregates. When added simultaneously with phage, P. aeruginosa was readily killed and aggregates were unable to form. When added after initial aggregate formation, phage were unable to eliminate all of the aggregates because of exopolysaccharide production; however, seeding of new aggregates by dispersed migrants was inhibited. We propose a model in which aggregates provide a mechanism that allows P. aeruginosa to tolerate phage therapy during chronic infection without the need for genetic mutation

Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries

Tooth decay (dental caries) is a widespread human disease caused by microbial biofilms. Streptococcus mutans, a biofilm-former, has been consistently associated with severe childhood caries; however, how this bacterium is spatially organized with other microorganisms in the oral cavity to promote disease remains unknown. Using intact biofilms formed on teeth of toddlers affected by caries, we discovered a unique 3D rotund-shaped architecture composed of multiple species precisely arranged in a corona-like structure with an inner core of S. mutans encompassed by outer layers of other bacteria. This architecture creates localized regions of acidic pH and acute enamel demineralization (caries) in a mixed-species biofilm model on human teeth, suggesting this highly ordered community as the causative agent. Notably, the construction of this architecture was found to be an active process initiated by production of an extracellular scaffold by S. mutans that assembles the corona cell arrangement, encapsulating the pathogen core. In addition, this spatial patterning creates a protective barrier against antimicrobials while increasing bacterial acid fitness associated with the disease-causing state. Our data reveal a precise biogeography in a polymicrobial community associated with human caries that can modulate the pathogen positioning and virulence potential in situ, indicating that micron-scale spatial structure of the microbiome may mediate the function and outcome of host-pathogen interactions

Genes contributing to Porphyromonas gingivalis fitness in abscess and epithelial cell colonization environments

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism

Co-infecting microbes dramatically alter pathogen gene essentiality during polymicrobial infection

Identifying genes required by pathogens during infection is critical for antimicrobial development. Here, we used a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data to determine the role of infection site and co-infecting microbes on the in vivo ‘essential’ genome of Staphylococcus aureus. We discovered that co-infection of murine surgical wounds with Pseudomonas aeruginosa results in conversion of ~25% of the in vivo S. aureus mono-culture essential genes to non-essential. Furthermore, 182 S. aureus genes are uniquely essential during co-infection. These “Community Dependent Essential” (CoDE) genes illustrate the importance of studying pathogen gene essentiality in polymicrobial communities

Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries

Tooth decay (dental caries) is a widespread human disease caused by microbial biofilms. Streptococcus mutans, a biofilm-former, has been consistently associated with severe childhood caries; however, how this bacterium is spatially organized with other microorganisms in the oral cavity to promote disease remains unknown. Using intact biofilms formed on teeth of toddlers affected by caries, we discovered a unique 3D rotund-shaped architecture composed of multiple species precisely arranged in a corona-like structure with an inner core of S. mutans encompassed by outer layers of other bacteria. This architecture creates localized regions of acidic pH and acute enamel demineralization (caries) in a mixed-species biofilm model on human teeth, suggesting this highly ordered community as the causative agent. Notably, the construction of this architecture was found to be an active process initiated by production of an extracellular scaffold by S. mutans that assembles the corona cell arrangement, encapsulating the pathogen core. In addition, this spatial patterning creates a protective barrier against antimicrobials while increasing bacterial acid fitness associated with the disease-causing state. Our data reveal a precise biogeography in a polymicrobial community associated with human caries that can modulate the pathogen positioning and virulence potential in situ, indicating that micron-scale spatial structure of the microbiome may mediate the function and outcome of host–pathogen interactions