Kangmin Duan

Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication

The change in gene expression patterns in response to host environments is a prerequisite for bacterial infection. Bacterial diseases often occur as an outcome of the complex interactions between pathogens and the host. The indigenous, usually non‐pathogenic microflora is a ubiquitous constituent of the host. In order to understand the interactions between pathogens and the resident microflora and how they affect the gene expression patterns of the pathogens and contribute to bacterial diseases, the interactions between pathogenic Pseudomonas aeruginosa and avirulent oropharyngeal flora (OF) strains isolated from sputum samples of cystic fibrosis (CF) patients were investigated. Animal experiments using a rat lung infection model indicate that the presence of OF bacteria enhanced lung damage caused by P. aeruginosa. Genome‐wide transcriptional analysis with a lux reporter‐based promoter library demonstrated that ≈ 4% of genes in the genome responded to the presence of OF strains using an in vitro system. Characterization of a subset of the regulated genes indicates that they fall into seven functional classes, and large portions of the upregulated genes are genes important for P. aeruginosa pathogenesis. Autoinducer‐2 (AI‐2)‐mediated quorum sensing, a proposed interspecies signalling system, accounted for some, but not all, of the gene regulation. A substantial amount of  AI‐2  was  detected  directly  in  sputum  samples from CF patients and in cultures of most non‐pseudomonad bacteria isolated from the sputa. Transcriptional profiling of a set of defined P. aeruginosa virulence factor promoters revealed that OF and exogenous AI‐2 could upregulate overlapping subsets of these genes. These results suggest important contributions of the host microflora to P. aeruginosa infection by modulating gene expression via interspecies communications.

A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. A modest number of bacterial pathogens have been correlated with pulmonary function decline; however, microbiological and molecular evidence suggests that CF airway infection is polymicrobial. To obtain a more complete assessment of the microbial community composition and dynamics, we undertook a longitudinal study by using culture-independent and microbiological approaches. In the process, we demonstrated that within complex and dynamic communities, the Streptococcus milleri group (SMG) can establish chronic pulmonary infections and at the onset of 39% of acute pulmonary exacerbations, SMG is the numerically dominant pathogen. We report the comprehensive polymicrobial community dynamics of a CF lung infection in a clinically relevant context. If a given organism, such as Pseudomonas aeruginosa, becomes resistant to antibiotic therapy, an alternative treatment avenue may mediate the desired clinical response by effectively managing the composition of the microbial community.

Environmental Regulation of Pseudomonas aeruginosa PAO1 Las and Rhl Quorum-Sensing Systems

The lasI-lasR and the rhlI-rhlR quorum-sensing systems in Pseudomonas aeruginosa regulate the expression of numerous cellular and secreted virulence factor genes and play important roles in the development of biofilms. The las and rhl systems themselves are known to be directly or indirectly regulated by a number of transcriptional regulators, and consequently, their expression is sensitive to environmental conditions. In this report, the activities of these two quorum-sensing systems have been examined systematically under 46 growth conditions, and the regulation by environmental conditions has been investigated. The relative timing and strength of expression of these two systems varied significantly under different conditions, which contrasts with the notion of a preset hierarchy with these two systems in P. aeruginosa. Depending on the growth conditions, the correlation between each synthase and its cognate transcriptional regulator also varied, suggesting that the transcription of these genes independently allows for further fine tuning of each system. Finally, we observe that the activities of both the lasI-lasR and the rhlI-rhlR quorum-sensing systems were dramatically enhanced in the presence of extracts of sputum samples from cystic fibrosis patients.

The YebC Family Protein PA0964 Negatively Regulates the Pseudomonas aeruginosa Quinolone Signal System and Pyocyanin Production

Bacterial pathogenicity is often manifested by the expression of various cell-associated and secreted virulence factors, such as exoenzymes, protease, and toxins. In Pseudomonas aeruginosa, the expression of virulence genes is coordinately controlled by the global regulatory quorum-sensing systems, which includes the las and rhl systems as well as the Pseudomonas quinolone signal (PQS) system. Phenazine compounds are among the virulence factors under the control of both the rhl and PQS systems. In this study, regulation of the phzA1B1C1D1E1 (phzA1) operon, which is involved in phenazine synthesis, was investigated. In an initial study of inducing conditions, we observed that phzA1 was induced by subinhibitory concentrations of tetracycline. Screening of 13,000 mutants revealed 32 genes that altered phzA1 expression in the presence of subinhibitory tetracycline concentrations. Among them, the gene PA0964, designated pmpR (pqsR-mediated PQS regulator), has been identified as a novel regulator of the PQS system. It belongs to a large group of widespread conserved hypothetical proteins with unknown function, the YebC protein family (Pfam family DUF28). It negatively regulates the quorum-sensing response regulator pqsR of the PQS system by binding at its promoter region. Alongside phzA1 expression and phenazine and pyocyanin production, a set of virulence factors genes controlled by both rhl and the PQS were shown to be modulated by PmpR. Swarming motility and biofilm formation were also significantly affected. The results added another layer of regulation in the rather complex quorum-sensing systems in P. aeruginosa and demonstrated a clear functional clue for the YebC family proteins.

Hybrid sensor kinase PA1611 in Pseudomonas aeruginosa regulates transitions between acute and chronic infection through direct interaction with RetS

Pseudomonas aeruginosa causes serious acute and chronic infections in humans. Major differences exist in disease pathogenesis, clinical treatment and outcomes between acute and chronic infections. P. aeruginosa acute infection characteristically involves the type III secretion systems (T3SS) while chronic infection is often associated with the formation of biofilms, a major cause of difficulties to eradicate chronic infections. The choice between acute and chronic infection or the switch between them by P. aeruginosa is controlled by regulatory pathways that control major virulence factors and genes associated with biofilm formation. In this study, we characterized a hybrid sensor kinase PA1611 that controls the expression of genes associated with acute and chronic infections in P. aeruginosa PAO1. Expression of PA1611 completely repressed T3SS and swarming motility while it promoted biofilm formation. The protein PA1611 regulates two small RNAs (sRNAs), rsmY and rsmZ which in turn control RsmA. Independent of phosphate relay, PA1611 interacts directly with RetS in vivo. The positive effect of RetS on factors associated with acute infection could presumably be restrained by PA1611 when chronic infection conditions are present. This RetS–PA1611 interaction, together with the known RetS–GacS interaction, may control disease progression and the lifestyle choice of P. aeruginosa.

Modulation of secreted virulence factor genes by subinhibitory concentrations of antibiotics in Pseudomonas aeruginosa

Recent studies have shown that subinhibitory antibiotics play important roles in regulating bacterial genes including virulence factor genes. In this study, the expression of 13 secreted virulence related gene clusters of Pseudomonas aeruginosa, an important opportunistic pathogen, was examined in the presence of subinhibitory concentrations of 4 antibiotics: vancomycin, tetracycline, ampicilin and azithromycin. Activation of gene expression was observed with phzAl, rhlAB, phzA2, lasB, exoY, and exoS. Subinhibitory concentrations of vancomycin resulted in more than 10-fold increase of rhlAB and phzA2 transcription. Both rhamnolipid production and pyocyanin production were significantly elevated, correlating phenotypes with the increased transcription. P. aeruginosa swarming and swimming motility also increased. Similar results were observed with subinhibitory tetracycline, azithromycin and ampicillin. These results indicate that the antibiotics at low concentrations can up-regulate virulence factors and therefore influence bacterial pathogenesis.

Chemical Interactions between Organisms in Microbial Communities

Bacteria live almost exclusively in communities with other microorganisms, and often in association with multicellular hosts. These communities are capable of maintaining complex structural and functional stability over time, and exhibit fascinating properties of resiliency in response to environmental changes. This is a result of interactions between microbes and the environment and amongst members of the community. A multitude of chemical interactions occur in microbial communities where primary and secondary metabolites contribute to a wealth of interactions between organisms. The chemicals include a variety of nutrients, toxic or neutral metabolic byproducts, antibiotics, and cell-cell signaling molecules. These chemical and physical signals facilitate microbial relationship that can be competitive, cooperative or neutral, and thus are responsible for determining community structure. In turn, the surrounding community changes the microenvironment of individual cells who respond to chemical and environmental cues in a combinatorial manner. Current laboratory understanding of the genetics and mechanisms of interactions between microbes has the power to help us understand how complex microbial communities behave in the natural environment. In this chapter we review the current understanding of microbial communication, from the genetic and molecular aspects, to our current understanding of their ecological role.

Identification of mutants with altered phenazine production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes serious and chronic infections. Many secondary metabolites are secreted throughout its growth, among which phenazine is a known virulence factor and signalling molecule. Phenazine is coordinately controlled by the global regulatory quorum-sensing (QS) systems. Despite the detailed understanding of phenazine biosynthesis pathways in P. aeruginosa, the regulatory networks are still not fully clear. In the present study, the regulation of the phzA1B1C1D1E1F1G1 operon (phzA1) has been investigated. Screening of 5000 transposon mutants revealed 14 interrupted genes with altered phzA1 expression, including PA2593 (QteE), which has been identified as a novel regulator of the QS system. Overexpression of qteE in P. aeruginosa significantly reduced the accumulation of homoserine lactone signals and affected the QS-controlled phenotypes such as the production of pyocyanin, rhamnolipids and LasA protease and swarming motility. Indeed, overexpression of qteE in P. aeruginosa attenuated its pathogenicity in the potato and fruit fly infection models. These findings suggest that qteE plays an important role in P. aeruginosa pathogenicity and is part of the regulatory networks controlling phenazine production.

PqsR-dependent and PqsR-independent regulation of motility and biofilm formation by PQS in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors including swarming motility and biofilm formation. Swarming motility plays an important role in the bacteriums spread to new environments, attachment to surfaces, and biofilm formation. Bacterial biofilm is associated with many persistent infections and increased resistance to antibiotics. In this study, we tested the effect of a 2‐alkyl‐4(1H)‐quinolone (AHQ) signal, the Pseudomonas quinolone signal (PQS) on P. aeruginosa swarming and biofilm formation. Our results show that PQS repressed the swarming motility of P. aeruginosa PAO1. Such repression was independent of its cognate receptor PqsR and was not related to changes in the flagellae, type IV pili or the production of the surface‐wetting agent rhamnolipid surfactant. While PQS did not affect twitching motility in PAO1, a pqsR deletion abolished twitching motility, indicating that pqsR is required for twitching motility. Our results also indicate that the enhancement of biofilm formation by PQS is at least partially dependent on the GacAS‐Rsm regulatory pathway but does not involve the las or rhl QS systems.

Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence

Resistance-Nodulation-Cell Division (RND) pumps play important roles in bacterial resistance to antibiotics. Pseudomonas aeruginosa is an important human pathogen which exhibits high level resistance to antibiotics. There are total of 12 RND pumps present in the P. aeruginosa PAOl genome. The recently characterized MuxABC-OpmB system has been shown to play a role in resistance to novobiocin, aztreonam, macrolides, and tetracycline in a multiple knockout mutation. In this study, we examined the expression levels of all the 12 RND pump gene clusters and tested the involvement of MuxABC-OpmB in pathogenicity. The results indicated that in addition to the four known constitutively expressed RND pumps, mexAB-oprM, mexGHI-opmD, mexVW, and mexXY, relatively high levels of expression were observed with mexJK and muxABC-opmB in the conditions tested. Inactivation of muxA in the muxABC-opmB operon resulted in elevated resistance to ampicillin and carbenicillin. The mutant also showed attenuated virulence in both Brassica rapa pekinensis and Drosophila melanogaster infection models. The decreased virulence at least in part was due to decreased twitching motility in the mutant. These results indicate that the RND pump MuxABC-OpmB is associated with ampicillin and carbenicillin susceptibility and also involved in pathogenesis in P. aeruginosa.