An Amino Acid Substitution in Elongation Factor EF-G1A Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa LasR-Null Mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

Cross-species activation of hydrogen cyanide production by a promiscuous quorum-sensing receptor promotes Chromobacterium subtsugae competition in a dual-species model.

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen.

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Tobramycin Adaptation Enhances Policing of Social Cheaters in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa LasR-LasI (LasR-I) quorum sensing system regulates secreted proteases that can be exploited by cheaters, such as quorum sensing receptor-defective (lasR) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy. These mutants are exploitative cheaters because they avoid the substantial cost of engaging in quorum sensing. Previous studies showed that quorum sensing increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppressed the emergence of lasR mutants in casein-passaged populations. Several mutations accumulated in those populations, indicating evidence of antibiotic adaptation. We found that mutations in one gene, ptsP, increased antibiotic resistance and also pleiotropically increased production of a quorum sensing-controlled phenazine, pyocyanin. When passaged on casein, ptsP mutants suppressed cheaters in a manner that was tobramycin independent. We found that the mechanism of cheater suppression in ptsP mutants relied on pyocyanin, which acts as a policing toxin by selectively blocking growth of cheaters. Thus, tobramycin suppresses lasR mutants through two mechanisms: first, through direct effects on cheaters and, second, by selecting mutations in ptsP that suppressed cheating in a tobramycin-independent manner. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during interspecies competition or infections. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa is a model for understanding quorum sensing, a type of cell-cell signaling important for cooperation. Quorum sensing controls production of cooperative goods, such as exoenzymes, which are vulnerable to cheating by quorum sensing-defective mutants. Because uncontrolled cheating can ultimately cause a population to collapse, much focus has been on understanding how P. aeruginosa can control cheaters. We show that an antibiotic, tobramycin, can suppress cheaters in cooperating P. aeruginosa populations. Tobramycin suppresses cheaters directly because the cheaters are more susceptible to tobramycin than cooperators. Tobramycin also selects for mutations in a gene, ptsP, that suppresses cheaters independent of tobramycin through pleiotropic regulation of a policing toxin, pyocyanin. This work supports the idea that adaptation to antibiotics can have unexpected effects on the evolution of quorum sensing and has implications for understanding how cooperation evolves in dynamic bacterial communities.

Burkholderia thailandensis Methylated Hydroxyalkylquinolines: Biosynthesis and Antimicrobial Activity in Cocultures.

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In coculture experiments, B. thailandensis strain E264 secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis strain 168. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC, and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576 ("hmqL"). The results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NOs). Synthetic HMAQ-NO is active against B. subtilis 168, showing ∼50-fold more activity than HMAQ. Both the methyl group and the length of the carbon side chain account for the high activity of HMAQ-NO. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensisIMPORTANCE The soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones that are mostly methylated 4-hydroxyalkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa Several of the methylated 4-hydroxyalkenylquinolines have antimicrobial activity against other species. We show that Bacillus subtilis strain 168 is particularly susceptible to N-oxidated methylalkenylquinolines (HMAQ-NOs). We confirmed that HMAQ-NO biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxyalkenylquinolines, which are unique to B. thailandensis This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

The Chemistry and Biology of Bactobolin: A 10-Year Collaboration with Natural Product Chemist Extraordinaire Jon Clardy.

Bactobolin is a hybrid natural product with potent cytotoxic activity. Its production from Burkholderia thailandensis was reported as part of a collaboration between the Greenberg and Clardy laboratories in 2010. The collaboration sparked a series of studies leading to the discovery of new analogues and associated structure-activity relationships, the identification of the bactobolin biosynthetic gene cluster and assembly of its unusual amino acid building block, the molecular target of and resistance to the antibiotic, and finally an X-ray crystal structure of the ribosome-bactobolin complex. Herein, we review the collaborations that led to our current understanding of the chemistry and biology of bactobolin.

Secondary metabolites from the Burkholderia pseudomallei complex: structure, ecology, and evolution.

Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model.

Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.

Malleilactone Is a Burkholderia pseudomallei Virulence Factor Regulated by Antibiotics and Quorum Sensing.

Burkholderia pseudomallei, the causative agent of melioidosis, encodes almost a dozen predicted polyketide (PK) biosynthetic gene clusters. Many of these are regulated by LuxR-I-type acyl-homoserine (AHL) quorum-sensing systems. One of the PK gene clusters, the mal gene cluster, is conserved in the close relative Burkholderia thailandensis The B. thailandensis mal genes code for the cytotoxin malleilactone and are regulated by a genetically linked LuxR-type transcription factor, MalR. Although AHLs typically interact with LuxR-type proteins to modulate gene transcription, the B. thailandensis MalR does not appear to be an AHL receptor. Here, we characterize the mal genes and MalR in B. pseudomallei We use chemical analyses to demonstrate that the B. pseudomallei mal genes code for malleilactone. Our results show that MalR and the mal genes contribute to the ability of B. pseudomallei to kill Caenorhabditis elegans In B. thailandensis, antibiotics like trimethoprim can activate MalR by driving transcription of the mal genes, and we demonstrate that some of the same antibiotics induce expression of B. pseudomallei malR We also demonstrate that B. pseudomallei MalR does not respond directly to AHLs. Our results suggest that MalR is indirectly repressed by AHLs, possibly through a repressor, ScmR. We further show that malleilactone is a B. pseudomallei virulence factor and provide the foundation for understanding how malleilactone contributes to the pathology of melioidosis infections.IMPORTANCE Many bacterially produced polyketides are cytotoxic to mammalian cells and are potentially important contributors to pathogenesis during infection. We are interested in the polyketide gene clusters present in Burkholderia pseudomallei, which causes the often-fatal human disease melioidosis. Using knowledge gained by studies in the close relative Burkholderia thailandensis, we show that one of the B. pseudomallei polyketide biosynthetic clusters produces a cytotoxic polyketide, malleilactone. Malleilactone contributes to B. pseudomallei virulence in a Caenorhabditis elegans infection model and is regulated by an orphan LuxR family quorum-sensing transcription factor, MalR. Our studies demonstrate that malleilactone biosynthesis or MalR could be new targets for developing therapeutics to treat melioidosis.

Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production.

UNLABELLED: Members of the genus Burkholderia are known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterized Burkholderia thailandensis biofilm development under flow conditions and sought to determine whether QS contributes to this process. B. thailandensis biofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by "dome" structures filled with biofilm matrix material. We showed that this process was dependent on QS. B. thailandensis has three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the three B. thailandensis QS systems, we show that QS-1 is required for proper biofilm development, since a btaR1 mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. The btaR1 mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions. IMPORTANCE: The saprophyte Burkholderia thailandensis is a close relative of the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms, B. thailandensis is an ideal model organism for investigating questions in Burkholderia physiology. In this study, we characterized B. thailandensis biofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows that B. thailandensis produces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience of B. thailandensis biofilms against changes in the nutritional environment.

A Burkholderia thailandensis Acyl-Homoserine Lactone-Independent Orphan LuxR Homolog That Activates Production of the Cytotoxin Malleilactone.

UNLABELLED: Burkholderia thailandensis has three acyl-homoserine lactone (AHL) LuxR-LuxI quorum-sensing circuits and two orphan LuxR homologs. Orphans are LuxR-type transcription factors that do not have cognate LuxI-type AHL synthases. One of the orphans, MalR, is genetically linked to the mal gene cluster, which encodes enzymes required for production of the cytotoxic polyketide malleilactone. Under normal laboratory conditions the mal gene cluster is silent; however, antibiotics like trimethoprim induce mal transcription. We show that trimethoprim-dependent induction of the mal genes requires MalR. MalR has all of the conserved amino acid residues characteristic of AHL-responsive LuxR homologs, but in B. thailandensis, MalR activation of malleilactone synthesis genes is not responsive to AHLs. MalR can activate transcription from the mal promoter in E. coli without addition of AHLs or trimethoprim. Expression of malR in B. thailandensis is induced by trimethoprim. Our data indicate that MalR binds to a lux box-like element in the mal promoter and activates transcription of the mal genes in an AHL-independent manner. Antibiotics like trimethoprim appear to activate mal gene expression indirectly by somehow activating malR expression. MalR activation of the mal genes represents an example of a LuxR homolog that is not a receptor for an AHL quorum-sensing signal. Our evidence is consistent with the idea that mal gene activation depends solely on sufficient transcription of the malR gene. IMPORTANCE: LuxR proteins are transcription factors that are typically activated by acyl-homoserine lactone (AHL) signals. We demonstrate that a conserved LuxR family protein, MalR, activates genes independently of AHLs. MalR is required for transcription of genes coding for synthesis of the cytotoxic polyketide malleilactone. These genes are not expressed when cells are grown under normal laboratory conditions. In laboratory culture, MalR induction of malleilactone requires certain antibiotics, such as trimethoprim, which increase malR expression by an unknown mechanism. At sufficient levels of malR expression, MalR functions independently of any external signal. Our findings show that MalR is an activator of the silent malleilactone biosynthesis genes and that MalR functions independently of AHLs.

Quorum Sensing Protects Pseudomonas aeruginosa against Cheating by Other Species in a Laboratory Coculture Model.

UNLABELLED: Many species of bacteria use a cell-cell communication system called quorum sensing (QS) to coordinate group activities. QS systems frequently regulate the production of exoproducts. Some of these products, such as proteases, are "public goods" that are shared among the population and vulnerable to cheating by nonproducing members of the population. Because the QS system of the opportunistic pathogen Pseudomonas aeruginosa regulates several public goods, it can serve as a model for studying cooperation. Bacteria also commonly regulate antimicrobial production through QS. In this study, we focused on the hypothesis that QS-regulated antimicrobials may be important for P. aeruginosa to protect against cheating by another bacterial species, Burkholderia multivorans. We assessed laboratory cocultures of P. aeruginosa and B. multivorans and investigated the importance of three P. aeruginosa QS-regulated antimicrobials, hydrogen cyanide, rhamnolipids, and phenazines, for competition. We found that P. aeruginosa dominates cocultures with B. multivorans and that the three antimicrobials together promote P. aeruginosa competitiveness, with hydrogen cyanide contributing the greatest effect. We show that these QS-regulated antimicrobials are also critical for P. aeruginosa to prevent B. multivorans from cheating under nutrient conditions where both species require a P. aeruginosa quorum-regulated protease for growth. Together our results highlight the importance of antimicrobials in protecting cooperating populations from exploitation by other species that can act as cheaters. IMPORTANCE: Cooperative behaviors are threatened by social cheating, wherein individuals do not produce but nonetheless benefit from shared public goods. Bacteria have been shown to use several genetic mechanisms to restrain the emergence of cheaters from within the population, but public goods might also be used by other bacterial species in the vicinity. We demonstrate that a public good produced by Pseudomonas aeruginosa can be used by another species, Burkholderia multivorans, to obtain carbon and energy. We also show that P. aeruginosa antimicrobials that are coregulated with the public good prevent invasion by the cheating species. Our results demonstrate that cross-species cheating can occur and that coregulation of public goods with antimicrobials may stabilize cooperative behavior in mixed microbial communities.

Bacterial quorum sensing, cooperativity, and anticipation of stationary-phase stress.

Acyl-homoserine lactone-mediated quorum sensing (QS) regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. The acyl-homoserine lactones are synthesized by members of the LuxI signal synthase family and are detected by cognate members of the LuxR family of transcriptional regulators. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in three related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary-phase stress. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example of QS serving as a means to anticipate stationary phase or life at the carrying capacity of a population by activating the expression of cytoplasmic enzymes, altering cellular metabolism, and producing a shared resource or public good, oxalate.

Characterization of natural product inhibitors of quorum sensing reveals competitive inhibition of Pseudomonas aeruginosa RhlR by ortho-vanillin.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde, and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also cause mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR-specific inhibitory activities over a range of concentrations below that causing toxicity. ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR. IMPORTANCE: Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho-vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.

Characterization of natural product inhibitors of quorum sensing in Pseudomonas aeruginosa reveals competitive inhibition of RhlR by ortho-vanillin.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. Ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR.

Regulation of an antibiotic resistance efflux pump by quorum sensing and a TetR-family repressor in Chromobacterium subtsugae.

The soil bacterium Chromobacterium substugae uses a single LuxI-R-type quorum-sensing system, CviI-R, to regulate genes in a cell density-dependent manner. CviI synthesizes the signal N-hexanoyl-homoserine lactone (C6-HSL) and CviR is a C6-HSL-responsive cytoplasmic transcription regulator. C6-HSL-bound CviR activates dozens of genes, for example the cdeAB-oprM cluster coding for an efflux pump conferring antibiotic resistance. The cdeAB-oprM genes are also regulated by an antibiotic-responsive transcription factor, CdeR, which represses expression of these genes. We are interested in understanding how C. subtsugae integrates different environmental cues to regulate antibiotic resistance. In this study, we sought to delineate the mechanism of regulation of the cdeAB-oprM genes by CviR and CdeR. In recombinant E. coli, the cdeA promoter is activated by CviR and repressed by CdeR. We identify non-overlapping sequence elements in the cdeA promoter that are required for CviR activation and CdeR repression, respectively. We also examined the role of CdeR in modulating cdeA activation by C6-HSL in C. subtsugae. We show that CviR and CdeR can independently modulate transcription from the cdeA promoter in C. subtsugae, consistent with the conclusion that CviR and CdeR regulate the cdeAB-oprM genes by interacting directly with different binding sites in the cdeA promoter. These results contribute to a molecular understanding of how the cdeAB-oprM genes are regulated and provide new insight into how C. subtsugae integrates different environmental cues to regulate antibiotic resistance.

Tobramycin adaptation alters the antibiotic susceptibility of Pseudomonas aeruginosa quorum sensing-null mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum sensing system to increase resistance to the aminioglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism allowing the lasR-null mutants to persist under tobramycin selection. We hypothesized that the effects of inactivating lasR on tobramycin resistance might be dependent on the presence or absence of other gene mutations in that strain, a phenomenon known as epistasis. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. We show that the effects of ΔlasR on tobramycin resistance are strain dependent. The effects can be attributed to a point mutation in the gene encoding the translation elongation factor fusA1 (G61A nucleotide substitution), which confers a strong selective advantage to lasR-null PA14 under tobramycin selection. This fusA1 G61A mutation results in increased activity of the MexXY efflux pump and expression of the mexXY regulator ArmZ. The fusA1 mutation can also modulate ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results demonstrate the importance of epistatic gene interactions on antibiotic susceptibility of lasR-null mutants. These results support of the idea that gene interactions might play a significant role in the evolution of quorum sensing in P. aeruginosa.

Engineering peptide-polymer hybrids for targeted repair and protection of cervical lesions.

By 2060, nearly 100 million people in the U.S. will be over age 65 years. One-third of these older adults will have root caries, and nearly 80% will have dental erosion. These conditions can cause pain and loss of tooth structure that interfere with eating, speaking, sleeping, and quality of life. Current treatments for root caries and dental erosion have produced unreliable results. For example, the glass-ionomer-cement or composite-resin restorations used to treat these lesions have annual failure rates of 44% and 17%, respectively. These limitations and the pressing need to treat these conditions in the aging population are driving a focus on microinvasive strategies, such as sealants and varnishes. Sealants can inhibit caries on coronal surfaces, but they are ineffective for root caries. For healthy, functionally independent elders, chlorhexidine varnish applied every 3 months inhibits root caries, but this bitter-tasting varnish stains the teeth. Fluoride gel inhibits root caries, but requires prescriptions and daily use, which may not be feasible for some older patients. Silver diamine fluoride can both arrest and inhibit root caries but stains the treated tooth surface black. The limitations of current approaches and high prevalence of root caries and dental erosion in the aging population create an urgent need for microinvasive therapies that can: (a) remineralize damaged dentin; (b) inhibit bacterial activity; and (c) provide durable protection for the root surface. Since cavitated and non-cavitated root lesions are difficult to distinguish, optimal approaches will treat both. This review will explore the multi-factorial elements that contribute to root surface lesions and discuss a multi-pronged strategy to both repair and protect root surfaces. The strategy integrates engineered peptides, novel polymer chemistry, multi-scale structure/property characterization and predictive modeling to develop a durable, microinvasive treatment for root surface lesions.

Pseudomonas aeruginosa Bacterioferritin Is Assembled from FtnA and BfrB Subunits with the Relative Proportions Dependent on the Environmental Oxygen Availability.

Ferritins are iron storage proteins assembled from 24 subunits into a spherical and hollow structure. The genomes of many bacteria harbor genes encoding two types of ferritin-like proteins, the bacterial ferritins (Ftn) and the bacterioferritins (Bfr), which bind heme. The genome of P. aeruginosa PAO1 (like the genomes of many bacteria) contains genes coding for two different types of ferritin-like molecules, ftnA (PA4235) and bfrB (PA3531). The reasons for requiring the presence of two distinct types of iron storage protein in bacterial cells have remained largely unexplained. Attempts to understand this issue in P. aeruginosa through the recombinant expression of the ftnA and bfrB genes in E. coli host cells, coupled to the biochemical and structural characterization of the recombinant 24-mer FtnA and 24-mer BfrB molecules, have shown that each of the recombinant molecules can form an Fe3+-mineral core. These observations led to the suggestion that 24-mer FtnA and 24-mer BfrB molecules coexist in P. aeruginosa cells where they share iron storage responsibilities. Herein, we demonstrate that P. aeruginosa utilizes a single heterooligomeric 24-mer Bfr assembled from FtnA and BfrB subunits. The relative content of the FtnA and BfrB subunits in Bfr depends on the O2 availability during cell culture, such that Bfr isolated from aerobically cultured P. aeruginosa is assembled from a majority of BfrB subunits. In contrast, when the cells are cultured in O2-limiting conditions, the proportion of FtnA subunits in the isolated Bfr increases significantly and can become the most abundant subunit. Despite the variability in the subunit composition of Bfr, the 24-mer assembly is consistently arranged from FtnA subunit dimers devoid of heme and BfrB subunit dimers each containing a heme molecule.

Inhibiting Iron Mobilization from Bacterioferritin in Pseudomonas aeruginosa Impairs Biofilm Formation Irrespective of Environmental Iron Availability.

Although iron is essential for bacteria, the nutrient presents problems of toxicity and solubility. Bacteria circumvent these problems with the aid of iron storage proteins where Fe3+ is deposited and, when necessary, mobilized as Fe2+ for metabolic requirements. In Pseudomonas aeruginosa, Fe3+ is compartmentalized in bacterioferritin (BfrB), and its mobilization as Fe2+ requires specific binding of a ferredoxin (Bfd) to reduce the stored Fe3+. Blocking the BfrB-Bfd complex leads to irreversible iron accumulation in BfrB and cytosolic iron deprivation. Consequently, given the intracellular iron sufficiency requirement for biofilm development, we hypothesized that blocking the BfrB-Bfd interaction in P. aeruginosa would impair biofilm development. Our results show that planktonic and biofilm-embedded cells where the BfrB-Bfd complex is blocked exhibit cytosolic iron deficiency, and poorly developed biofilms, even in iron-sufficient culture conditions. These results underscore inhibition of the BfrB-Bfd complex as a rational target to dysregulate iron homeostasis and possibly control biofilms.