Biology subjects

Chandler, J. R.

Publications and source records attributed to Chandler, J. R..

9 recordsLinked to original sources

bioRxiv2025

Quorum sensing regulation by the nitrogen phosphotransferase system in Pseudomonas aeruginosa

In the opportunistic pathogen Pseudomonas aeruginosa, the nitrogen-related phosphotransferase system (PTSNtr) influences multiple virulence behaviors. The PTSNtr is comprised of three enzymes: first PtsP, then the PtsO phosphocarrier, and the final PtsN phosphoacceptor. We previously showed that ptsP inactivation increases LasI-LasR quorum sensing, a system by which P. aeruginosa regulates genes in response to population density. LasI synthesizes a diffusible autoinducer that binds and activates the LasR receptor, which activates a feedback loop by increasing lasI expression. In this study, we examined the impact of the PTSNtr on quorum sensing. Disruption of ptsP increased the expression of some, but not all, tested quorum-controlled genes, including lasI, phzM (pyocyanin biosynthesis), hcnA (hydrogen cyanide biosynthesis), and, to a lesser extent, rsaL (quorum sensing regulator). Expression of these genes remained dependent on LasR and the autoinducer, whether provided endogenously or exogenously. Increased lasI expression in {Delta}ptsP (or {Delta}ptsO) cells was partly due to the presence of unphosphorylated PtsN, which alone was sufficient to elevate lasI expression. However, we observed residual increases in {Delta}ptsP or {Delta}ptsO cells even in the absence of PtsN, suggesting that PtsP and PtsO can regulate gene expression independent of PtsN. Indeed, genetically disrupting the PtsO phosphorylation site impacted gene expression in the absence of PtsN, and transcriptomic evidence suggested that PtsO and PtsN have distinct regulons. Our results expand our view of how the PTSNtr components function both within and apart from the classic phosphorylation cascade to regulate key virulence behaviors in P. aeruginosa. IMPORTANCEPseudomonas aeruginosa often causes severe and difficult-to-treat infections. P. aeruginosa virulence requires the nitrogen-related phosphotransferase system (PTSNtr), which comprises the phosphocarrier proteins PtsP and PtsO and the final phosphoacceptor, PtsN. The PTSNtr is known to modulate quorum sensing, but little is known about the mechanism of regulation. Here, we examined quorum sensing regulation by the PTSNtr. We showed that the PTSNtr increases quorum sensing-mediated activation of certain genes through the additive effects of both PtsO and PtsN. We also used transcriptomics to determine the regulons of PtsO and PtsN and found that they are largely nonoverlapping. The results position PtsO and PtsN as independent effectors in the Nitro-PTS and shed new light on virulence regulation in this important pathogen.

microbiology↗

bioRxiv2024

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. IMPORTANCE Quorum sensing (QS) regulates many aspects of pathogenesis in bacteria and has attracted interest as a target for anti-virulence therapies. As QS is regulated by low molecular weight chemical signals, the development of chemical strategies that can interfere with this cell-cell communication pathway has seen considerable scrutiny over the past 25 years. Much of this research has focused on common human pathogens, including the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective chemical agents capable of blocking the activity of these receptors 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 QS control in P. aeruginosa. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.

microbiology↗

bioRxiv2023

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. ImportanceMany bacteria regulate antibiotic resistance in response to antibiotics and other cues from the environment. In many cases the regulatory mechanisms are best understood in the context of clinical isolates where mutations frequently emerge in resistance regulation pathways. However, an understanding of the role of antibiotic resistance regulators in integrating environmental information is less well understood. In the soil bacterium Chromobacterium subtsugae, an antibiotic-resistance gene cluster is regulated by population density and antibiotics through two different transcription factors; the quorum sensing signal receptor CviR and an antibiotic-responsive transcription factor CdeR. In this study, we show that these factors independently modulate the transcription of the antibiotic resistance genes and coordinate to ensure sensitive responses to changes in cell density. The results give new insight into antibiotic resistance regulation in C. subtsugae and contribute to a broader understanding of how bacteria optimize the regulation of antibiotic resistance in response to changes in the environment.

microbiology↗

bioRxiv2023

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 aminoglycoside 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 {Delta}lasR on tobramycin resistance are strain dependent, which is due to a single mutation in the fusA1 gene encoding the translation elongation factor EF-G1A (G61A nucleotide substitution). The fusA1 G61A mutation confers a strong selective advantage to {Delta}lasR mutants under tobramycin treatment. The effects of fusA1 G61A on {Delta}lasR-dependent tobramycin resistance are dependent on the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulates {Delta}lasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results provide a possible explanation for the emergence of lasR-null mutants in clinical isolates and illustrate the importance of epistatic gene interactions in the evolution of quorum sensing.

microbiology↗

bioRxiv2022

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. IMPACT STATEMENTIn quorum sensing, population density-dependent changes in gene regulation are the result of a cytoplasmic transcription regulator binding to a quorum sensing signal. The signal-receptor interaction is considered to be specific to ensure fidelity of the system. However, some quorum-sensing receptor proteins have relaxed specificity and can recognize and respond to a range of signals. These promiscuous receptors might provide some benefit by enabling interspecies activation of quorum sensing by "eavesdropping," although the potential benefits of eavesdropping are not well studied. The current study utilizes a dual-species laboratory competition model, where one species has a promiscuous signal receptor and can respond to signals produced by the other species. In our study, we identify the signals that enable quorum sensing cross talk and show that cross talk promotes competition by inducing hydrogen cyanide production. Our results highlight how quorum sensing-enabled interspecies cross talk might provide an advantage during competition and provide a new basis for understanding how receptor-signal pairs might evolve in natural environments. DATA SUMMARYThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2020

Tobramycin adaptation enhances policing of social cheaters in Pseudomonas aeruginosa

The Pseudomonas aeruginosa LasR-I quorum sensing (QS) system regulates secreted proteases that can be exploited by cheaters, such as QS receptor-defective (lasR) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy and increase in the population because they do not incur the substantial cost of engaging in QS. QS also increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppresses the emergence of lasR mutants in casein-passaged populations. We also identify several mutations that accumulate in those populations indicating evidence of antibiotic adaptation. Mutations in one gene, ptsP, increase activity of the LasR-I system and production of a QS-controlled phenazine, pyocyanin. We find that mutations in ptsP lead to suppression of cheaters independent of tobramycin. Cheater suppression relies on pyocyanin, which acts as a policing toxin by targeting cheaters. These results show that tobramycin suppresses lasR mutants through two mechanisms: first, by directly acting on tobramycin-susceptible cheaters and second, by selecting mutations in ptsP that lead to pyocyanin-dependent policing. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during infections.

microbiology↗

bioRxiv2020

Burkholderia thailandensis methylated hydroxy-alkylquinolines: biosynthesis and antimicrobial activity in co-culture experiments

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 liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. 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. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is [~]50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. 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. thailandensis. IMPORTANCEThe soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones, mostly methylated 4-hydroxy-alkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxy-alkenylquinolines have antimicrobial activity against other species. We show that N-oxidated methyl-alkenylquinolines are particularly antimicrobial and sufficient to kill Bacillus subtilis in co-cultures. We confirmed their 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-hydroxy-alkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

microbiology↗

bioRxiv2019

Two resistance nodulation division-family efflux pumps in Chromobacterium species and their role in antibiotic resistance and tolerance

Very little is known of the antibiotic resistance mechanisms of members of the Chromobacterium genus. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM). Here, we show the cdeAB-oprM genes are widely distributed in members of the Chromobacterium genus. We use antimicrobial susceptibility testing with a CV017 cdeAB-oprM mutant to show the products of these genes confers resistance to a variety of antibiotics including ciprofloxacin, a clinically important antibiotic. We also identified a related RND-family pump, cseAB-oprN, in the genome of CV017 and other C. subtsugae species, that is not present in other members of the Chromobacterium genus. We demonstrate that CdeAB-OprM and CseAB-OprN are both transcriptionally induced in CV017 cells treated with sub-lethal antibiotic concentrations and they are important for induction of tolerance to different antibiotics. While CdeAB-OprM has a broad antibiotic specificity, the CseAB-OprN system is highly specific for a ribosome-targeting antibiotic produced by the saprophytic bacterium Burkholderia thailandensis, bactobolin. Finally, we use a previously developed B. thailandensis-C. subtsugae CV017 co-culture model to demonstrate that adding sub-lethal bactobolin at the beginning of co-culture growth increases the ability of CV017 to compete with B. thailandensis in a manner that is dependent on the CseAB-OprN system. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps during competition with other bacterial species. IMPORTANCEThis study describes two closely related efflux pumps in members of the Chromobacterium genus, which includes opportunistic but often-fatal pathogens and species with highly versatile metabolic capabilities. Efflux pumps remove antibiotics from the cell and are important for antibiotic resistance. One of these pumps is broadly distributed in the Chromobacterium genus and increases resistance to clinically relevant antibiotics. The other efflux pump is present only in Chromobacterium subtsugae and is highly specific for bactobolin, an antibiotic produced by the soil saprophyte Burkholderia thailandensis. We demonstrate these pumps can be activated to increase resistance by their antibiotic substrates, and that this activation is important for C. subtsugae to survive in a laboratory competition experiment with B. thailandensis. These results have implications for managing antibiotic-resistant Chromobacterium infections, bioengineering of Chromobacterium species, and for understanding the evolution of efflux pumps.

microbiology↗

bioRxiv2018

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. Previous work has described a regularly-timed pH change in the intestine of Caenorhabditis elegans called the pH wave. To characterize this wave and its function in the worm, we developed a novel fluorescent dye (KR35) that accumulates in the intestine and sensitively responds to dynamic changes in pH. 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 pathogens completely neutralize the pH in the intestine of wild type, but not 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 permit pH changes in the intestine that are important for fighting pathogens.\n\nAuthor SummaryInnate immunity is critical for host defense against pathogens. However, questions remain about how the host senses and responds to pathogen invasion. Using a pH-sensitive fluorescent dye and a Caenorhabditis elegans pathogen infection model we show that pathogens induce changes in pH of the worm intestine. We also show that intestinal pH directly affects production of reactive oxygen species (e.g. H2O2) important for pathogen defense. Our results show that pH regulation is an important component of the innate immune response to pathogens.

microbiology↗