• Single-nucleotide polymorphism-based genetic diversity analysis of clinical Pseudomonas aeruginosa isolates.

      Muthukumarasamy, Uthayakumar; Preusse, Matthias; Kordes, Adrian; Koska, Michal; Schniederjans, Monika; Khaledi, Ariane; Häussler, Susanne; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Oxford Academic, 2020-03-20)
      Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Since different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single nucleotide polymorphisms (SNPs) that are the basis for branching of the phylogenetic tree. We also identified SNPs that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. While our results demonstrate that the P. aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.
    • Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility.

      Bense, Sarina; Bruchmann, Sebastian; Steffen, Anika; Stradal, Theresia E B; Häussler, Susanne; Düvel, Juliane; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-03-12)
      The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.
    • Targeting Bacterial Gyrase with Cystobactamid, Fluoroquinolone, and Aminocoumarin Antibiotics Induces Distinct Molecular Signatures in Pseudomonas aeruginosa.

      Franke, Raimo; Overwin, Heike; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ASM, 2021-07-13)
      The design of novel antibiotics relies on a profound understanding of their mechanism of action. While it has been shown that cellular effects of antibiotics cluster according to their molecular targets, we investigated whether compounds binding to different sites of the same target can be differentiated by their transcriptome or metabolome signatures. The effects of three fluoroquinolones, two aminocoumarins, and two cystobactamids, all inhibiting bacterial gyrase, on Pseudomonas aeruginosa at subinhibitory concentrations could be distinguished clearly by RNA sequencing as well as metabolomics. We observed a strong (2.8- to 212-fold) induction of autolysis-triggering pyocins in all gyrase inhibitors, which correlated with extracellular DNA (eDNA) release. Gyrase B-binding aminocoumarins induced the most pronounced changes, including a strong downregulation of phenazine and rhamnolipid virulence factors. Cystobactamids led to a downregulation of a glucose catabolism pathway. The study implies that clustering cellular mechanisms of action according to the primary target needs to take class-dependent variances into account. IMPORTANCE Novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Bacterial pathogens possess few privileged targets for a successful therapy: the majority of existing antibiotics as well as current candidates in development target the complex bacterial machinery for cell wall synthesis, protein synthesis, or DNA replication. An important mechanistic question addressed by this study is whether inhibiting such a complex target at different sites with different compounds has similar or differentiated cellular consequences. Using transcriptomics and metabolomics, we demonstrate that three different classes of gyrase inhibitors can be distinguished by their molecular signatures in P. aeruginosa. We describe the cellular effects of a promising, recently identified gyrase inhibitor class, the cystobactamids, in comparison to those of the established gyrase A-binding fluoroquinolones and the gyrase B-binding aminocoumarins. The study results have implications for mode-of-action discovery approaches based on target-specific reference compounds, as they highlight the intraclass variability of cellular compound effects.
    • Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria.

      Donnert, Monique; Elsheikh, Sarah; Arce-Rodriguez, Alejandro; Pawar, Vinay; Braubach, Peter; Jonigk, Danny; Haverich, Axel; Weiss, Siegfried; Müsken, Mathias; Häussler, Susanne; et al. (PLOS, 2020-12-22)
      Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.
    • Thermoplasmatales and sulfur-oxidizing bacteria dominate the microbial community at the surface water of a CO2-rich hydrothermal spring located in Tenorio Volcano National Park, Costa Rica

      Arce-Rodríguez, Alejandro; Puente-Sánchez, Fernando; Avendaño, Roberto; Martínez-Cruz, María; de Moor, J Maarten; Pieper, Dietmar H; Chavarría, Max; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2019-01-01)
      Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems.
    • Towards individualized diagnostics of biofilm-associated infections: a case study.

      Müsken, Mathias; Klimmek, Kathi; Sauer-Heilborn, Annette; Donnert, Monique; Sedlacek, Ludwig; Suerbaum, Sebastian; Häussler, Susanne; H-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      Organized within biofilm communities, bacteria exhibit resistance towards a broad spectrum of antibiotics. Thus, one might argue that bacteria isolated from biofilm-associated chronic infections should be subjected to resistance profiling under biofilm growth conditions. Various test systems have been developed to determine the biofilm-associated resistance; however, it is not clear to what extent the in vitro results reflect the situation in vivo, and whether the biofilm-resistance profile should guide clinicians in their treatment choice. To address this issue, we used confocal microscopy in combination with live/dead staining, and profiled biofilm-associated resistance of a large number (>130) of clinical Pseudomonas aeruginosa isolates from overall 15 cystic fibrosis patients. Our results demonstrate that in addition to a general non-responsiveness of bacteria when grown under biofilm conditions, there is an isolate-specific and antibiotic-specific biofilm-resistance profile. This individual resistance profile is independent on the structural properties of the biofilms. Furthermore, biofilm resistance is not linked to the resistance profile under planktonic growth conditions, or a mucoid, or small colony morphology of the tested isolates. Instead, it seems that individual biofilm structures evolve during biofilm-associated growth and are shaped by environment-specific cues. In conclusion, our results demonstrate that biofilm resistance profiles are isolate specific and cannot be deduced from commonly studied phenotypes. Further clinical studies will have to show the added value of biofilm-resistance profiling. Individualized diagnosis of biofilm resistance might lead to more rational recommendations for antimicrobial therapy and, thus, increased effectiveness of the treatment of chronically infected patients.
    • The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression.

      Hirschmann, Stephanie; Gómez-Mejia, Alejandro; Mäder, Ulrike; Karsunke, Julia; Driesch, Dominik; Rohde, Manfred; Häussler, Susanne; Burchhardt, Gerhard; Hammerschmidt, Sven; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-02-24)
      Streptococcus pneumoniae two-component regulatory systems (TCSs) are important systems that perceive and respond to various host environmental stimuli. In this study, we have explored the role of TCS09 on gene expression and phenotypic alterations in S. pneumoniae D39. Our comparative transcriptomic analyses identified 67 differently expressed genes in total. Among those, agaR and the aga operon involved in galactose metabolism showed the highest changes. Intriguingly, the encapsulated and nonencapsulated hk09-mutants showed significant growth defects under nutrient-defined conditions, in particular with galactose as a carbon source. Phenotypic analyses revealed alterations in the morphology of the nonencapsulated hk09- and tcs09-mutants, whereas the encapsulated hk09- and tcs09-mutants produced higher amounts of capsule. Interestingly, the encapsulated D39∆hk09 showed only the opaque colony morphology, while the D39∆rr09- and D39∆tcs09-mutants had a higher proportion of transparent variants. The phenotypic variations of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 are in accordance with their higher numbers of outer membrane vesicles, higher sensitivity against Triton X-100 induced autolysis, and lower resistance against oxidative stress. In conclusion, these results indicate the importance of TCS09 for pneumococcal metabolic fitness and resistance against oxidative stress by regulating the carbohydrate metabolism and thereby, most likely indirectly, the cell wall integrity and amount of capsular polysaccharide.
    • Unravelling post-transcriptional PrmC-dependent regulatory mechanisms in Pseudomonas aeruginosa.

      Krueger, Jonas; Pohl, Sarah; Preusse, Matthias; Kordes, Adrian; Rugen, Nils; Schniederjans, Monika; Pich, Andreas; Häussler, Susanne; Twincore, Zentrum für experimentelle und klinische Infektionsforschung GmbH, Feodor-Lynen Str. 7, 30625 Hannover, Germany. (2016-10)
      Transcriptional regulation has a central role in cellular adaptation processes and is well investigated. In contrast, the importance of the post-transcriptional regulation on these processes is less well defined. The technological advancements have been critical to precisely quantify protein and mRNA level changes and hold promise to provide more insights into how post-transcriptional regulation determines phenotypes. In Pseudomonas aeruginosa the methyltransferase PrmC methylates peptide chain release factors to facilitate translation termination. Loss of PrmC activity abolishes anaerobic growth and leads to reduced production of quorum sensing-associated virulence factors. Here, by applying SILAC technology in combination with mRNA-sequencing, they provide evidence that the P. aeruginosa phenotype can be attributed to a change in protein to mRNA ratios of selected protein groups. The UAG-dependent translation termination was more dependent on PrmC activity than the UAA- and UGA-dependent translation termination. Additionally, a bias toward UAG stop codons in global transcriptional regulators was found. The finding that this bias in stop codon usage determines the P. aeruginosa phenotype is unexpected and adds complexity to regulatory circuits. Via modulation of PrmC activity the bacterial cell can cross-regulate targets independently of transcriptional signals, a process with an underestimated impact on the bacterial phenotype.
    • Untargeted LC-MS Metabolomics Differentiates Between Virulent and Avirulent Clinical Strains of Pseudomonas aeruginosa

      Depke, Tobias; Thöming, Janne Gesine; Kordes, Adrian; Häussler, Susanne; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-07-13)
      Pseudomonas aeruginosa is a facultative pathogen that can cause, inter alia, acute or chronic pneumonia in predisposed individuals. The gram-negative bacterium displays considerable genomic and phenotypic diversity that is also shaped by small molecule secondary metabolites. The discrimination of virulence phenotypes is highly relevant to the diagnosis and prognosis of P. aeruginosa infections. In order to discover small molecule metabolites that distinguish different virulence phenotypes of P. aeruginosa, 35 clinical strains were cultivated under standard conditions, characterized in terms of virulence and biofilm phenotype, and their metabolomes were investigated by untargeted liquid chromatography-mass spectrometry. The data was both mined for individual candidate markers as well as used to construct statistical models to infer the virulence phenotype from metabolomics data. We found that clinical strains that differed in their virulence and biofilm phenotype also had pronounced divergence in their metabolomes, as underlined by 332 features that were significantly differentially abundant with fold changes greater than 1.5 in both directions. Important virulence-associated secondary metabolites like rhamnolipids, alkyl quinolones or phenazines were found to be strongly upregulated in virulent strains. In contrast, we observed little change in primary metabolism. A hitherto novel cationic metabolite with a sum formula of C12H15N2 could be identified as a candidate biomarker. A random forest model was able to classify strains according to their virulence and biofilm phenotype with an area under the Receiver Operation Characteristics curve of 0.84. These findings demonstrate that untargeted metabolomics is a valuable tool to characterize P. aeruginosa virulence, and to explore interrelations between clinically important phenotypic traits and the bacterial metabolome.
    • Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa.

      van Duuren, Jozef B J H; Müsken, Mathias; Karge, Bianka; Tomasch, Jürgen; Wittmann, Christoph; Häussler, Susanne; Brönstrup, Mark (2017-07-12)
      Impedance spectroscopy has been applied in prokaryotic and eukaryotic cytometry as a label-free method for the investigation of adherent cells. In this paper, its use for characterizing the growth dynamics of P. aeruginosa biofilms is described and compared to crystal violet staining and confocal microscopy. The method allows monitoring the growth of biofilm-forming P. aeruginosa in a continuous and label-free manner over a period of 72 h in a 96 well plate format. Impedance curves obtained for P. aeruginosa PA14 wild type and mutant strains with a transposon insertion in pqsA and pelA genes exhibited distinct phases. We propose that the slope of the declining curve following a maximum at ca. 35-40 h is a measure of biofilm formation. Transplant experiments with P. aeruginosa biofilms and paraffin suggest that the impedance also reflects pellicle formation at the liquid-air interface, a barely considered contributor to impedance. Finally, the impairment of biofilm formation upon treatment of cultures with L-arginine and with ciprofloxacin, tobramycin and meropenem was studied by single frequency impedance spectroscopy. We suggest that these findings qualify impedance spectroscopy as an additional technique to characterize biofilm formation and its modulation by small molecule drugs.
    • Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis.

      Kayser, Hartmut; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2016-01)
      Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.
    • The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways.

      Malone, Jacob G; Jaeger, Tina; Manfredi, Pablo; Dötsch, Andreas; Blanka, Andrea; Bos, Raphael; Cornelis, Guy R; Häussler, Susanne; Jenal, Urs; Biozentrum of the University of Basel, Basel, Switzerland. (2012-06)
      The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.