The Putative De-N-acetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones

Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target

Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512

The rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Genome-wide detection of predicted non-coding RNAs in Rhizobium etli expressed during free-living and host-associated growth using a high-resolution tiling array

Non-coding RNAs (ncRNAs) play a crucial role in the intricate regulation of bacterial gene expression, allowing bacteria to quickly adapt to changing environments. In the past few years, a growing number of regulatory RNA elements have been predicted by computational methods, mostly in well-studied gamma-proteobacteria but lately in several alpha-proteobacteria as well. Here, we have compared an extensive compilation of these non-coding RNA predictions to intergenic expression data of a whole-genome high-resolution tiling array in the soil-dwelling alpha-proteobacterium Rhizobium etli.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Image-based dynamic phenotyping reveals genetic determinants of filamentation-mediated beta-lactam tolerance

Antibiotic tolerance characterized by slow killing of bacteria in response to a drug can lead to treatment failure and promote the emergence of resistance. beta-lactam antibiotics inhibit cell wall growth in bacteria and many of them cause filamentation followed by cell lysis. Hence delayed cell lysis can lead to beta-lactam tolerance. Systematic discovery of genetic factors that affect beta-lactam killing kinetics has not been performed before due to challenges in high-throughput, dynamic analysis of viability of filamented cells during bactericidal action. We implemented a high-throughput time-resolved microscopy approach in a gene deletion library of Escherichia coli to monitor the response of mutants to the beta-lactam cephalexin. Changes in frequency of lysed and intact cells due to the antibiotic action uncovered several strains with atypical lysis kinetics. Filamentation confers tolerance because antibiotic removal before lysis leads to recovery through numerous concurrent divisions of filamented cells. Filamentation-mediated tolerance was not associated with resistance, and therefore this phenotype is not discernible through most antibiotic susceptibility methods. We find that deletion of Tol-Pal proteins TolQ, TolR, or Pal but not TolA, TolB, or CpoB leads to rapid killing by beta-lactams. We also show that the timing of cell wall degradation determines the lysis and killing kinetics after beta-lactam treatment. Altogether, this study uncovers numerous genetic determinants of hitherto unappreciated filamentation-mediated beta-lactam tolerance and support the growing call for considering antibiotic tolerance in clinical evaluation of pathogens. More generally, the microscopy screening methodology described here can easily be adapted to study lysis in large numbers of strains

Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli

Background: The alarmone (p) ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p) ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p) ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p) ppGpp-regulon in an alpha-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. Results: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p) ppGpp targets, including small non-coding RNAs. The majority of the 834 (p) ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p) ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p) ppGpp during active growth. Furthermore, we identified two (p) ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. Conclusions: The regulatory role of (p) ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in alpha-proteobacteria

Multiplex STR amplification sensitivity in a silicon microchip

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (mu PCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 mu l each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the mu PCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both mu PCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis

Silicon µPCR chip for forensic STR profiling with hybeacon probe melting curves

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-ona-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 mu l each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated onchip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis

Antibacterial activity of 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol against antibiotic-resistant strains of diverse bacterial pathogens, biofilms and in pre-clinical infection models

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa.

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies

Functional characterisation of the Rhizobium etli type III secretion system

Rhizobium etli is een Gram-negatieve bodembacterie die een stikstoffixerende symbiose kan aangaan met Phaseolus vulgaris L., de gewone boonplant. Een uitgebreide signaal­uitwisseling leidt tot de infectie van de wortels en de vorming van gespecialiseerde plantenorganen, de wortelnodules. Bacteriële signalen die de specificiteit van de interactie bepalen zijn onder meer Nod factoren, oppervlaktepolysachariden en gesecreteerde proteïnen. Het belang van gespecialiseerde proteïnesecretiesystemen die zogeheten effectoren rechtstreeks in het cytosol van gastheercellen kunnen transloceren, wordt steeds duidelijker. Een dergelijk secretiemechanisme, dat intensief bestudeerd wordt, is het type III secretiesysteem (T3SS). Deze macromoleculaire machine is essentieel voor de virulentie van vele pathogenen van planten en dieren. Een tiental jaar geleden werd aangetoond dat T3SS ook een rol kunnen spelen in de symbiose tussen rhizobia en vlinderbloemige planten. Analyse van de in 2003 gepubliceerde DNA-sequentie van het symbiotische plasmide van R. etli duidde op de aanwezigheid van een T3SS. Dit vormde de aanleiding voor nader onderzoek van de rol die het R. etli T3SS speelt in de symbiotische interactie met P. vulgaris. Een gedetailleerde in silico analyse van de R. etli T3SS-regio leidde tot de identificatie van alle zogenaamde Rhizobium geconserveerde (rhc), structurele genen, op één na. Er werd een genomische kernregio afgebakend die in verscheidene R. etli stammen van diverse geografische origine geconserveerd is. Drie genen die coderen voor kandidaat-helperproteïnen werden gevonden: hrpW, hrpK1 en hrpK2. De laatste twee hebben kenmerken van translocatorproteïnen en zouden dus kunnen bijdragen tot de translocatie van effectorproteïnen naar het gastheercelcytosol. Er werden ook kenmerken van de R. etli T3SS-regio afgeleid die merkelijk verschillen van de overeenkomstige loci in andere rhizobia. Zo ontbreken een homoloog voor een geconserveerd buitenmembraansecretine en verscheidene regulatorische elementen. Dit wijst op aanzienlijke divergentie binnen rhizobiële T3SS. Een fylogenetische analyse bevestigde deze stelling.De symbiotische rol van het R. etli T3SS werd geanalyseerd door het symbiotisch fenotype van P. vulgaris te bepalen na inoculatie met ofwel de wildtype stam, ofwel een rhcN mutant. Door het screenen van genetisch diverse P. vulgaris variëteiten kon een gastheer­specifiek effect op nodulatie vastgesteld worden bij een beperkt aantal boonvariëteiten. Enkele wilde en onkruidachtige variëteiten vormden beduidend meer pseudonodules na inoculatie met een rhcN mutant, terwijl sommige gecultiveerde variëteiten omgekeerd reageerden. Expressie van de R. etli T3SS-genen werd bestudeerd op transcriptioneel niveau door gebruik te maken van een promotorloos gusA gen als reporter. Ondanks het testen van een grote verscheidenheid aan experimentele condities en stimuli kon geen duidelijke inductie waargenomen worden. Secretie werd nagegaan op translationeel niveau door een proteoomanalyse van eiwit­fracties geïsoleerd uit cultuursupernatans. Er konden geen verschillen waargenomen worden tussen de wildtype stam en de rhcN mutant.Ten slotte werd R. etli HrpW onderzocht. Dit proteïne is een mogelijk substraat voor het T3SS en werd tot dusver uitsluitend in plantpathogene bacteriën aangetroffen. Het eiwit werd tot overexpressie gebracht, opgezuiverd en biochemisch gekarakteriseerd. Er werd enzymatische activiteit aangetoond voor het carboxyterminaal gelegen pectaat­lyasedomein. Dit is, voor zover wij weten, de eerste keer voor een HrpW-achtig proteïne.I.The role of rhizobial protein secretion in the Rhizobium-legume symbiosis II.In silico analysis of the Rhizobium etli type III secretion system region III.The role of the Rhizobium etli type III secretion system in symbiosis with Phaseolus vulgaris L. IV.Expression analysis of the Rhizobium etli type III secretion system V.In search of substrates for the Rhizobium etli type III secretion system VI.Characterisation of the Rhizobium etli candidate T3SS substrate HrpW VII.General conclusions and perspectivesstatus: publishe