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TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa Neidig, Anke; Yeung, Amy T; Rosay, Thibaut; Tettmann, Beatrix; Strempel, Nikola; Rueger, Martina; Lesouhaitier, Olivier; Overhage, Joerg Apr 9, 2013

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RESEARCH ARTICLE Open AccessTypA is involved in virulence, antimicrobialresistance and biofilm formation in PseudomonasaeruginosaAnke Neidig1, Amy TY Yeung2, Thibaut Rosay3, Beatrix Tettmann1, Nikola Strempel1, Martina Rueger1,Olivier Lesouhaitier3 and Joerg Overhage1*AbstractBackground: Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult totreat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections andbroad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified tomodulate virulence in enteric Gram-negative pathogens.Results: Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocyticamoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cellattachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracyclineand antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, ofimportant virulence-related genes such as those involved in regulation and assembly of the Type III secretionsystem, consistent with the observed phenotypes and role in virulence of P. aeruginosa.Conclusions: These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and isinvolved in multiple virulence-related characteristics.Keywords: Pseudomonas aeruginosa, Pathogen, TypA, Type III secretion system, Virulence, Dictyosteliumdiscoideum, Macrophage, Biofilm, ResistanceBackgroundPseudomonas aeruginosa is a ubiquitous environmentalGram-negative soil bacterium that is also an importantopportunistic human pathogen causing a variety of differ-ent nosocomial infections including pneumonia, catheterand urinary tract infections as well as sepsis in burnwound and immunocompromised patients [1]. Moreover,P. aeruginosa is the most prevalent and significant pul-monary pathogen in patients with cystic fibrosis causingeventually fatal lung disease [2]. The inability to success-fully clear P. aeruginosa infections through antibiotictreatment is a major contributor to the complicated andoften severe outcome of P. aeruginosa infections [3]. Itdemonstrates high intrinsic resistance to antibiotics andan ability to develop even higher resistance through muta-tion, acquisition of genetic elements, and adaptation toenvironmental conditions, e.g. through biofilm formationon surfaces.P. aeruginosa also possesses a large arsenal of virulence-related factors. Among others are a type II, III and VIsecretion system and their associated effector proteins suchas extracellular proteases and phospholipases and the TypeIII secreted toxins ExoU, S, T and Y. In addition, they haveflagella and type IV pili that are involved in motility andhost cell adhesion [4-6]. P. aeruginosa also regulates thegene expression of most virulence factors including genesinvolved in iron acquisition (e.g. pyoverdine), toxin produc-tion (hydrogen cyanide), exopolysaccharide biosynthesis orbiofilm formation in a cell density dependant mannertermed quorum sensing mediated by the two master regu-lators LasR and RhlR [4,7,8]. Although some virulencefactors seem to be host or site specific, the majority are* Correspondence: joerg.overhage@kit.edu1Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, POBox 3640, 76021, Karlsruhe, GermanyFull list of author information is available at the end of the article© 2013 Neidig et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Neidig et al. BMC Microbiology 2013, 13:77http://www.biomedcentral.com/1471-2180/13/77involved in multi-host infections in a variety of differentnon-mammalian and mammalian organisms includingamoebae, flies, nematodes, rodents and humans [9-11].The coordinated control of the production of virulenceand antibiotic resistance factors and the ability to adapt tovarious environmental changes is a likely and importantreason that P. aeruginosa is a successful and commonpathogen. The genome sequence of this microorganismrevealed that more than 500 genes, representing nearly10% of the genome, have a putative role in regulation [1].In addition to conventional regulators involved in transcrip-tion of particular genes, e.g. sigma factors, repressors, activa-tors or two-component response regulators, P. aeruginosapossesses several additional proteins that modulate transla-tion, protein biosynthesis and degradation, etc. Here we havedefined the role of the GTPase TypA in the lifestyle ofP. aeruginosa.TypA, also named BipA, belongs to a superfamily ofribosome-binding GTPases within the TRAFAC class(translation factors) of GTPases [12-14]. GTPases arewidely distributed molecular switches found across allbacterial species, and generally cycle between a GDP-bound “off” state and a GTP-bound “on” state [14,15].Collectively they are involved in the regulation of mul-tiple cellular processes and can play important roles intranslation, ribosome biogenesis and assembly, tRNAmodification, protein translocation, cell polarity, cell div-ision and signaling events [14,16]. Since GTPases arewidely conserved in prokaryotes and play an essentialrole in many important bacterial processes, they are anattractive target for novel antibiotic development [17].TypA is highly conserved in bacteria and sharessequence homologies to other GTPases like elongationfactor G. It is found in many pathogens of significantpublic health importance including Vibrio cholera,Yersinia pestis and Mycobacterium tuberculosis [13].Although its precise function is still poorly understood,TypA has been suggested to be involved in the regula-tion of virulence and stress responses in pathogenicEscherichia coli [18,19] and Salmonella enterica SerovarTyphimurium [15], and stress responses in non-pathogenicSinorhizobium meliloti [20] and Bacillus subtilis [21]. Openreading frame PA5117 is annotated as the GTPase TypA,exhibits 75% sequence homology to TypA/BipA from E.coli [13], and plays a role in swarming motility and biofilmformation in P. aeruginosa PAO1 [22]. However, the roleof TypA in pathogenesis of P. aeruginosa is still unknown.Here we constructed a knock-out mutant of typA in P.aeruginosa PA14 and demonstrated the involvement ofTypA in the pathogenesis of P. aeruginosa using differ-ent in vitro and in vivo infection model systems. Consist-ent with these data, we showed using gene expressionanalysis that several virulence-associated genes weredown-regulated in a TypA mutant during host-pathogeninteraction. We also found that TypA plays a role inantibiotic resistance to a variety of different antibioticsand initial attachment leading to subsequent biofilmformation in P. aeruginosa PA14.ResultsTypA is involved in P. aeruginosa virulencePreviously, we showed that a mutation in the typA geneled to a defect in particular virulence-associated featuressuch as swarming and biofilm formation in P. aeruginosaPAO1 [22]. To further investigate the involvement ofTypA in the pathogenesis of P. aeruginosa, we constructeda site-directed typA knock-out mutant in P. aeruginosastrain PA14. Strain PA14 is capable of infecting a widerange of organisms including the amoeba D. discoideum[23,24] and the nematode C. elegans [4] and was thereforemore suitable for virulence analysis using in vivo modelsystems in comparison to strain PAO1.Detailed analyses of virulence attenuation of the PA14typA mutant using the unicellular eukaryotic model or-ganism D. discoideum revealed a consistent, statisticallysignificant (P < 0.001 by Mann Whitney test) 2-foldreduction in the numbers of amoebae required to form aplaque when compared to wild type strain PA14(Figure 1). The virulence phenotype could be completelyrestored to wild type level by heterologous expression ofthe cloned typA gene in strain PA14 typA::ptypA+. Incomparison, a similar 2-fold reduction in numbers ofamoebae was determined when analyzing PA14 trans-poson mutant ID29579 obtained from the Harvard PA14mutant library [25] with a defect in the pscC gene, whichis an essential part of the Type III secretion systemmachinery [26], as a control (Figure 1). To exclude thefact that a simple growth deficiency of the typA mutantis responsible for the attenuated virulence phenotype ofPA14 typA, we performed growth analyses at 23°C and37°C in M9 minimal medium using a Tecan plate readerunder shaking conditions. At both temperatures nosignificant growth defect was observed (data not shown).Since phagocytosis of pathogens by macrophages is acrucial factor in the human immune defense system, wequantitatively analyzed in vitro uptake of PA14 WT andrespective mutant strains using human macrophages ina gentamicin protection assay. We determined a morethan 2-fold increase in internalization of the typA andthe pscC mutant strain in comparison to cells of PA14WT and complemented strain PA14 typA::ptypA+(Figure 2). This result was in accord with the virulencedefect observed in the amoeba model of infection, whichis similarly based on phagocytic killing of bacterial cells.To better understand the mechanism of virulence defi-ciency in the typA mutant, we additionally determinedvirulence in a nematode infection model using C. elegansas host organism under slow killing conditions. In contrastNeidig et al. BMC Microbiology 2013, 13:77 Page 2 of 10http://www.biomedcentral.com/1471-2180/13/77to the Type III secretion based killing of unicellulareukaryotic hosts like amoebae or macrophages, nematodekilling is rather dependent on quorum sensing relatedvirulence features in P. aeruginosa [4,27]. When feedingC. elegans with PA14 wild type, typA mutant andcomplemented PA14 typA::ptypA+ strain, we observed asimilar worm killing rate for all tested strains with onlymarginal differences between PA14 wild type and typAknock-out mutant at day 4 of the incubation time(Figure 3).TypA is involved in rapid attachment and biofilmformationThe ability to form biofilms is a known and important fac-tor in the pathogenesis of P. aeruginosa. To assess theability of the typA mutant to develop biofilms, static mi-crotiter assays were performed to show that PA14 typAdisplayed with approximately 20% reduction a statistically0200040006000800010000PA14 pscCPA14 typA::ptypA+PA14 typAamoeba cell countsPA14 WT******Figure 1 D. discoideum plate killing assay. Each point represents the number of amoebae required to form a plaque on the bacterial lawn ofP. aeruginosa PA14 strains after 5 days of incubation. The typA and pscC mutants had a major defect in this virulence model of infection, whichwas statistically significant as measured with the Mann Whitney test (*** p < 0.001, n = 9).Figure 2 Uptake of P. aeruginosa by human macrophages.Strains were incubated with 1.5 × 105 cells/ml macrophages for 1 hat an MOI of 10. Subsequently, extracellular and attached bacteriawere killed by treatment with gentamicin, and macrophages werelysed with Triton X-100 and lysates plated to enumerate viableintracellular bacteria. Results are expressed as the percentage ofintracellular bacteria that were recovered relative to the PA14 WT.The box plots (median, thick line in the box) represent the mean of3 independent biological repeats, each assayed minimum induplicate (n =≥6). *** indicates a statistically significant difference(p < 0.001), between the typA and pscC mutant and PA14 WT asdetermined by Whitney Mann test.Figure 3 P. aeruginosa virulence towards C. elegans worms.(a) Slow killing: Kaplan-Meier survival plots of worms fed with P.aeruginosa PA14 control (n = 320) (squares), PA14 typA mutant (n = 277)(diamonds) and the complemented strain PA14 typA::ptypA+ mutant(n = 319) (triangles). Each value reported for the assay is the mean ofmeasurements of nine samples from three independent experiments.Neidig et al. BMC Microbiology 2013, 13:77 Page 3 of 10http://www.biomedcentral.com/1471-2180/13/77significant (P < 0.001 by Mann Whitney test) impairmentin biofilm formation at 24 hours (Figure 4) in comparisonto the PA14 WT. This biofilm defect could becomplemented by heterologous expression of wild typetypA in strain PA14 typA::ptypA+. To analyze whether thisbiofilm formation phenotype emerged during the initialadherence phase or later during biofilm growth, a rapidattachment assay was carri d out. The mutant PA14 typAexhibited with approximately 20% reduction a statisticallysignificant (P < 0.001 by Mann Whitney test) defect inadherence which was similar to the biofilm phenotype.However, the investigation of flagellum-mediated swim-ming and swarming motility as well as the type IV pilus-mediated twitching motility, which are all involved inattachment and subsequent biofilm development, revealedno differences between mutant and wild type strain (datanot shown) ruling out defects in the biosynthesis andfunction of these cellular appendages in the typA mutant.Antibiotic susceptibility testingSince recent studies have demonstrated a role for TypAin multidrug resistance in E. coli [28], we studied the im-pact of the typA gene in antibiotic resistance of P.aeruginosa against a variety of different antimicrobialcompounds. As shown in Table 1, the typA mutantexhibited a consistent 2-fold increase in susceptibility to0.10.20.30.40.50.60.70.80.9PA14 typA PA14 typA::ptypA+Biomass[OD 595nm]***PA14 WTB)A)0.00.20.40.60.81.01.2PA14 typA::ptypA+PA14 typABiomass[OD 595nm]PA14 WT***Figure 4 Defects in attachment and biofilm formation in the typA mutant. (A) Requirement for typA in rapid attachment. Attachment wasdetermined using diluted overnight cultures for 60 min at 37°C. Adhered cells were stained with crystal violet. (B) Requirement for typA in staticbiofilm formation. Cells were grown for 24 h at 37°C in polystyrene microtiter plates containing BM2 medium with 0.5% (w/v) casamino acids.Microtiter wells were washed several times during incubation to remove planktonic bacteria. Adherent biofilms were stained with crystal violet,followed by ethanol solubilization of the crystal violet and quantification (A595nm) of stained wells. The box plots (median, thick line in the box)represent the mean of 3 independent biological repeats, each assayed in quintuplicate (n = 15). *** indicates a statistically significant difference(p < 0.001), between the typA mutant and PA14 WT as determined by Whitney Mann test.Neidig et al. BMC Microbiology 2013, 13:77 Page 4 of 10http://www.biomedcentral.com/1471-2180/13/77the cationic peptides polymyxin B and colistin, the ß-lactam antibiotics ceftazidime and meropenem, as wellas tetracycline in comparison to the parent strain. Thisaltered susceptibility could be complemented by intro-ducing wild type copies of typA into the mutant strain.No change in susceptibility was observed regarding thefluoroquinolone ciprofloxacin, the aminoglycoside tobra-mycin, and the cationic host defence peptide LL-37(Table 1).Reduced virulence of PA14 typA due to down-regulationof the Type III secretion systemPrevious studies have shown, that uptake by and killing ofeukaryotic host cells is highly dependent on the Type IIIsecretion system in P. aeruginosa [5,29,30]. To analyze thepotential molecular basis for reduced virulence of the typAmutant observed in our experiments, we investigated geneexpression of known virulence-associated genes in P.aeruginosa using qRT-PCR on bacterial RNA of wild typeand typA mutant strain isolated during host-pathogeninteraction with D. discoideum. These studies revealedthat under the tested conditions, genes coding for the syn-thesis, function and regulation of the Type III secretionsystem, were significantly down-regulated in the typAmutant compared to wild type (Table 2). This observeddown-regulation of important virulence-related genes isconsistent with the noticed virulence defects in the cellu-lar infection studies with D. discoideum and human mac-rophages as hosts.DiscussionIn this study, we have shown that TypA is involved invirulence of P. aeruginosa by analyzing the consequencesof a typA knock-out on phagocytic amoebae and humanmacrophages as well as the interaction with thenematode C. elegans. Moreover, TypA also contributesto resistance to different antibiotics as well as attach-ment and biofilm formation in P. aeruginosa.TypA is a highly conserved prokaryotic GTPaseexhibiting structural homologies to translation factorGTPases such as EF-G and LepA and is described toassociate with the ribosomes under normal bacterialgrowth [15,31]. In enteropathogenic E. coli (EPEC),TypA co-ordinates the expression of key stress and viru-lence factors including flagella, Type III secretion systemas well as the LEE and the espC pathogenicity islands[18,32] by regulating gene expression of major regulatorssuch as Ler, which in turn controls these respectivepathogenicity islands. Consequently, it has been sug-gested that TypA is on a relatively high level in the com-plex regulatory hierarchy of virulence regulation in thisorganism [18,32]. In contrast, analysis in Mycobacteriumtuberculosis revealed that TypA does not act as a viru-lence regulator in this human pathogen, ruling out ageneral involvement of this protein in virulence regula-tion in pathogenic bacteria [33]. However, our resultsdemonstrate that TypA plays a role in the pathogenesisof P. aeruginosa. The typA knock-out mutant exhibiteda significant virulence deficiency in both the amoebaeinfection model and the macrophage uptake studies.These defects were comparable to a pscC mutant with adisrupted Type III secretion system and consistent withthe down-regulation of Type III secretion genes duringhost-pathogen interaction. The Type III secretion systemof Gram-negative bacteria is an important factor ofpathogenesis and is involved in manipulating eukaryoticcells by injecting effector proteins into the host [27] andimpacts diretly on bacterial uptake by phagocytic cells[30]. In P. aeruginosa, this complex, needle-like machin-ery is encoded by 36 genes and an important factor forthe survival during interaction with phagocytic amoebaeor human macrophages, among others [5,29,30]. UsingqRT-PCR, we observed the down-regulation of selectedgenes participating in different aspects of Type III secre-tion, including pscC (outer membrane ring), pscJ (basalsubstructure), pscT (translocation), and pcrV (needle-tipTable 1 MICs of different antibiotics towardsP. aeruginosa PA14 WT, PA14 typA mutant andcomplemented mutant PA14 typA::ptypA+aMIC (μg/ml)Antibiotic PA14 WT PA14 typA PA14 typA::ptypA+Ciprofloxacin 0.03 0.03 0.03Meropenem 2 1 2Ceftazidime 4 2 4Tetracycline 8 4 8Tobramycin 0.25 0.25 0.25Polymyxin B 0.5 0.25 0.5Colistin 0.25 0.125 0.25LL-37 16 16 16aMICs were determined by serial 2-fold dilutions in MH-medium. The MICrepresents the concentration at which no growth was visually observed after24 h of incubation at 37°C. The values shown are the modes of 4 to 6independent experiments.Table 2 Gene expression of selected Type III secretiongenes in the typA mutant compared to that in wild typePA14 using RT-qPCRGene Fold change in gene expression in the typA mutantrelative to wild typeaT3SSexsA −3.1 ± 0.5pscC −2.3 ± 0.4pscJ −3.5 ± 0.3pscT −5.1 ± 0.3pcrV −5.8 ± 0.6Neidig et al. BMC Microbiology 2013, 13:77 Page 5 of 10http://www.biomedcentral.com/1471-2180/13/77complex). The transcription of Type III secretion genesis tightly regulated by ExsA in P. aeruginosa. This mas-ter regulator controls both, the synthesis of the secretionsystem as well as effector protein production, and inter-acts in concert with the global cyclic AMP and Gacregulatory systems [5,34]. Our studies showed that inaddition to genes involved in assembly of the secretionapparatus, expression of exsA was also significantlydown-regulated in the typA mutant compared to wildtype cells. To identify, if increasing Type III secretionactivity is sufficient to complement our virulence pheno-type, we heterologously expressed the exsA gene usingplasmid pUCP20::exsA+ in the typA mutant and obtainedan identical number of amoebae required for plaque for-mation in both mutant and wild type PA14 harboringpUCP20::exsA (data not shown). These findings suggestthat, like in E. coli, TypA is part of the complex regulatorycascade involved in controlling Type III secretion in P.aeruginosa by impacting expression of genes involved inregulation and assembly of the secretion machinery. SinceTypA is a GTPase associated with the ribosomes, a furtherdown-regulation of the Type III secretion machinery atthe translational level might also be possible; this couldresult in an even stronger impairment of the Type IIIsecretion system.Previously, it has been shown that the Type III secre-tion system including its associated virulence effectorsdoes not play a noticeable role in nematode killing[4,35], which is rather dependent on quorum sensingrelated virulence factors such as RhlR and LasR [27,36].Thus, it is not surprising, that a mutation in typA with adown-regulation in the Type III secretion system did notresult in significant virulence attenuation in our studiedinfection model. Additional analyses of quorum sensingdependent production of the extracellular protease LasBand toxin pyocyanin did not reveal a significant differ-ence between wild type and mutant strain (data notshown) demonstrating that TypA does, most likely, notaffect quorum sensing in P. aeruginosa PA14.TypA was first described to be involved in human bac-tericidal/permeability-increasing protein BPI, a cationichost defence peptide from human neutrophils, resistancein S. typhimurium and E. coli [37,38]. Although we werenot able to detect any differences regarding resistance tocationic human host defence peptide LL-37, we found thatTypA is also participating in resistance against a variety ofclinically important antibiotics such as ß-lactam, tetracyc-line and peptides antibiotics in P. aeruginosa. Due to thiswide range of different antimicrobials with unrelatedmodes of action, it is likely that the involvement of TypAin antibiotic stress resistance is rather unspecific andcould be based on the fact that TypA is part of a moregeneral stress response resulting in resistance. This wouldbe in accordance with earlier studies showing theinvolvement of TypA in a wide variety of very differentstress responses in a number of pathogenic and non-pathogenic microorganisms, among other stress factorswere antimicrobials, low pH, oxidative or detergent stress[20,37,38].Biofilm formation is a crucial factor in the pathogen-esis of P. aeruginosa and is involved in many chronicinfections including chronic lung infections of cysticfibrosis patients or foreign body part infections [39]. Bio-film development is a sequential process initiated by theattachment of planktonic cells to a surface, followed byformation of microcolonies and biofilm maturation. Bac-teria grown in biofilms exhibit high resistance againstantimicrobial agents, are protected from the host im-mune response and are notoriously difficult to eradicate[39-41]. Although the typA mutant was able to formbiofilms, we observed a more than 20% reduction in bio-film mass compared to wild type cells. By analyzing theinitial adhesion phase of biofilm development, we identi-fied that this reduction in biofilm is, at least in parts,due to a significant impairment in rapid attachment ofthe typA mutant in the respective microtiter plate assay.This impairment in attachment results in less bacterialcells initiating biofilm formation and subsequently lowerbiofilm growth, which could not be restored to wild typelevels during further biofilm development. Interestingly,it was shown previously that TypA is involved in adher-ence to biotic surfaces and interaction of enteropatho-genic E. coli with epithelial cells [19] and the symbioticinteraction of S. meliloti with the nodules of the legumeMedicago truncatula [20] indicating a role of TypA incell-cell contact. Biofilm initiation and cell adhesion arerather complex processes influenced by a large numberof proteins and factors, among others are flagellum- andtype IV pilus-mediated bacterial motility and attach-ment, respectively. Although we have recently shown,that TypA is involved in swarming motility in P.aeruginosa strain PAO1 [22], we did not observe anyimpairment in swimming, swarming or twitching motilityin the PA14 typA mutant suggesting a mechanism not re-lated to a defect in flagella or type IV pili biogenesis andfunction, respectively, is responsible for the impairment inadhesion and biofilm initiation in this mutant.ConclusionsIn this study, we were able to demonstrate the involve-ment of TypA in the pathogenesis of P. aeruginosa by ana-lyzing the consequences of a typA knock-out. This typAmutant exhibited reduced virulence towards phagocyticamoebae and increased uptake by human macrophages,impaired cell attachment and subsequent biofilm forma-tion and a reduction in antimicrobial resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics.The typA mutant exhibited a dysregulation of genesNeidig et al. BMC Microbiology 2013, 13:77 Page 6 of 10http://www.biomedcentral.com/1471-2180/13/77involved in regulation and assembly of the Type III secre-tion system, consistent with the observed phenotypes androle in virulence regulating Type III secretion system.MethodsOrganisms, plasmids, primers, and growth conditionsThe organisms and plasmids used in this study are listedin Table 3 and include P. aeruginosa PA14 [25] andDictyostelium discoideum Ax2 [24]. The sequences ofDNA primers (Eurofins MWG Operon, Germany) usedin these studies are available upon request. E. coli wasroutinely grown in Luria-Bertani (LB) broth, P.aeruginosa in M9 [23], LB or BM2 [44] medium, and D.discoideum in HL5 broth medium [45]. D. discoideumwas incubated in cell culture flasks (Greiner Bio One,Frickenhausen, Germany) at 22.5°C and sub-culturedtwice a week. When required for plasmid or resistancegene selection or maintenance, gentamicin, ampicillinand carbenicillin were added at final concentrations of30, 100 and 200 μg/ml, respectively.Amoeba plaque assayIn this cellular model system, a more virulent P.aeruginosa strain will limit the ability of the amoebae toform a plaque on a bacterial lawn to a greater extentthan a less virulent strain. The assay was performedaccording to the method described previously [23].Briefly, 50 μl of overnight cultures grown in LB mediumwere mixed with 200 μl PBS buffer and plated on M9agar plates. Plates were dried on a laminar flow benchfor 15 min to obtain a dry, even bacterial lawn. Amoe-bae grown for 2 to 4 days in the respective mediumwere harvested by centrifugation at 510 x g for 10 -minutes, washed and resuspended in PBS buffer. Cellswere adjusted to 8 × 106 cells per ml and kept on ice.This stock solution was serially diluted and used toprepare droplets of 5 μl containing between 5 and20,000 amoebae, which were subsequently spottedonto the bacterial lawn. Plates were incubated for 5days at 22.5°C and the highest dilution at whichgrowth of the amoebae caused a visible plaque of bac-terial clearance was reported. Three independent ex-periments performed at least in duplicate were carriedout for each bacterial strain.Gentamicin protection assayIn vitro internalization of PA14 WT and mutant cells byhuman macrophages derived from monocytes (MDM)was performed as previously described [46] with modifi-cations. Briefly, mid-logarithmic phase cultures of P.aeruginosa were washed with complete RPMI mediumand resuspended in 1 ml of the medium. The resuspendedbacteria were added to 1.5 x 105 MDM cells/ml, at amultiplicity of infection (MOI) of 10, and incubated for1 h at 37°C. Subsequently, cells were washed withcomplete RPMI and incubated with 400 μg/ml of gentami-cin for 30 min at 37°C to kill the extracellular andattached bacteria. After gentamicin treatment, MDM cellswere washed and lysed with 0.1% Triton X-100. Lysateswere plated onto LB agar and incubated overnight at37°C. The next day, colonies were counted and relativephagocytic uptake was determined by CFU counts. Threeindependent experiments with at least duplicates in eachexperiment were performed for each bacterial strain.Caenorhabditis elegans synchronization and virulence assayThe C. elegans wild-type Bristol strain N2 was obtainedfrom the Caenorhabditis Genetics Center (Minneapolis,MN, USA). C. elegans were maintained under standardculturing conditions at 22°C on nematode growthTable 3 Strains and plasmids used in this studyStrain or plasmid Description and characteristicsa ReferenceStrainsP. aeruginosaPA14 WT Wild type P. aeruginosa PA14 [25]PA14 typA typA insertion mutant of PA14, Gmr This studyPA14 typA::ptypA+ Complemented mutant PA14 typA harboring plasmid pUCP20::typA+; Gmr, Cbr This studyPA14 pscC pscC transposon mutant ID29579 of the Harvard PA14 mutant library [25]E. coliDH5α F–φ80lacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK– mK+) supE44λ– thi-1 gyrA96 relA InvitrogenPlasmidspUCP20 E. coli – Pseudomonas shuttle vector for constitutive expression of cloned genes, Cbr [42]pEX18Ap Suicide vector for mutant regeneration in Pseudomonas, Ampr/Cbr [43]pUCP20::typA+ pUCP20 containing the cloned typA gene; Ampr/Cbr This studypUCP20::exsA+ pUCP20 containing the cloned exsA gene; Ampr/Cbr This studya Antibiotic resistance phenotypes: Ampr, ampicillin resistance for E. coli; Cbr, carbenicillin resistance for P. aeruginosa; Gmr, gentamicin resistance.Neidig et al. BMC Microbiology 2013, 13:77 Page 7 of 10http://www.biomedcentral.com/1471-2180/13/77medium (NGM: 3 g NaCl, 2.5 g peptone, 17 g agar, 5 mgcholesterol, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 25 ml1 M KH2PO4, H2O to 1 liter) agar plates with E. coliOP50 as a food source [47]. Synchronous cultures ofworms were generated after worm adult populationexposure to a sodium hypochlorite/sodium hydroxidesolution as previously described [48] and adapted [49].The resulting eggs were incubated at 22°C on an E. coliOP50 lawn until the worms reached the L4 (48 hours)life stage (confirmed by light microscopy). Bacteriallawns used for C. elegans survival assays were preparedby spreading 50 μl of P. aeruginosa strains on 35 mmNGM conditioned Petri dishes supplemented with0.05 mg ml−1 5-fluoro-20-deoxyuridine. This nucleotideanalog blocks the development of the next C. elegansgeneration by inhibition of DNA synthesis. The plateswere incubated overnight at 37°C and then placed atroom temperature for 4 h. Fifteen to twenty L4 synchro-nized worms were harvested by resuspension in M9buffer (3 g KH2PO4, 6 g NaHPO4, 5 g NaCl, 1 ml 1 MMgSO4, H2O to 1 liter), plated on the 35 mm assay Petridishes and incubated at 22°C. Worm survival was scoredafter 1 h, 24 h and on each subsequent day, using anAxiovert S100 optical microscope (Zeiss, Oberkochen,Germany) equipped with a Nikon digital Camera DXM1200 F (Nikon Instruments, Melville, NY, USA). Wormswere considered dead when they remained static withoutgrinder movements for 20 s. The results were expressedas the percentage of living worms and were the average ofthree independent assays performed in triplicate.Growth curvesOvernight cultures grown in LB medium were dilutedinto M9 medium to obtain equal starting optical dens-ities at 600 nm (OD600). Five-μl portions of thesecultures were added to 195 μl of fresh M9 medium in96-well microtiter plates. The growth of the cultures at37°C and 23°C under shaking conditions was monitoredwith a Tecan Infinite F200 Pro.Plasmid and typA knock-out mutant generationFor the construction and complementation of a typAknock-out mutant in P. aeruginosa PA14 the typA gene(gene number PA_67560) was amplified by PCR usingEcoRI and HindIII flanked oligonucleotides, respectively,and subsequently cloned behind the lac promoter in thebroad host range vector pUCP20, resulting in pUCP20::typA+. For the heterologous expression of the exsA gene,exsA was amplified by PCR using EcoRI and XbaIflanked oligonucleotides, respectively, and subsequentlycloned into pUCP20, resulting in pUCP20::exsA+. Theseplasmids were then transferred into E. coli DH5α bytransformation or P. aeruginosa by electroporation. Theknock-out mutant was obtained according to themethods described previously [43]. Briefly, the hybridplasmid pUCP20::typA+ was digested with SmaI todelete a 1.1 kb fragment from the typA gene, which wassubsequently replaced with a Ω gentamicin resistancegene cassette for selection. The disrupted typAΩGmgene was amplified by PCR and cloned into the suicidevector pEX18Ap [43] and transferred into P. aeruginosaPA14 to generate the typA knock-out mutant named P.aeruginosa PA14 typA by allelic exchange.MIC determinationMICs were measured using standard broth microdilutionprocedures [50] in Mueller Hinton (MH) medium.Growth was scored following 24 h of incubation at 37°C.Motility, biofilm and rapid attachment assaysSwimming, swarming and twitching motility were evalu-ated as described previously [44]. The abiotic solid sur-face assay was used to measure biofilm formationaccording to the previously described method with thefollowing modifications [51]. Overnight cultures werediluted 1:100 in BM2 containing 0.5% (w/v) casaminoacids and inoculated into 96-well polystyrene microtiterplates and incubated at 37°C for 60 min without shakingto allow bacterial cell adhesion. Subsequently, the micro-titer wells were washed twice to remove planktonic cellsand new biofilm growth medium was added. This wash-ing step was repeated after 4 and 16 hours of incubation.After 24 h, the biofilm was staining using crystal violetand the absorbance was measured at 595 nm using aTecan Infinite F200 Pro.Rapid attachment of bacterial cells to a surface wasanalyzed as described previously [44]. Briefly, overnightcultures grown in BM2-medium were washed and di-luted in BM2 medium containing 0.1% (w/v) casaminoacids (CAA) to an OD595nm of 1.0. One hundred μl ofthis suspension was used to inoculate each well of amicrotiter plate. Cells were allowed to adhere for 60 minat 37°C prior to staining with crystal violet.RNA extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR)For analysis of virulence gene expression, overnight cul-tures of P. aeruginosa PA14 WT and the typA mutantwere washed twice and resuspended in PBS buffer andadjusted to an OD600 of 2.0. For each bacterial cell sus-pension, 10 μl was mixed with washed amoeba cells of2-day old D. discoideum cultures at a ratio of 3:1 bacteriato amoebae and the mixtures were plated on M9 agarplates. After incubation for 48 h at 22.5°C, cells wereharvested from the agar plate surface, using an inoculationloop, and were resuspended in M9 medium supplementedwith RNA protect reagent (Qiagen, Germany). To sep-arate cells of D. discoideum from the bacterial cells, theNeidig et al. BMC Microbiology 2013, 13:77 Page 8 of 10http://www.biomedcentral.com/1471-2180/13/77mixtures were centrifuged for 1 min at 1,000 rpm andthe supernatants containing the bacterial cells wereused for RNA extraction.RNA isolation, cDNA synthesis, and qRT-PCR analysiswere performed as described previously [52] using thePower SYBR Green PCR Master Mix in an Abi 7300 RealTime PCR System (Applied Biosystems). All reactionswere normalized to the house keeping gene rpsL. Experi-ments were repeated with three independent cultures.Competing interestsThe authors declare that they have no competing interest.Authors’ contributionsOL and JO designed the experiments, supervised the research and wrote thepaper. AN, ATYY, TR, BT, NS and MR did experiments and/or data analysis. Allauthors read and approved the final manuscript.AcknowledgementsWe gratefully acknowledge financial support by the BioInterfaces (BIF)Program of the Karlsruhe Institute of Technology (KIT) in the HelmholtzAssociation and by the “Concept for the Future” of the Karlsruhe Institute ofTechnology (KIT) within the German Excellence Initiative. ATYY receivedstudentships from Cystic Fibrosis Canada and the Natural Sciences andEngineering Research Council of Canada (NSERC). We thank Prof. M. Steinertfor kindly providing D. discoideum, Prof. G. Hänsch for help with thegentamicin protection assay, and Olivier Maillot and Magalie Barreau fortechnical assistance.Author details1Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, POBox 3640, 76021, Karlsruhe, Germany. 2Centre for Microbial Diseases &Immunity Research, University of British Columbia, 2259 Lower Mall,Vancouver, BC, Canada. 3Laboratory of Microbiology Signals andMicroenvironment, LMSM EA 4312, University of Rouen, 55 rue SaintGermain, 27000, Evreux, France.Received: 30 October 2012 Accepted: 4 April 2013Published: 9 April 2013References1. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ,Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, et al: Complete genomesequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.Nature 2000, 406(6799):959–964.2. Govan JR, Deretic V: Microbial pathogenesis in cystic fibrosis: mucoidPseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996,60(3):539–574.3. 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Microbiology 2007, 153(Pt 2):474–482.doi:10.1186/1471-2180-13-77Cite this article as: Neidig et al.: TypA is involved in virulence,antimicrobial resistance and biofilm formation in Pseudomonasaeruginosa. BMC Microbiology 2013 13:77.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitNeidig et al. BMC Microbiology 2013, 13:77 Page 10 of 10http://www.biomedcentral.com/1471-2180/13/77

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