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Contents lists available at SciVerse ScienceDi rect

Journ al of Inverte brate Pathology

journal homepage: www.elsevier .com/ locate/ j ip

Short Communication

Iron regulates Bacillus thuringiensis haemolysin hlyII gene expression during insect infection

Seav-Ly Tra n 1, Elisabeth Guillemet 1, Didier Lereclus, Nalini Ramarao ⇑INRA, Unité MICALIS UMR-1319, La Minière, 78280 Guyancourt, France

a r t i c l e i n f o

252627282930313233

Article history:Received 23 January 2013 Accepted 4 April 2013 Available online xxxx

Keywords:B. thuringiensis hlyIIGene regulation Fur Iron In vivo expression

0022-2011/$ - see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.jip.2013.04.001

⇑ Corresponding author. Address: INRA, Unité MICALa Minière, 78280 Guyancourt, France. Fax: +33 1 30

E-mail address: nalini.ramarao@jouy.inra.fr (N. Ra1 These authors contributed equally to this work.

Please cite this article in press as: Tra n, S.-L ., et aPatho l. (2013), http://dx.doi.org/1 0.1016/j.j ip.20

a b s t r a c t

Bacillus thuringiensis (Bt) is a spore-forming entomopathogen broadly used in agriculture crop . Thehaemolysin HlyII is an important Bt virulence factor respo nsible for insect death. In this work, we focused on the regulation of the hlyII gene throughout the bacterial growth in vitro and in vivo during insect infec- tion. We show that hlyII regulation depends on the global regulator Fur. This regulation occurs indepen- dently of HlyIIR, the other known regulator of hlyII gene expression. Moreover, we show that hlyII ishighly expressed when iron is depleted in vivo . As HlyII induces haemocyte and macrophage death, which are involved in the sequestration of iron upon infection, HlyII may induce host cell death to allow bacteria to gain access to iron.

� 2013 Published by Elsevier Inc.

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1. Introductio n

The insect pathogen Bacillus thuringie nsis (Bt) is a model ofchoice to study the time course of the infection process in insect.The mechanis m by which Bt infects and kills insect larvae has been extensively studied and mainly relies on the presence of plasmids encoding specific toxins (Schnepf et al., 1998 ). Bt spores and toxins are ingested by larvae, followed by toxin binding to specific recep- tors located on the midgut epithelial cells (Nielsen-L eRoux et al.,2012). The toxins induce cell lysis and create favorable conditions for germination and bacterial growth. Next, the vegetative bacteria multiply in the insect hemocoel and cause septicemia (Raymondet al., 2010; Salamitou et al., 2000 ). In addition to insecticidal tox- ins, Bt harbors a number of genes encoding exported factors such as enterotoxins , haemolysins, phospholipa ses and protease s that might contribute to virulence (Agaisse et al., 1999; Brillard et al.,2008; Gilois et al., 2007; Ramarao and Lereclus, 2006; Tran et al.,2010). Among these toxins, the haemolysin HlyII has been shown to be an important virulence factor in the B. cereus group (Cadotet al., 2010; Tran et al., 2011a ). HlyII has haemolytic properties (Andreeva et al., 2006; Miles et al., 2006 ), and induces apoptosis of host monocytes and macrophages in vivo (Tran et al., 2011a,b ).A precise knowledge of the conditions governing the expression of a gene improves our understa nding of its role in the functioning

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of the bacterial cell, but also during pathogenes is. We and others have shown that hlyII expression is negatively regulated by the transcrip tional regulator HlyIIR (Budarina et al., 2004; Guillemet et al., 2013 ), encoded by the gene located immedia tely down- stream of hlyII. In addition, the hlyII promoter region contains aFur (ferric uptake regulator) box (Harvie et al., 2005 ). Fur is a major factor implicate d in the regulation of systems involved in iron acquisition and metabolism, ensuring iron homeostas is. In the presence of iron, Fur binds to a Fur box located in the promoter re- gion of various genes, thus inhibiting their transcription. In vitro studies in the heterologou s systems E. coli and B. subtilis have con- sistently shown that hlyII expression is regulated by Fur (Sinevaet al., 2012 ).

In this study, we investigated the dual regulatory process ofhlyII expression in its natural host Bt, and showed the involvement of HlyIIR, iron and Fur in the time course of hlyII expression during host infection.

2. Material s and methods

2.1. Plasmid and mutant strain constructions

The fur gene was deleted as follows. BamHI-EcoRI (1000 bp) and SphI-HindIII (1000 bp) DNA fragments corresponding to upstream and downstre am regions of the fur gene were generated from the Bt407 chromosome by PCR using the primer pairs: fur-1 (50-CGCGGATC CCCCTTTCCTAT GC-3 0)-fur-2 (50-CGGAATTCGT ACAC AATGTGGTG -30) and fur-3 (50-ACATGC ATGCCCGATCTCT GGCG-3 0)-fur-4 (50-CCCAAG CTTCGCCGTTTTA TTAT-3 0). A Tet R cassette

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carrying a tet gene was purified from pHTS1 (Sanchis et al., 1996 )as a 1.6 Kb fragment. The amplified DNA fragments were digested with the appropriate enzymes and inserted in the pRN5101 plasmid (Villafane et al., 1987 ) with the selection marker cassette cloned in between the up- and downstre am chromosomal fragments of the fur gene. The resulting plasmid was introduced into Bt407 by electroporation (Arantes and Lereclus, 1991 ) and the fur gene was deleted by a double crossover event as previously described (Lereclus et al., 1992 ). Chromosom al allele exchange was confirmed by PCR with oligonucl eotide primers located upstream and downstre am from fur-1, and fur-4, respectively: fur-5 (50-AATCGCTTCCG TTGCTAAACTT C-3 0) and fur-6 (50-GTGTGAACGGAGGAAGAAA AAT-3 0). The insertion mutant strain was designated Bt407Dfur.

Transcriptional hlyII-lacZ and hlyIIR-lacZ fusions were con- structed by insertion of putative promote r regions of genes under investigatio n in front of a promoterles s lacZ reporter gene, encoding a b-galactos idase enzyme. The pHT304-P hlyII0Z and pHT304- PhlyIIR0Z plasmids (Guillemet et al., 2013 ) were introduced into Bt407 and into the mutant strains Bt407 Dfur and Bt407 DhlyIIR(Guillemet et al., 2013 ) by electropo ration. Transforma nts were named Bt407 [pHT-P hlyII0Z], Bt407 Dfur [pHT-PhlyII0Z], Bt407 DhlyIIR[pHT-PhlyII0Z], Bt407 [pHT-P hlyIIR0Z] and Bt407 Dfur [pHT-PhlyIIR0Z].

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2.2. ß-galactosidase assays

Strains harboring plasmid transcriptional lacZ fusions were grown in LB medium with the appropriate antibiotics at 37 �C un- der shaking. Samples were taken every hour from t � 1 h (t � 1) tot + 4 h (t4), t0 h (t0) corresponding to the transition state between the exponential and the stationary phases of the bacterial culture.Determinat ion of ß-galactosidase activity was achieved as previ- ously described (Bouillaut et al., 2005; Guillemet et al., 2010 ).When indicated, the iron chelator 2,2 0dipyridyl (Dip, Sigma), used at a final concentration of 0.2 mM and ferric chloride (FeCl3) at afinal concentratio n of 0.8 mM were added to the bacterial culture as described in (Fedhila et al., 2006 ) at t-1 and t0, respectively .

ß-galactosidase assays from infected insect larvae were per- formed as previously described (Guillemet et al., 2010 ). Briefly, in- sects were intra-haemo coelly infected (Ramarao et al., 2012 ) with 5 � 106 bacteria of the Bt407 [pHT304- hlyII0Z] strain (Guillemetet al., 2013 ). When indicated FeCl 3 or Dip were injected into the in- sect hemocoel before infection with the bacteria. At the indicated time points post infection, 6 larvae were crushed per condition sand bacteria were recovered by centrifugation and multi step washing. Bacteria were then lyzed and ß-galactosidase assays were performed. For in vitro assays, the specific activities are expressed in units of ß-galactosidase relative to the total bacterial protein amount (Miller units). For in vivo data, protein levels in the sam- ples were normalized to uninfected larvae and thus cannot becompared with in vitro data.

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3. Results

3.1. hlyII expression is negatively regulated by iron and Fur inB. thuringiensis

Very recent data show that Fur, when overexpresed in the het- erologous host B. subtilis , interfere with hlyII expression by binding to the Fur box and competing with the RNA polymerase binding tothe hlyII promoter (Sineva et al., 2012 ). Concomitan tly to these data, we studied the involvement of Fur in hlyII regulation in its na- tive host B. thuringiensis by constructi ng a Bt407 mutant deficientin the fur gene and by monitoring hlyII expression in this mutant by b-galactosidas e assay (Fig. 1A). On average, hlyII expression

Please cite this article in press as: Tra n, S.-L ., et al. Iron regulates Bacil lus thuri ngPathol. (2013), http://dx.doi.org/10.1 016/j.jip .2013.04.001

was 2–4-fold higher in the Dfur mutant than in the wt strain. Thus,Fur is a negative regulator of hlyII expression in Bt.

As Fur is generally controlle d by iron, we assessed hlyII expres-sion in a medium lacking free iron by the use of the iron chelator Dipyridyl , with or without iron in excess (Fig. 1B). Addition ofthe iron chelator led to a drastic increase in hlyII expression, show- ing that hlyII expression is inhibited by iron. Consistently, addition of iron in excess in the medium previousl y depleted in iron, in- duced hlyII expression levels to drop to levels observed in the con- trol medium. Thus hlyII expression was drastically increased iniron-dep leted conditions.

To determine whether Fur and iron were acting on hlyII expres-sion by acting via its repressor HlyIIR, hlyII expression was moni- tored in a DhlyIIR mutant in an iron-deplete d medium (Fig. 1C).hlyII expression was also higher without iron in a DhlyIIR mutantwith a 2–3-fold increase compared to control medium. The overall expression of hlyII was around 2-fold higher (with or without iron)in the DhlyIIR mutant (Fig. 1C) compared to the wt (Fig. 1B),consisten t with the regulator y role of HlyIIR in hlyII expression(Guillemet et al., 2013 ). In addition, the expression of the repressor hlyIIR was assessed in a Dfur mutant. As expected , hlyIIR expressionwas similar in the wild type and the Dfur mutant (not shown).Thus, the role of Fur and iron on hlyII expression is not mediated through HlyIIR.

3.2. hlyII expression during insect infection and responsiveness to iron

To assess the expression of hlyII in vivo and the influence of iron during infection, G. mellonella larvae were crushed after infection,bacteria were taken from infected insect, washed and hlyII expres-sion was monitored by ß-galactosidase assay (Fig. 2A), with protein content values normalized to the protein content of non- infected insects as described in (Guillemet et al., 2010 ). The hlyIIexpression was low during the first 6 h of infection and increased until 24 h post-infection. After insect death, usually occurring around 24 h post infection, hlyII expression decreased. The hlyIIgene was thus expresse d in vivo before insect death, highlight ing the role of HlyII during the bacterial virulence process. hlyII expres-sion was, on average, two fold higher in the DhlyIIR mutant than inthe wt strain (Fig. 2A) showing that HlyIIR also acted as a negative regulator of hlyII expression in vivo .

To assess the role of iron on hlyII expression in vivo , G. mellonell alarvae were crushed after infection, and hlyII expression was mon- itored by ß-galactosidase assay. hlyII expression was monitored after co-injection in the insects of bacteria and either iron or its chelator DIP. During the early steps of infection, addition of DIP led to an increase in hlyII expression, and addition of iron resulted in a reduction of hlyII expression (Fig. 2B). These data suggest that,at the beginning of the infection process, iron inhibits hlyII expres-sion and thus, chelating iron led to an increase in hlyII expression.Taken together, our data show that iron plays an important role inregulatin g hlyII expression in vivo .

4. Discussion

During infection, bacteria are confronted with a different envi- ronment. Their capacity to adapt to this usually hostile environment is a challenge that will determine their ability to survive and induce pathology . All bacteria require energy for growth. As such, iron is acrucial compound for bacterial multiplication and thus for their capacity to colonize their hosts. It has been reported that a common host defense mechanism relies on iron sequestra tion by immune cells (Ratledge and Dover, 2000; Weinberg, 2009 ) and imply that pathogen ic bacteria may acquire iron from the host by killing the cells. We consisten tly show that the hlyII gene, coding for a Bt

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Fig. 1. Fur and iron repress hlyII expression independently of HlyIIR. (A) Specific b-galactosidase activity (Miller units) of Bt407 (circle) and Bt407 Dfur (diamond) strains harboring the transcriptional hlyII0-lacZ fusion was measured at the indicated time points. Values are means of three independent experiments done in triplicate. The bacterial strains Bt407 (B) and Bt407 DhlyIIR (C) harboring the transcriptional fusion hlyII0-lacZ were grown at 37 �C in either LB medium (circle) or LB medium supplemented at t-1 with the iron chelator Dipyridyl at 0.2 mM (Dip) (square), or LB medium supplemented at t-1 with Dip and at t0 with ferric chloride (FeCl3) at 0.8 mM (cross). Specificb-galactosidase activity (Miller units) was measured at the indicated time points. Values are expressed as the mean of three independent experiments.

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haemolysin implicated in phagocyti c cell death, is negatively regulated by the presence of iron. hlyII expression is induced in aniron-deplete d medium and when iron is added in excess to this depleted medium hlyII expression is shut down again. These results indicate that iron concentr ation in the environm ent influences hlyIIexpression.

The importance of HlyII during vir ulence has been previously demonstrat ed in insect, mice and crustacean (Tran et al.,2011a,b; Sineva et al., 2009 ). We aimed to determine the importance of iron present in the environm ent during patho- genesis on regulation of hlyII expression. Our data show that during the early steps of infection, iron inhibits hlyII expression.Then, hlyII expression increases in vivo along the infectious pro- cess, possibly suggesting a limitation in iron. Thus, as long asthe bacteria have access to iron, for instance by scavenging itusing siderophores (Hotta et al., 2010 ) or other proteins such as IlsA (Daou et al., 2009 ), hlyII expression is maintained low by the activation of Fur. When iron gets sequestered by phago- cytic cells as a natural host defense, Fur gets inactivated and hlyII expression starts. HlyII is then produced by the bacteria,

Please cite this article in press as: Tra n, S.-L ., et al. Iron regulates Bacil lus thur ingPatho l. (2013), http://dx.doi.org/1 0.1016/j.j ip.2013.04.00 1

secreted and induces haemocytes and macrophage death (Tranet al., 2011a ). The cell content might then be released into the environment, providing the bacteria access to nutrients,allowing bacterial growth and promoting a new cycle of hlyIIgene inhibition/e xpression.

It has consistently been previously shown that an increase inproduction of host iron-bind ing proteins is observed following infection in order to limit bacterial growth (Ratledge and Dover,2000). To counteract this phenomenon, bacteria induce the expres- sion of genes that are negatively iron-regulated (Ratledge and Do- ver, 2000 ). These genes include those implicated in iron acquisition and several virulence factors (Daou et al., 2009; Fedhila et al., 2006;Harvie et al., 2005 ). Consistently, deletion of fur in Bt induces a de- crease in virulence, showing a link between virulence and iron metaboli sm (Harvie et al., 2005 ).

Taken together, our data provide new insights into the regula- tory pathways of hlyII, which play an important role during the Bt virulence process. As iron is an essential bacterial growth com- ponent, we believe that our data provide an important contribution to the understa nding of bacterial infection strategies.

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Fig. 2. (A) hlyII is expressed in vivo during infection. Galleria mellonella larvae were injected into their haemocoel with the Bt407 (circle) or Bt407 DhlyIIR (square) strains harboring the transcriptional hlyII0-lacZ fusion. b-galactosidase was measured at the indicated time points post infection. Values represent means of four independent experiments. (B) Iron regulates hlyII expression in vivo during infection. G. mellonella larvae were co-injected into their haemocoel with the Bt407 strain harboring the transcriptional hlyII0-lacZ fusion, and with Dip or FeCl 3. b-galactosidase was measured 4 h post infection. Values represent means of four independent experiments.

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Acknowledgmen ts

We wish to thank Alain Lerééc for excellent technical assis- tance. We thank Stéphane Perchat for very helpful computer support.

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