the solute-binding component of a putative mn(ii) abc transporter (mnta) is a novel bacillus...

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Molecular Microbiology (2005) 58(2), 533–551 doi:10.1111/j.1365-2958.2005.04848.x First published online 13 September 2005 © 2005 Blackwell Publishing Ltd No claim to original Israeli government works Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005 ? 2005582533551Original ArticleRole of MntA in anthrax pathogenesisO. Gat et al. Accepted 8 August, 2005. *For correspondence. E-mail [email protected]; Tel. (+972) 8 9381595; Fax (972) 8 9401404. The solute-binding component of a putative Mn(II) ABC transporter (MntA) is a novel Bacillus anthracis virulence determinant Orit Gat, 1 Itai Mendelson, 1 Theodor Chitlaru, 1 Naomi Ariel, 1 Zeev Altboum, 2 Haim Levy, 2 Shay Weiss, 2 Haim Grosfeld, 1 Sara Cohen 1 and Avigdor Shafferman 1 * Departments of 1 Biochemistry and Molecular Genetics and 2 Infectious Diseases, Israel Institute for Biological Research, PO Box 19, Ness Ziona, 74100, Israel. Summary Here we describe the characterization of a lipoprotein previously proposed as a potential Bacillus anthracis virulence determinant and vaccine candidate. This protein, designated MntA, is the solute-binding com- ponent of a manganese ion ATP-binding cassette transporter. Coupled proteomic-serological screen of a fully virulent wild-type B. anthracis Vollum strain, confirmed that MntA is expressed both in vitro and during infection. Expression of MntA is shown to be independent of the virulence plasmids pXO1 and pXO2. An mntA deletion, generated by allelic replace- ment, results in complete loss of MntA expression and its phenotypic analysis revealed: (i) impaired growth in rich media, alleviated by manganese sup- plementation; (ii) increased sensitivity to oxidative stress; and (iii) delayed release from cultured mac- rophages. The ΔmntA mutant expresses the anthrax- associated classical virulence factors, lethal toxin and capsule, in vitro as well as in vivo, and yet the mutation resulted in severe attenuation; a 10 4 -fold drop in LD 50 in a guinea pig model. MntA expressed in trans allowed to restore, almost completely, the virulence of the ΔmntA B. anthracis strain. We pro- pose that MntA is a novel B. anthracis virulence deter- minant essential for the development of anthrax disease, and that B. anthracis ΔmntA strains have the potential to serve as platform for future live attenu- ated vaccines. Introduction Bacillus anthracis is an endospore-forming toxin-produc- ing bacterium and the causative agent of anthrax, a seri- ous and often fatal disease of livestock and humans. Although primarily a disease of herbivores, anthrax can affect humans via contact with infected animals or upon direct exposure. B. anthracis is also considered a biolog- ical terror agent (CDC, 2005), because of its capability to cause lethal inhalational anthrax and the environmental persistence of its spores. During inhalation, dormant B. anthracis spores are engulfed by resident alveolar macrophages (Guidi-Ron- tani et al., 1999; 2001; Dixon et al., 2000). En route to the regional lymph nodes, the ingested spores germinate and probably multiply within the phagosome while producing virulence factors. Subsequently, the vegetative cells lyse the macrophages and escape into the blood stream, resulting in bacteraemia, sepsis and ensuing death. Bacillus anthracis virulence has been predominantly attributed to factors carried by two native plasmids; the tripartite toxin: protective antigen (PA), lethal factor (LF) and oedema factor (EF) (encoded by pagA, lef and cya genes, located on pXO1) and the antiphagocytic poly- γ-D- glutamic acid capsule (synthesis and degradation of which is encoded by capBCA and dep genes, located on pXO2). However, a battery of as yet undefined virulence factors probably reside on the B. anthracis chromosome (Cohen et al., 2000; Baillie and Read, 2001; Brossier et al., 2002; Ariel et al., 2003; Bourgogne et al., 2003). This is also suggested by the observed enhanced protective immunity conferred by live B. anthracis vaccines (Cohen et al., 2000) or by supplementing PA-based vaccines with for- malin-inactivated spores (Brossier et al., 2002). Recent availability of genome sequences of several members of the B. cereus group of organisms, which includes B. cereus, B. anthracis and B. thuringiensis, allows for application of global genome-based search strategies as well as for delineation of B. anthracis mech- anism of pathogenesis. These include computational (Ariel et al., 2002; 2003; Read et al., 2003) and proteomic (Chitlaru et al., 2004) analyses, complemented by high throughput expression screens (Bourgogne et al., 2003; Gat et al., 2005), targeted at identification of specific vir- ulence-related genes and potential vaccine candidates. In a recent computational screen of the B. anthracis Ames chromosome sequence, we have identified several classes of novel putative virulence factors, including SLH proteins, proteins containing repeat motifs, lipoproteins,

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Molecular Microbiology (2005)

58

(2), 533–551 doi:10.1111/j.1365-2958.2005.04848.xFirst published online 13 September 2005

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2005

? 2005

58

2533551

Original Article

Role of MntA in anthrax pathogenesisO. Gat

et al.

Accepted 8 August, 2005. *For correspondence. [email protected]; Tel. (

+

972) 8 9381595; Fax (972) 8 9401404.

The solute-binding component of a putative Mn(II) ABC transporter (MntA) is a novel

Bacillus anthracis

virulence determinant

Orit Gat,

1

Itai Mendelson,

1

Theodor Chitlaru,

1

Naomi Ariel,

1

Zeev Altboum,

2

Haim Levy,

2

Shay Weiss,

2

Haim Grosfeld,

1

Sara Cohen

1

and Avigdor Shafferman

1

*

Departments of

1

Biochemistry and Molecular Genetics and

2

Infectious Diseases, Israel Institute for Biological Research, PO Box 19, Ness Ziona, 74100, Israel.

Summary

Here we describe the characterization of a lipoproteinpreviously proposed as a potential

Bacillus anthracis

virulence determinant and vaccine candidate. Thisprotein, designated MntA, is the solute-binding com-ponent of a manganese ion ATP-binding cassettetransporter. Coupled proteomic-serological screen ofa fully virulent wild-type

B. anthracis

Vollum strain,confirmed that MntA is expressed both

in vitro

andduring infection. Expression of MntA is shown to beindependent of the virulence plasmids pXO1 andpXO2. An

mnt

A deletion, generated by allelic replace-ment, results in complete loss of MntA expressionand its phenotypic analysis revealed: (i) impairedgrowth in rich media, alleviated by manganese sup-plementation; (ii) increased sensitivity to oxidativestress; and (iii) delayed release from cultured mac-rophages. The

ΔΔΔΔ

mnt

A mutant expresses the anthrax-associated classical virulence factors, lethal toxinand capsule,

in vitro

as well as

in vivo

, and yet themutation resulted in severe attenuation; a 10

4

-folddrop in LD

50

in a guinea pig model. MntA expressed

in trans

allowed to restore, almost completely, thevirulence of the

ΔΔΔΔ

mnt

A

B. anthracis

strain. We pro-pose that MntA is a novel

B. anthracis

virulence deter-minant essential for the development of anthraxdisease, and that

B. anthracis

ΔΔΔΔ

mnt

A strains have thepotential to serve as platform for future live attenu-ated vaccines.

Introduction

Bacillus anthracis

is an endospore-forming toxin-produc-ing bacterium and the causative agent of anthrax, a seri-

ous and often fatal disease of livestock and humans.Although primarily a disease of herbivores, anthrax canaffect humans via contact with infected animals or upondirect exposure.

B. anthracis

is also considered a biolog-ical terror agent (CDC, 2005), because of its capability tocause lethal inhalational anthrax and the environmentalpersistence of its spores.

During inhalation, dormant

B. anthracis

spores areengulfed by resident alveolar macrophages (Guidi-Ron-tani

et al

., 1999; 2001; Dixon

et al

., 2000). En route to theregional lymph nodes, the ingested spores germinate andprobably multiply within the phagosome while producingvirulence factors. Subsequently, the vegetative cells lysethe macrophages and escape into the blood stream,resulting in bacteraemia, sepsis and ensuing death.

Bacillus anthracis

virulence has been predominantlyattributed to factors carried by two native plasmids; thetripartite toxin: protective antigen (PA), lethal factor (LF)and oedema factor (EF) (encoded by

pag

A,

lef

and

cya

genes, located on pXO1) and the antiphagocytic poly-

γ

-

D

-glutamic acid capsule (synthesis and degradation of whichis encoded by

cap

BCA and

dep

genes, located on pXO2).However, a battery of as yet undefined virulence factorsprobably reside on the

B. anthracis

chromosome (Cohen

et al

., 2000; Baillie and Read, 2001; Brossier

et al

., 2002;Ariel

et al

., 2003; Bourgogne

et al

., 2003). This is alsosuggested by the observed enhanced protective immunityconferred by live

B. anthracis

vaccines (Cohen

et al

.,2000) or by supplementing PA-based vaccines with for-malin-inactivated spores (Brossier

et al

., 2002).Recent availability of genome sequences of several

members of the

B. cereus

group of organisms, whichincludes

B. cereus

,

B. anthracis

and

B. thuringiensis

,allows for application of global genome-based searchstrategies as well as for delineation of

B. anthracis

mech-anism of pathogenesis. These include computational(Ariel

et al

., 2002; 2003; Read

et al

., 2003) and proteomic(Chitlaru

et al

., 2004) analyses, complemented by highthroughput expression screens (Bourgogne

et al

., 2003;Gat

et al

., 2005), targeted at identification of specific vir-ulence-related genes and potential vaccine candidates.

In a recent computational screen of the

B. anthracis

Ames chromosome sequence, we have identified severalclasses of novel putative virulence factors, including SLHproteins, proteins containing repeat motifs, lipoproteins,

534

O. Gat

et al.

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works,

Molecular Microbiology

,

58

, 533–551

adhesins and ATP-binding cassette (ABC) transporters(Ariel

et al

., 2003). The ABC transporters superfamily isubiquitous among living organisms and comprises one ofthe largest functionally diverse protein families catalysingvectorial uptake or efflux of specific molecules using ATPhydrolysis to energize the process (Schmitt and Tampe,2002; Garmory and Titball, 2004). In addition to nutrientand metal ion uptake, ABC transporters are central tomany physiological processes, such as secretion ofsignalling molecules, antibiotics and toxins, multidrug-resistance, translational regulation, competence and cellattachment.

Genetic and computational screens for virulence deter-minants of bacterial pathogens, coupled with multipleinfection models studies suggest that ABC cation trans-porters, specific for zinc, iron or manganese, may playeither a direct or an indirect role in microbial virulence andhuman disease (Brown and Holden, 2002; Janulczyk

et al

., 2003; Paik

et al

., 2003; Garmory and Titball, 2004).ABC transporters are contained within operons of threeto four genes. These operons include genes for an ATP-binding protein, an integral membrane permease and asolute-binding receptor, which determines the specificity.The solute-binding component of manganese ion in theseABC transporter systems (named MntA for ‘Mn transport’)belongs to a family of proteins termed LraI (lipoproteinreceptor antigen), originally recognized in oral

strepto-cocci

(Jenkinson, 1994), and have since been discoveredin other genera. The family was thus extended andrenamed cluster IX (Claverys, 2001), which was furthersubdivided into two subclusters with specificity for eitherzinc (

adc

-like) or manganese ions (

psa

-like) (Dintilhac

et al

., 1997). In addition to ABC transporters, a secondtype of import system, which belongs to a family of diva-lent metal transition ion transporters, termed Nramp (nat-ural resistance-associated macrophage protein, MntH), isknown to transport Mn

2

+

in bacteria (Cellier

et al

., 2001;Forbes and Gros, 2001).

The

B. anthracis

genome harbours several putativemetal cation ABC transporter systems (Ariel

et al

., 2003;Read

et al

., 2003), including a chromosomal orthologueof the

Streptococcus pneumoniae

LraI protein, PsaA(Accession No. NP_845499, Ames strain). In the currentstudy we describe results of a genetic and biochemicalanalysis of this

B. anthracis

PsaA-orthologue, namedherein MntA, which was proposed as a potential virulencedeterminant and vaccine candidate (Ariel

et al

., 2003).Phenotypic analysis of a

B. anthracis

virulent strain lack-ing MntA revealed impaired growth

in vitro

and in mac-rophage cultures. Moreover, although the mutant strainexhibited unimpaired expression of the classical

B. anthracis

virulence factors (toxins and capsule), it wassignificantly attenuated for virulence. The

B. anthracis

MntA identified in this study thus constitutes a major

B. anthracis

virulence determinant, other than the tripar-tite toxin and the antiphagocytic capsule.

Results

Bacillus anthracis

MntA is a member of the LraI/cluster IX family of ABC transporters

A bioinformatic search of the

B. anthracis

Ames straingenome for new virulence determinants and vaccine can-didates (Ariel

et al

., 2003) resulted in identification ofan open reading frame (ORF) encoding a putative311-amino-acid lipoprotein, an orthologue of the

S. pneumoniae

surface antigen PsaA. The ORF-productexhibits 40–50% overall sequence identity with the sol-ute-binding components of the multiple-metal ion ABCtransporter superfamily LraI/cluster IX. The

B. anthracis

ORF-product harbours all common general features listedfor a prototypical LraI/cluster IX solute-binding proteincomponent (Claverys, 2001; Elsner

et al

., 2002). Basedon the analyses described below, we designate this

B. anthracis

PsaA-orthologue, MntA.The

B. anthracis

MntA (ORF GBAA3189, Fig. 1A) islocated within a terminator bound operon and is precededby two ORFs. The first ORF of this operon (GBAA3191)encodes a putative 249-residue ATP-binding protein witha typical consensus nucleotide-binding site and domain-linker motif. The second ORF (GBAA3190) encodes a288-residue putative transmembrane protein with a signa-ture sequence characteristic of hydrophobic membraneproteins. In the parallel

S. pneumoniae

operon, a thiolperoxireductase encoding gene (

psa

D) is located down-stream of the

psa

BCA locus, whereas the

B. anthracismnt

operon is followed by an

α

/

β

hydrolase gene(GBAA3187, Fig. 1A). Upstream of the

B. anthracis mnt

operon, a protein belonging to the ThiJ/PfpI family(GBAA3192), harbouring a DNA binding motif, isencoded, and thus could potentially act as a transcriptionregulator.

The MntA sequence displays the conserved metalbinding site identified in the X-ray structure of the

S. pneumoniae

orthologue PsaA, the best characterizedmember of this transporter group, namely: conservedHis

67

(H69, MntA), His

139

(H141, MntA), Glu

205

(E207,MntA) and Asp

280

(D282, MntA) residues (Fig. 1B,Lawrence

et al

., 1998). The MntA sequence also har-bours: the PsaA domain (CDD, NCBI); multiple copies ofa PsaA-like derived protein adhesin motif (Interpro entryIPR006128, Fig. 1B); the characteristic fingerprint signa-ture sequence of LraI/cluster IX manganese (

psa

-like)or zinc (

adc

-like) ABC transporters (Fig. 1C). Thus,

B. anthracis

MntA may be classified as a

bona fide

mem-ber of the LraI/cluster IX metal ion ABC transporters sol-ute-binding component.

Role of MntA in anthrax pathogenesis

535

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works,

Molecular Microbiology

,

58

, 533–551

Fig. 1.

Bacillus anthracis

MntA belongs to LraI/Cluster IX family of ABC transporters.A. Schematic depiction of the

mnt

A operon (black block arrows) and adjacent genes (white block arrows). The genes, marked according to GBAA numbers (locus tag), code for the following products: 3187,

α

/β hydrolase; 3189, the PsaA-like lipoprotein designated here MntA; 3190, permease; 3191, ATPase; 3192, ThiJ/PfpI family protein. Terminators are marked as stem-loops and the chromosomal location (B. anthracis Ames strain) is indicated above the line.B. Domain and motif architecture of the MntA protein. A bold line represents the signal peptide, harbouring a lipobox sequence LXXC. Boxes (located between residues: 1, 34–52; 2, 65–78; 3, 79–95; 4, 196–217; 5, 249–267; 6, 275–294) mark adhesin lipoprotein motifs IPR 006128 (according to InterPro, EBI). Residues 18–309 comprise the PsaA domain (CDD, NCBI). The putative metal binding residues according to the Streptococcus PsaA are marked under the line.C. Conservation of the Cluster IX signature motif (Claverys, 2001) in MntA of B. anthracis [versus sequence-related proteins, identified by BLAST (NCBI) searches followed by multiple alignment of top hits using T-Coffee]. Location of the conserved putative metal-binding residue His69 is marked by an arrow. In the multiple alignment, residues conserved throughout are marked by black background. A grey background denotes similar residues. The gi accession numbers corresponding to the listed species are: 47528481 (B. anthracis, Ames ancestor); 52005059 (B. lichiniformis, ATCC 14580); 10173129 (B. halodurans C-125); 15613079 (B. halodurans C-125); 56965719 (B. clausii KSM-K16); 16080129 (B. subtilis 168); 8925939 (S. mutans); 29375189 (E. faecalis V583); 7446934 (S. gordonii); 7920458 (S. pneumoniae NA-1064/97); 7579029 (S. mitis); 7579031 (S. oralis); 50913738 (S. pyogenes MGAS10394); 32718581 (S. agalactiae); 44004523 (B. cereus ATCC 10987); 42781169 (B. cereus ATCC 10987); 47566802 (B. cereus G9241); 52143391(B. cereus ZK); 49481239 (B. thuringiensis kokukian 97-27); 30020158 (B. cereus ATCC 14579). Note that all B. cereus lack the Cluster IX psa-like motif although these proteins are all orthologues of B. anthracis MntA.

GXDPHEYEPXPXDVKKIXXADLIVYNGLXLEsignature

MntA

B. lichiniformisB. haloduransB. haloduransB. clausiiB. subtilisS. mutansE. faecalisS. gordoniiS. pneumoniaeS. mitisS. oralisS. pyogenesS. agalactiaeB. cereus 10987B. cereus 10987B. cereus G9241B. cereus ZKB. thuringiensisB. cereus 14579

A

B

C

2940k2937k

mntA3187 3189 3190 3191 3192 3193

L18TACH69 H141 E207 D282

PsaA domain (CDD)

Putative metal-binding site

1 2 3 4 5 6

69

536 O. Gat et al.

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works, Molecular Microbiology, 58, 533–551

The closest known protein exhibiting sequence homol-ogy to B. anthracis MntA (58% identity), which is alsopart of an ABC transporter operon, is MntA fromB. lichiniformis ATCC 14580, harbouring all the conservedresidues and motifs described above. Another MntAorthologue appears to be the surface adhesin ofB. halodurans C-125, which exhibits 40% sequence iden-tity and most of the prototypical signature sequence, yetit is not part of an ABC transporter system. The B. subtilisMntA is only 30% identical and lacks most of the LraIsignature sequence, as well as the part of the conservedmetal binding site (Fig. 1C). Most surprisingly, allsequenced genomes of other members of the phylogenet-ically related B. cereus family of microorganisms, namelyB. thuringiensis serovar kokukian, B. cereus 14579,B. cereus 10987 and the most closely related toxin-pro-ducing human pathogen B. cereus G9241 (Rasko et al.,2005), exhibit overall extensive sequence similarity toB. anthracis MntA; however, they seem to lack the psa-like signature but rather harbour an adc-like solute-bindingcomponent (Fig. 1C).

Expression of MntA, in vitro and during infection, is independent of the presence of the virulence plasmids pXO1 and pXO2

In order to determine the pattern of expression of the MntAprotein as well as of other ABC transporters in B. anthracis,a proteomic survey, which entailed protein separation bytwo-dimension electrophoresis (2-DE) gels and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identification of membranal andsecreted proteins, was carried out. This was part of a largerstudy that involved inspection of the protein signature ofeither wild-type B. anthracis (strain Vollum, harbouringboth pXO1 and pXO2 virulence plasmids) or non-virulentbacteria (ΔVollum, cured of the virulence plasmids), cul-tured in a variety of growth conditions (Chitlaru et al., 2004and T. Chitlaru and A. Shafferman, unpubl. data). Usingthis approach, protein spots corresponding to MntA wererecognized in the proteome of B. anthracis grown in richmedium (brain–heart infusion, BHI), during the logarithmicas well as the stationary growth phases. This observationis exemplified in the 2-DE separations of membrane-asso-ciated proteins harvested from BHI-grown cells at thebeginning of the stationary phase (see Fig. 2A; proteinspots representing MntA are boxed in all gels). The identityof MntA was unequivocally established by the character-istic fingerprint of the trypsin-digested 2-DE-separatedprotein spots, obtained by MALDI-TOF analysis (boxedspots in Fig. 2). Three MntA-identified protein spots arereproducibly detected on the 2-DE gels suggesting thatMntA exists in three forms differing in their isoelectric point.Occurrence of multiple spots of the same protein is a

common phenomenon on 2-DE gels, and has been dis-cussed in a previous report pertaining to B. anthracis pro-teomic analysis (Chitlaru et al., 2004). The identity of theseprotein spots was also confirmed by their distinct recog-nition on 2-DE-derived Western blots (Fig. 2B) by specificanti-MntA antibodies [prepared in mice by DNA-mediatedimmunization employing the MntA gene coding sequencecloned in a eukaryotic expression vector (see Experimentalprocedures)].

Notably, the MntA protein could be detected both incultures of the fully virulent Vollum strain of B. anthracisand in ΔVollum cultures (Fig. 2A), at comparable levels.This indicates that the in vitro expression of the MntAprotein is not dependent upon the presence of pathoge-nicity-related or regulatory factors encoded by thevirulence plasmids pXO1 or pXO2, unlike otherchromosomally encoded B. anthracis potential virulence-associated proteins (Bourgogne et al., 2003).

To further examine MntA expression pattern, bacterialpellets were collected at different time points during growthof a ΔVollum culture, and the MntA protein presence wasprobed on SDS-PAGE blots using the anti-MntA specificantibodies. While the protein was detected in the cell-associated fraction throughout the exponential phase ofgrowth, it reached a higher level in early stationary phase(Fig. 2C) and started to accumulate also in the culturemedium during the stationary phase (data not shown).

In order to determine whether the MntA protein isexpressed in vivo, the 2-DE-derived Western blots wereprobed with antisera collected from guinea pigs infectedwith virulent B. anthracis spores (according to the protocoldetailed in Experimental procedures). The observationthat MntA protein reacts with this anti-B. anthracis antise-rum (Fig. 2D), indicates not only that MntA is antigenic butalso, more importantly, that it is expressed by the bacteriain vivo during infection. Furthermore, the MntA proteinspots exhibited seroreactivity when probed with serumobtained from animals infected with spores of an avirulentB. anthracis strain, cured of the virulence plasmids (datanot shown). Thus, MntA expression in vitro, as well as invivo, is not dependent upon the presence of factorsencoded by the pXO1 or pXO2 plasmids.

Generation of the ΔmntA mutant

Proteins belonging to the LraI/cluster IX family, to whichMntA exhibits extensive homology, were reported to beinvolved in the virulence of several pathogenic bacteria.Mutations of the LraI-homologue genes caused alteredpathogenesis and reduced virulence of the correspondingpathogens (Paik et al., 2003). Interestingly, the LraI-homo-logue of S. pneumoniae, PsaA, elicits a protective antip-neumococcal immune response and represents acandidate for vaccine development (Areas et al., 2004;Miyaji et al., 2002).

Role of MntA in anthrax pathogenesis 537

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works, Molecular Microbiology, 58, 533–551

Fig. 2. Identification of MntA by a proteomic-serological analysis.A. Membranal fractions were prepared from cultures of the virulent B. anthracis Vollum strain and the pXO1 and pXO2 cured (ΔVollum) strain (grown on BHI), separated by 2-DE and detected by Coomassie blue staining. The three isoforms/spots representing the MntA protein are boxed in all panels.B. Identification of MntA by MALDI-TOF analysis of protein tryptic digests, and Western blot analysis of the ΔVollum 2-DE separated proteins using mouse anti-MntA antibodies generated by mntA-DNA immunization. The MntA derived peptides recognized in the spectra are indicated by their amino acid sequence superimposed on the respective MALDI-TOF peaks, and are underlined in the MntA sequence.C. In vitro expression of MntA. A B. anthracis ΔVollum culture (BHI) was sampled at the indicated times and the cell pellets were separated from the growth media and resuspended in Tris buffer (50 mM, pH 6.8) to equivalent turbidity. Equal volumes were boiled for 5 min in SDS-PAGE sample buffer, and then separated by PAGE. Western blot was probed with the specific mouse anti-MntA antibodies. The corresponding growth curve is depicted in the right.D. Western blot analysis of the ΔVollum 2-DE separated proteins, probed with sera collected from guinea pigs infected with the virulent Vollum strain (see Experimental procedures).

94A pH

MW

Vollum (wt)(pXO1+ pXO2+)

DVollum(pXO1- pXO2-)

25

32.5

47.5

62

83

LHDETVNR

FLISSEGAFK

RFLISSEGAFK

VPALFVETSVDR

VPALFVETSVDRR

TGYIWEINSENQGTPDQIR

VEIHSLVPIGANPHEYDPLPK

B

MKFKNVVLSILCIFVFALTACSSNTNGKEEGSGKLKVVTTYSIIYDMVKQIGGEKVEIHSLVPIGANPHEYDPLPKDVMKMTDADMVLYNGLNLEEGGAWFKKLLKTANKSEKDAPVYKVSEGVEAIYLETKGLEKEPDPHAWMNIKNGILYAENVKKALIKEDPKNKEFYTKNADNYVAELQKLHDETVNRIHQIPEEKRFLISSEGAFKYFGKAYDIKTGYIWEINSENQGTPDQIRDVVSVIQTNKVPALFVETSVDRRSMETVSKETNVPIAGTIFTDSLGKSGEDGDTYLKMMKW NIDTIINGLQK

Anti-MntA Serum

D

3 4 5 6 97 10 11 12

Time (hour)

Hyperimmune Serum

C

0.1

1

10

0 2 4 6 8 10 12

Time (hour)

Tur

bid

ity

(OD

)

94

538 O. Gat et al.

© 2005 Blackwell Publishing LtdNo claim to original Israeli government works, Molecular Microbiology, 58, 533–551

In order to study the role of the MntA protein inB. anthracis pathogenesis, a specific deletion of the mntAgene was introduced by allelic replacement into its corre-sponding gene in the fully virulent B. anthracis Vollumwild-type (wt) strain. The ΔmntA mutant was generated byinsertion of a kanamycin resistance gene replacing thewild-type allele by homologous recombination (Fig. 3Aand B), resulting in a complete loss of the MntA proteinproduct expression, as verified by using the specific anti-MntA antibodies (Fig. 3C). The ΔmntA mutant exhibitedtypical colony morphology when grown on LB agar plates;however, the colonies were slightly smaller compared withthose of the parental wt strain (not shown). To furtheranalyse the phenotype associated with the MntA disrup-tion, the ΔmntA mutated strain was compared with the wtstrain in: (i) growth characteristics; (ii) metal ions require-ments for growth; (iii) resistance to oxidative stress; (iv)intracellular germination and growth in a cultured mac-rophage cells model; (v) ability to express the anthrax-associated native virulence factors; and (vi) virulence inan animal model, as described below.

The ΔmntA mutant exhibits an impaired growth in vitro, restored by addition of Mn2+ and Fe2+ ions

Based on the sequence and motif similarity of theB. anthracis MntA to the Streptococcal manganese trans-porter PsaA, we tested whether the in vitro growth of theΔmntA mutant is affected and dependent on Mn2+ supple-mentation. Culturing the wt and the ΔmntA mutant strainsin rich BHI broth under aerobic conditions revealed adiscrete growth difference between the two strains, sug-gesting a defect in the mutant growth (Fig. 4A). The cal-culated doubling time of the mutant is 49 min (growth rateof 0.87 h−1), compared with 30 min (1.4 h−1) for the wt.Towards the stationary phase, the mutant reaches theplateau level of the wt strain (after 12 h of growth, datanot shown). Supplementation of Mn2+ complemented thegrowth defect of the mutant strain, while growth of the wtstrain was unaffected by addition of 10 μM MnSO4. Theobservation that the mutant strain growth rate was restoredto levels comparable to that of the parental strain (Fig. 4A),indicates that exogenous Mn2+ is necessary and sufficientto restore optimal growth of the mutant in this medium.

The dependence of ΔmntA growth on Mn2+ and addi-tional metal cations was studied in Dulbecco’s modifiedEagle medium (DMEM) culture media containing 10%fetal calf serum. In this serum-based medium, growthdefects resulting from metal ion deficiency are expectedto be more pronounced because of the chelating effect ofserum proteins (Krachler et al., 1999), and indeed theΔmntA mutant strain was unable to be propagated in thismedium. In a series of such studies it was found thatgrowth could be supported by supplementing the medium

with Mn2+ and Fe2+, but not Zn2+, Mg2+, Ca2+ or Cu2+ (notshown). The impact of manganese on the ΔmntA mutantgrowth was further investigated by decreasing the con-centration (1.0–0.1 μM) of supplementary Mn2+ (Fig. 4B).In these experiments, partial reconstitution of growth was

Fig. 3. Construction of the ΔmntA mutant.A. Schematic representation of the pEO-mntA plasmid used for mntA gene disruption. Upon homologous recombination, a KmR gene is inserted into the mntA coding region, producing a truncation between nucleotides 299–579.B. PCR products amplified from chromosomal DNA preparations with primers that span the mntA gene, confirming insertion of the KmR gene in the ΔmntA mutant (see Table 1 for the location of the primers used).C. Coomassie blue stained SDS-PAGE gel and corresponding West-ern blot, probed with the specific mouse anti-MntA serum, of wt and ΔmntA mutant total bacterial pellets collected from cultures grown overnight in BHI.

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observed with as little as 0.1 μM MnSO4. Full restorationwas detected with 0.5 μM MnSO4. For the Fe2+, muchhigher concentrations were needed.

Thus, the B. anthracis MntA appears to be important forgrowth under aerobic conditions, and the growth defect ofthe mntA-deficient mutant strain may be corrected by addi-tion of manganese or, to a lesser extent, by ferrous ions.

The ΔmntA mutant exhibits increased susceptibility to oxidative stress

Manganese has been shown to be essential in protect-

ing cells from the deleterious effects associated with oxi-dative stress, preventing the propagation of free radicalchain reactions, by either (i) serving as a cofactor forsuperoxide dismutase (SOD); (ii) quenching; or (iii) com-peting for metal binding sites on proteins with otherreactive metal ions, Fe2+ and Cu2+ (Herbig and Helmann,2001; Tseng et al., 2002). The observed reduced growthrate of the ΔmntA mutant in an aerobic environmentmay suggest that this strain has increased sensitivity tooxidative stress, which is an inevitable consequence ofaerobic growth. Under aerobic conditions, reactive oxy-gen species (ROS) are generated and their accumula-

Fig. 4. Dependence of the growth of B. anthracis ΔmntA mutant on Mn2+ and Fe2+ ions.A. Growth of wt and ΔmntA mutant strains in BHI medium, at 37°C with vigorous agitation. Absence or presence of Mn2+ (10 μM MnSO4) is denoted by open and filled diamonds (wt) and squares (ΔmntA). The error bars show standard deviations.B. Growth of the ΔmntA mutant in modified DMEM (containing 10% fetal calf serum), with different concentration of added MnSO4 or FeSO4, performed and monitored in a 96-well microtitre plate incubated at 37°C in a plate reader.

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tion has the potential of causing damage to nucleicacids, proteins and lipids.

Susceptibility to oxidative stress of the ΔmntA mutantwas compared with that of the wt strain, in the presenceof hydrogen peroxide or methyl viologen (paraquat). Bothtreatments can generate a state of oxidative stress (Tsenget al., 2002; Johnston et al., 2004; McAllister et al., 2004).While H2O2 acts externally, paraquat is a redox compoundthat is reduced by low-potential electron donors inside thecells, leading to generation of superoxide radicals in thecytoplasm.

Mid-exponential growth phase bacterial cells wereexposed to increased concentrations (0.1–25 mM) ofhydrogen peroxide for 30 min, and their survival wasdetermined by viable counts (Fig. 5A). This experimentclearly demonstrates that the ΔmntA mutant cells are sig-nificantly more susceptible to peroxide. Unlike the wt, themutant did not survive at concentrations equal to or higherthan 2.5 mM H2O2. The enhanced susceptibility of themutant strain was apparent also when oxidative stresswas induced by treatment with paraquat. Cultures were

grown in the presence of 75 mM paraquat for 2 h(Fig. 5B). While the wt strain was moderately affected bythe presence of paraquat, the growth of the ΔmntA mutantwas strongly inhibited (Fig. 5B). Furthermore, the treat-ment had a definite impact upon colony morphology.While the wt morphology was essentially unaffected bythe presence of paraquat, the colonies formed by theparaquat-treated mutant cells were significantly smaller,transparent and exhibited a heterogeneous irregularshape (data not shown).

Growth of ΔmntA mutant bacteria in macrophage culture

The initial stages of the anthrax infection are believedto occur within phagocytic cells (Guidi-Rontani et al.,1999; 2001; Dixon et al., 2000). Following inhalation,B. anthracis endospores undergo engulfment by alveolarmacrophages, which then migrate to the regional lymphnodes. The spores germinate inside the phagocytes, andthere, the vegetative bacilli presumably start replicationand toxin production.

We have followed the fate of wt and ΔmntA spores incultured RAW264.7 macrophages (Fig. 6). The outline ofthe experimental design was based on addition of 5 × 105

spores per ml to 5 × 105 cultured cells per ml, followed by1 h incubation (t = −2.5), extensive washing, 2.5 h incuba-tion in the presence of gentamycin and extensive washing(t = 0). Both wt and ΔmntA spores were equally engulfedby the macrophages, as 20–25% of either strain wasdetected by monitoring total counts at t = 0 versus t = −2.5. However, following a heat-shock treatment of the cul-ture at t = 0, it was found that 8.4% of the wt bacteriaremained in the form of spores, while 48.6% of the ΔmntAmutant bacteria were still in the form of spores. This maybe because of a germination defect of the ΔmntA mutant.Indeed, in separate experiments following in vitro growthinduction of endospores by BHI (Fig. 7) or alanine (notshown), a germination defect was again detected for theΔmntA mutant. This defect was manifested by a delay inconversion to heat sensitivity (Fig. 7), as well as by aprolonged period of outgrowth as vegetative cells in BHI,detected by the increase in the turbidity of the sporesuspension (1 h for the mutant as compared with 30 minfor the wt).

Propagation of the engulfed bacteria and appearanceof extracellular vegetative bacilli were further monitored atdifferent time points from t = 0. Up to 4 h, the total bacte-rial counts were similar for both strains. From 8 h (t = 8),a marked difference in extracellular bacterial counts andthe concomitant lysis of the infected macrophages wasobserved (Fig. 6A–C). While for the wt culture completemacrophage lysis occurred at t = 10 (Fig. 6A and B), themutant showed a similar effect only after 20 h. This delayin growth and macrophage lysis, observed for the mutant

Fig. 5. Effect of the ΔmntA mutation on oxidative stress response.A. wt (grey bars) and ΔmntA mutant (black bars) vegetative cultures were incubated with indicated concentrations of H2O2 in BHI medium for 30 min and the number of viable bacteria was determined by plating.B. wt (grey diamond) and ΔmntA mutant (black square) vegetative cultures, in the presence (filled shapes) or absence (open shapes) of 75 mM paraquat.

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strain, could be relieved by addition of 5 μM MnSO4 to theRAW264.7 culture infected with the ΔmntA mutant sporesor by mntA complementation, as described in the nextsection (Fig. 6A–C).

MntA is a critical virulence determinant in vivo

Guinea pigs represent a more appropriate animal modelto study B. anthracis infection in a host system, as com-pared with the widely used murine model. While mice

develop severe septicaemia following exposure to thecapsulated bacteria even in the absence of the plasmid-encoded toxins, in larger animals such as guinea pigs,rabbits and monkeys, as well as humans, developmentof the anthrax disease is dependent upon toxin produc-tion in addition to the capsule (Welkos and Friedlander,1988; Welkos and Marrero, 1996; Fellows et al., 2001;Altboum et al., 2002; Brossier et al., 2002). In order toexamine whether the mntA mutation affects B. anthracisvirulence, guinea pigs were initially infected with doses

Fig. 6. Behaviour of the ΔmntA mutant in macrophage culture. Cells of the RAW264.7 macrophage line were infected either with wt (white bars) or ΔmntA mutant spores, without (light grey bars) or with (dark grey bars) addition of 5 μM MnSO4, or with ΔmntA/pMntA complemented strain spores (black bars). Samples were withdrawn at the indicated times, and evaluated by visualization of the fixed cell cultures by light microscopy (A), macrophage (MΦ) lysis measured by LDH release to the culture growth medium (B) and amount of free bacteria released from the cells as measured by viable plate counting (C). Note the appearance of bacilli at t = 10 h in wt-infected macrophages and the concomitant macrophage lysis, which is not observed in the ΔmntA mutant infected culture at this time point. This delay of the mutant macrophage-dependent growth was overcome by addition of Mn2+ or by trans-complementation of mntA.

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of 10–104 spores per animal, of either wt or mutantspores. All guinea pigs infected with more than 2 × 102

spores of the parental wt strain died, while all animalsinoculated with 104 spores of the mutant strain, survived(Fig. 8C). Only at doses equal to or higher than 105, wasdeath of guinea pigs infected with the ΔmntA mutant

spores observed. The estimated lethal dose 50 (LD50) is102 spores for the wt and 8 × 105 spores for the mutantstrain. These results are consistent with the requirementof MntA for full virulence of B. anthracis. It should benoted that at such high infective spore dose, even thevaccine Sterne strain, entirely lacking the virulence plas-mid pXO2, is lethal to guinea pigs (Ivins et al., 1990;Cohen et al., 2000).

To confirm that the phenotype associated with theΔmntA mutation is indeed attributed to the absence of theMntA protein, rather than an indirect effect (e.g. attributedto polar mutation), we introduced a plasmid expressingthe MntA protein in trans, into the ΔmntA mutant. UsingWestern blot analysis, we show that the ΔmntA-comple-mented strain, ΔmntA/pMntA, indeed expresses MntA atlevels comparable to that of the wt (Fig. 8A). Moreover, incontrast to ΔmntA, the ΔmntA/pMntA complementedstrain grows in BHI medium without added manganeseand in macrophages, similarly to the wt (Figs 8B and 6A–C). Most notably, the mntA trans-complementationresulted in a restored virulence (LD50 of 4 × 102 spores ascompared with 8 × 105 in the ΔmntA mutant, Fig. 8C),clearly confirming that the significant decrease in the vir-ulence of the MntA-deficient strain is directly associatedwith the lack of MntA activity.

Fig. 7. In vitro germination deficiency of the ΔmntA mutant. Loss of heat resistance of germinated spores of the wt (grey diamond) and the ΔmntA mutant (black squares) monitored in vitro using modified BHI broth as germinant following heat-shock treatment.

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Fig. 8. Comparison of B. anthracis wt, its ΔmntA mutant and ΔmntA/pMntA trans-comple-mented strain.A. Western blot analysis of total cell lysates of the wt, ΔmntA mutant and ΔmntA/pMntA trans-complemented strains, using the specific mouse anti-MntA antibodies. Equal amounts of cell pellets were loaded on the SDS-PAGE gel.B. Growth rates, calculated from the logarithmic phase of cultures grown in BHI medium (per-formed as in Fig. 4A), in the presence (filled bars) or absence (open bars) of 10 μM MnSO4.C. Virulence of the three strains, wt (diamonds), ΔmntA (squares) and ΔmntA/pMntA (triangles), in the guinea pig model. Animals were injected subcutaneously with 0.1 ml spore suspension at the indicated doses, and followed daily for survival. The dotted lines indicate the LD50 spore dose values.

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A decline in B. anthracis virulence, such as documentedhere for the ΔmntA mutant, has been reported so far onlyto be associated with the loss of one of the major virulencefactors, the lethal toxin (LT) or the capsule production(Cataldi et al., 2000; Drysdale et al., 2005). To rule out thepossibility that the decrease in the virulence of the ΔmntAmutant was attributed to any indirect effect on expressionof the anthrax-associated known virulence factors, wedetermined the expression of these virulence factors, bothin vitro and in vivo. The estimated levels of the LT proteins,PA and LF, secreted in vitro (Fig. 9A) and the poly-γ-D-glutamic acid capsule synthesis (Fig. 9B), were found tobe similar in the mutant and the wt strains, when grownunder the same conditions to equivalent cell densities.Furthermore, the toxin produced by both strains exhibitedcomparable LT-specific activity. This was demonstrated byquantification of the bacterial-secreted toxin componentsin the conditioned culture medium, assaying the cytotoxicactivity on J774A.1 macrophage cells (Fig. 9C). Further-

more, the cytotoxicity of both LT preparations was neutral-ized equally well by specific anti-PA antibodies (Fig. 9C).

To determine the ability of the ΔmntA mutant strain toexpress the LT in vivo during its development in the host,an indirect approach, based on measuring specific anti-toxin antibodies, was undertaken. To this end, animalswere infected with mutant spores at a dose of 106 cfu peranimal, equivalent to the LD50 (Fig. 8). Indeed, at thisdose, 10 out of the 24 animals survived (Fig. 10A). Nineout of these 10 surviving animals exhibited unusuallyhigh antibody titers against PA and LF (Fig. 10B), clearlyindicating that the mutant cells retain the ability toexpress the toxin involved in anthrax-associated patho-genesis. The antibody levels generated by the ΔmntAmutant strain are comparable to those obtained by a sim-ilar infection dose with the Sterne vaccine strain (Cohenet al., 2000).

On day 42 following injection of ΔmntA, the 10 survivinganimals were challenged with 60 LD50 of the virulent Vol-lum strain (Fig. 10A). All guinea pigs survived this chal-lenge and exhibited antibody titers 103−105 of either anti-PA or anti-LF (Fig. 10B). It therefore appears that theΔmntA strain could serve as a useful platform for a futurelive vaccine, provided that measures are taken to manip-ulate it further for safe use.

The altered virulence associated with the mntA muta-tion, documented here, represents the first instance of aB. anthracis strain, carrying both pXO1 and pXO2 viru-lence plasmids, which maintains full capability to expressthe anthrax-associated classical virulence genes, and yetexhibits a remarkable diminution in pathogenesis.

Discussion

The B. anthracis virulence factors currently known to beinvolved in the course of the anthrax disease are primarilyattributed to genes encoded by the native plasmids, pXO1and pXO2 (Bourgogne et al., 2003; Mock and Mignot,2003). pXO1 encodes for the toxin components PA, LFand EF, a germination operon and a central regulator,AtxA. pXO2 harbours genes associated with synthesisand disassembly of the antiphagocytic capsule and a sec-ond regulator, AcpA. The most studied B. anthracisstrains, Sterne and Pasteur, lack either pXO2 or pXO1,respectively, and are virulent only in rodent models.Recent studies, applying high-throughput searches ofgene expression patterns, have suggested that pXO1-and pXO2-encoded regulators control genes located onboth the plasmids and the chromosome, other than theknown virulence factors (Bourgogne et al., 2003), and thata cross-talk exists between the chromosome and the plas-mids (Mock and Mignot, 2003). However, the identity ofadditional genes, which may be involved in anthrax patho-genesis, is still unknown.

Fig. 9. Comparison of production levels of the virulence factors, lethal toxin and capsule, by the wt and ΔmntA mutant.A. Supernatant samples collected from cultures grown in NBY-Bicar-bonate broth of wt and ΔmntA mutant cells were fractionated by SDS-PAGE and analysed by Western blots probed with anti-PA and anti-LF antibodies.B. India ink staining of the poly-γ-D-glutamic acid capsule of wt and ΔmntA mutant cells grown in NBY-Bicarbonate broth.C. Cytotoxic activity determined from equal volumes of the bacterial supernatants collected from wt (diamonds) and ΔmntA mutant (squares) cells grown in NBY-Bicarbonate broth, by the J774A.1 mac-rophage lysis assay in the presence (open symbols) or absence (dark symbols) of anti-PA antibodies.

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An in silico search of the B. anthracis Ames straingenome for novel virulence factors identified a solute-binding lipoprotein belonging to the LraI/cluster IX familyof ion receptors (Ariel et al., 2003), designated hereinMntA. The B. anthracis mntA gene is located within athree-gene operon, encoding for an ABC transporter(Fig. 1). Extensive sequence, motif and domain similari-ties exist between the B. anthracis MntA and PsaA, thesolute-binding component of a manganese ABC trans-porter and a virulence factor of the human pathogenS. pneumoniae (Fig. 1). Expression analysis by pro-teomic studies demonstrated that the B. anthracis MntAprotein is expressed both in vitro and in vivo (Fig. 2).Unlike the toxin-encoding genes, MntA expression is notunder the regulatory control of pXO1 or pXO2 virulenceplasmids, as it is produced in strains cured of the plas-

mids (Fig. 2). Given the location of the mntA gene ofB. anthracis within a putative ABC transporter operonand its homology to manganese import genes, weinvestigated a ΔmntA mutant strain (Fig. 3) with respectto functions that are assumed to be affected by Mn2+

ions.

Reduced growth rate and dependence on Mn2+ and Fe2+ ions in vitro

The ΔmntA mutant has a growth defect in rich BHImedium, which can be overcome by addition of Mn2+ ions(Fig. 4). Ions other than Mn2+ or Fe2+ do not restore thegrowth limitation of the ΔmntA mutant. In the ΔmntA/pMntA trans-complemented strain, the stringent require-ment for Mn2+ is relieved (Fig. 8B). All these studies are

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Fig. 10. Consequences of infection of guinea pigs with a sublethal dose of the ΔmntA mutant spores.A. A group of 24 guinea pigs were injected subcutaneously with 106 ΔmntA spores (1 LD50) and monitored for mortality. On day 42, the 10 surviving animals were challenged with a lethal spore dose (60 LD50) of the virulent Vollum strain. All 10 animals survived the challenge, while in a parallel group of eight naïve animals (control group), all animals died within 3 days from the challenge (not shown).B. Anti-PA and anti-LF ELISA antibody titres in blood samples collected from surviving animals at day 28 post ΔmntA spores inoculation and at day 63 (21 days post the Vollum lethal chal-lenge).

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consistent with the conclusion that, in B. anthracis, MntAactivity is associated with Mn2+ transport.

Among the Bacillus spp., the B. subtilis manganeseimport systems have been extensively studied. Two activeMn2+ transport operons were identified: MntH, which is aproton-dependent Mn2+ importer (of the Nramp family),and the MntABCD, which is an ABC transporter (Que andHelmann, 2000). It was suggested that these transportersmay either function as high- and low-affinity manganeseion uptake systems, or alternatively, be differentiallyexpressed at a particular growth phase (Que and Hel-mann, 2000; Guedon et al., 2003). Unlike the B. anthracisΔmntA mutant, where growth is not severely impairedand can be restored by addition of as little as 0.1 μMMnSO4 (Fig. 4), the metal requirements for growth ofS. pneumoniae psaA mutant are much more stringent(Dintilhac et al., 1997; Marra et al., 2002a,b). Similarly,growth of mtsA mutant of S. pyogenes was restored bymanganese supplementation only at much higher concen-trations (Janulczyk et al., 2003). This observation can berationalized by the presence of a compensating mntHgene-product in B. anthracis (Read et al., 2003), docu-mented as absent from these Streptococcus spp. (Janul-czyk et al., 2003; McAllister et al., 2004).

As mentioned, ferrous ions can also alleviate theB. anthracis ΔmntA mutant growth deficiency, but at ahigher concentration range (Fig. 4). A dual specificity inmetal ion ABC transporters has been suggested in otherstudies. For example, investigation of metal ion transport-deficient mutants in S. gordonii (scaCBA), S. pneumoniae(psaBCA), S. mutans (sloABC) and S. parasanguis (fim-CBA) (Dintilhac et al., 1997; Kolenbrander et al., 1998;Oetjen et al., 2002; Janulczyk et al., 2003; Paik et al.,2003) showed that both manganese and ferrous ions canbe bound and transported. These two ions are requiredby bacteria as enzyme cofactors and as structural com-ponents of proteins, but only manganese is known toparticipate in oxidative stress defence (Jakubovics andJenkinson, 2001). Thus, the growth deficiency of themutant may be a result of an impaired metabolism and/orreduced capacity to withstand oxidative stress generatedby metabolism.

Hypersensitivity to oxidative stress of the ΔmntA mutant

Oxidative stress results from the generation of internaldeleterious ROS, such as superoxide radicals, hydrogenperoxide and hydroxyl radicals. Furthermore, pathogensinvading host systems also have to face external ROS,which play a major role in the host defence against bac-terial infections (Raupach and Kaufmann, 2001).

The B. anthracis ΔmntA mutant is indeed highly sensi-tive to killing by superoxide compared with the parentalcells (Fig. 5), implying that MntA plays a key role in the

protection of the bacterial cell against oxidative killing, aspreviously reported for the MntA-orthologues ofS. pneumoniae, S. mutans and S. pyogenes (Tseng et al.,2002; Johnston et al., 2004; McAllister et al., 2004). Mn2+

protects cells from superoxide toxicity by at least twomechanisms: (i) functioning as an antioxidant, whichdetoxifies ROS; and (ii) as a cofactor for the activity ofMnSOD. It has been reported that the reduced Mn2+

uptake by the S. pneumoniae psaA mutant causes adecrease in MnSOD activity and thus renders the bacteriasensitive to killing by oxidative stress (Yesilkaya et al.,2000). Mn2+ uptake has also been linked to oxidative stressresistance in Salmonella typhimurium (Kehres et al.,2000). The involvement of SOD in oxidative stressresponse in B. anthracis is yet to be resolved. However,addressing this issue experimentally may be quite com-plicated because of the presence of four different SODparalogues in the B. anthracis genome (Read et al., 2003).

The altered phenotype of ΔmntA in macrophage culture

The interaction of B. anthracis spores with phagocyticcells during infection is a critical step in anthrax pathogen-esis (Moayeri and Leppla, 2004). The disease is initiatedby uptake of invading spores by the host macrophages.Germination of the spores and initiation of toxin expres-sion occur within the cells. Finally, the bacilli lyse themacrophages and multiply extracellularly.

In the RAW264.7 macrophage cell line model, a markeddelay in the ΔmntA mutant growth and concomitant mac-rophage lysis, were observed (Fig. 6). This defect wasrelieved either by supplementing Mn2+ at appropriate con-centration to the culture medium or by mntA trans-com-plementation (Fig. 6). This behaviour of the ΔmntA mutantcould be attributed to the limited defect in germination(Fig. 7), and/or to its reduced growth rate as a conse-quence of limited availability of manganese (Fig. 4), aswell as to its enhanced sensitivity to ROS (Fig. 5) formedby the phagocytic cells.

In this context, it may be relevant to refer to the work ofCendrowski et al. (2004), who documented the implicationof iron acquisition by siderophores on B. anthracis patho-genesis. Deletion of a predicted siderophore gene, absA,resulted in reduced virulence of a B. anthracis Sternestrain (pXO1+ pXO2–) in mice and complete inability togrow in RAW264.7 macrophages. Unlike this dominanteffect, the ΔmntA mutant (pXO1+ pXO2+) retains ability togerminate, replicate and lyse the phagocytic cell host.

Mutation in MntA results in loss of virulence with essentially unimpaired expression of classical anthrax virulence factors

Production of the poly-γ-D-glutamic acid capsule and the

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LT components (PA and LF) by the ΔmntA mutant strain,appears to be unaffected by the mutation (Fig. 9). Thehigh levels of anti-PA and anti-LF antibodies, induced dur-ing infection of guinea pigs with the ΔmntA mutant spores,suggest that the toxin is also produced in vivo (Fig. 10).All these studies indicate that the B. anthracis ΔmntA isnot impaired in production of the well-characterized clas-sical virulence factors and therefore it was quite remark-able to find out that disruption of mntA resulted in thepronounced attenuation of the bacteria (a drop of fourorders of magnitude in virulence, Fig. 8). The findings thatthe guinea pigs surviving the infection at a dose of 106

spores had generally high titers to both PA and LF(Fig. 10), and that all were protected from a lethal chal-lenge of 60 LD50 of the virulent Vollum strain, suggest thatΔmntA could be a platform for development of a future liveattenuated vaccine. However, some further manipulationsof the ΔmntA may be required before it could serve as asufficiently safe vaccine (Aloni-Grinstein et al., 2005; Men-delson et al., 2005).

The observation that virulence of the ΔmntA mutant canbe restored almost completely, both in vitro and in vivo,by trans-complementation with a vector expressing onlythe B. anthracis MntA polypeptide (Figs 6 and 8), clearlydemonstrates that the loss of virulence in the mntA dele-tion mutant is not attributed to some polar effect but rathera direct consequence of the loss of functional MntA. It isquite plausible that the combination of the observed phe-notypic defects in MntA could be responsible for its atten-uation in spite of its ability to express the toxin and thecapsule. It is interesting that MntA-orthologues were notidentified in any of the B. cereus genomes published todate, including the genomic sequence of the very closerelative B. cereus G9241, which harbours a copy of theB. anthracis pXO1 plasmid. This may imply that the MntA-dependent function has evolved late during divergence ofthe B. cereus group, providing B. anthracis with a selec-tive advantage in pathogenesis in comparison with its lessvirulent relatives.

Experimental procedures

Computational analyses

MntA was first identified by computational analysis of theB. anthracis Ames strain draft genome sequence, asdescribed previously (Ariel et al., 2003). In view of databaseupdates, the B. anthracis Ames Ancestor orthologue, gi47528481 (Read et al., 2003), was re-analysed against boththe non-redundant and the microbial genomes databases(NCBI), in search for novel orthologues, using BLAST andPSI-BLAST (Altschul et al., 1997). Domain analysis was car-ried out against CDD database (Marchler-Bauer et al.,2005) and motif analysis against Interpro database (Zdob-nov and Apweiler, 2001). Top BLAST hits were aligned usingthe web-based T-coffee multiple sequence aligning algo-

rithm (Poirot et al., 2003) and visualized using the BOX-SHADE viewer (http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html).

Bacterial strains, media and growth conditions

Bacterial strains used in this study are listed in Table 1.B. anthracis Vollum (pXO1+ pXO2+) wt and ΔVollum (pXO1–

pXO2–) strains were cultivated in BHI broth (Becton, Dickin-son and Company) at 37°C, with vigorous shaking, 250 r.p.m.(aerobic conditions). For induction of toxins and capsule pro-duction, NBY-Bicarbonate broth (Welkos and Marrero, 1996)was used. Sporulation was carried out using G broth, asdescribed (Kobiler et al., 2002). Germination was monitoredby incubating heat-activated (70°C, 20 min) spores in germi-nation buffer, 10 mM Na-phosphate, 100 mM NaCl, pH 7.8(Ireland and Hanna, 2002). After 10 min of pre-incubation,germination was initiated by the addition of BHI broth (to 5%)or L-alanine (to 100 mM). Spores were monitored by viablecounts following heat-shock treatment to kill germinatedspores, and by detecting the changes of optical density(OD600) associated with outgrowth to the vegetative state.Where indicated, MnSO4 was added to the growth media, toa final concentration of 10 μM. For enhancement of metal iondeficiencies, a modified DMEM [supplemented with 10% fetalcalf serum, 4 mM L-Glutamine, 1 mM Sodium pyruvate, 1%non-essential amino acids (Biological Industries, Beit-Hae-mek, Israel)] cell-line maintenance medium was used as thebacterial growth medium. Growth was initiated and carriedout with 5 × 105 cfu ml−1 divided in a 96-well microtitre plateincubated at 37°C in a Sunrise plate reader (TECAN). Cultureturbidity was monitored every 1 h at 630 nm. The followingdivalent cations (by added compound) were supplemented:Mn2+ (MnSO4), Fe2+ (FeSO4), Zn2+ (ZnCl2), Mg2+ (MgSO4),Ca2+ (CaCl2), Cu2+ (CuSO4). All experiments were performedat least in triplicates.

Escherichia coli strains (Table 1) were used for facilitationof plasmid construction. Antibiotic concentrations used forselection in LB agar/broth (Difco) were: for E. coli strains,ampicillin (Ap, 100 μg ml−1); for B. anthracis strains, kanamy-cin (Km, 10 μg ml−1), chloramphenicol (Cm, 7.5 μg ml−1) anderythromycin (Em, 5 μg ml−1).

Plasmid and strain construction

Plasmids and oligonucleotide primers used in this study aresummarized in Table 1. The oligonucleotide primers weredesigned according to the genomic sequence of B. anthracisAmes strain and prepared using the Expedite synthesizer(Applied Biosystems). Genomic DNA (containing the chro-mosomal DNA and the native plasmids, pXO1 and pXO2) forcloning of the mntA gene or related gene fragments, wasextracted from the Vollum strain, as described (Levy et al.,2005). Polymerase chain reaction (PCR) amplifications wereperformed using the Taq (Qiagen) or Expand High Fidelity(Roche) systems. DNA sequences were determined with theABI rhodamine termination reaction kit (ABI310 Genetic Ana-lyzer, Applied Biosystems).

For generation of specific anti-MntA antibodies via DNAimmunization, the plasmid pCI-mntA was constructed. Prim-

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ers DNA1 (forward) and DNA2 (reverse) were used to amplifyan 867 bp fragment of the mntA gene, excluding the 66 bppredicted leader sequence. The PCR product was digestedwith KpnI and NotI, and cloned in the eukaryotic expressionvector pCI, carrying both the T7 promoter for in vitro expres-sion and the eukaryotic cytomegalovirus promoter for in vivoexpression. Large-scale production of pCI-mntA for DNAimmunization by gene gun was performed by alkali lysisfollowed by CsCl gradient centrifugation (Gat et al., 2005).The plasmid preparation was suspended at a concentrationof 1 mg ml−1 in distilled pyrogen-free water and stored at−20°C.

The vector used for disruption of the mntA gene by allelicreplacement, pEO-mntA (Table 1), was constructed in four

steps: (i) an intermediate vector pEO (5.3 kb) was con-structed by joining the 4.4 kb SacII-PstI fragment of plasmidpHV1249 (containing a temperature-sensitive origin of repli-cation in Bacillus), 0.78 kb PstI-HindIII fragment of pBR322and a 0.2 kb HindIII-SacII synthetic DNA multiple cloning site(HindIII-NotI-SnaBI-SpeI-StuI-SalI-AscI-KpnI-SacII); (ii) aKmR gene from pDG782 (Guerout-Fleury et al., 1995), wasinserted as a blunt-ended StuI-SmaI 1.5 kb cassette into theStuI site of pEO; (iii) into the vector from step ii, a NotI-SpeI334 bp restriction fragment of the mntA 5′ end derived byPCR using MUT1 (forward) and MUT2 (reverse) primers, wascloned; and (iv) finally, a SalI-AscI 299 bp restriction fragmentof the mntA-3′ end-derived PCR product (MUT3 and MUT4)was introduced.

Table 1. Bacterial strains, plasmids and oligonucleotide primers used in this study.

Description/characteristics Source

StrainB. anthracis strains

Vollum Parental (wt) strain. PA+ LF+ EF+ (pXO1+) Cap+ (pXO2+) ATCC14578ΔVollum Vollum cured of both virulence plasmids. PA– LF– EF– (pXO1–)

Cap– (pXO2–)IIBR stock

ΔmntA mntA in frame deletion mutant of Vollum This studyΔmntA/pMntA Complementation of ΔmntA by mntA expression in trans,

from pMntAThis study

E. coli strainsDH5α endA1 recA1 ClontechGM2929 dam13::Tn9 (CmR) dcm-6 NEB

PlasmidpCI E. coli-eukaryotic shuttle vector. ApR PromegapCI-mntA DNA immunization vector. pCI containing a PCR-amplified mntA,

without the leader sequence, downstream of T7 and CMV promotersThis study

pHV1249 E. coli-Bacillus shuttle vector. pBR322 ori pE194ts ori ApR EmR BGSCpBR322 E. coli vector. ApR NEBpDG782 Source of KmR gene BGSCpEO Derivative of pHV1249 in which the mini Tn10 and transposase

gene were deletedThis study

pEO-mntA Allelic replacement vector. pEO containing two PCR-amplified mntA fragments (nucleotides 42–300 and 580–879), and the KmR gene from pDG782 cloned between the two fragments

This study

pASC-α E. coli-Bacillus expression vector, carrying the pagA gene under the control of the Bacillus amyloliqifaciens α-amylase promoter. ApR CmR pC194 ori

Cohen et al. (2000)

pMntA MntA expression vector. pASC-α containing a PCR-amplified mntA gene replacing the pagA gene, downstream of the pagA ribosome binding site and the α-amylase promoter

This study

Primers Sequencea

DNA1 5′-tataggtaccaccatgAGTAACACACAAATGGAAAAGAAGAGG-3′DNA2 5′-gaagatctgcggccgcccgggTTACTTTTGTAACCCATTAATAATGGT-3′MUT1 5′-gacgcgcggccgcCGTATTTGCATTAACAGCGTGTT-3′MUT2 5′-ggactagtCCACGCTCCACCTTCTTCTAGGTT-3′MUT3 5′-ggcggtcgacCATCAAATCCCTGAGGAGAAACGG-3′MUT4 5′-ttggcgcgccCGTATCTCCATCCTCACCTGATTTACC-3′COM1 5′-gtaaATGAAATTTAAAAATGTTGTATTATCG-3′COM2 5′-cgcggatccTT ACTTTTGTAACCCATTAATAATGG-3′MNT1 5′-CAGCAGAGTTGTCATAATTGATTGG-3′MNT2 5′-CACATGAGTTGGCTCTTCCTTACC-3′PAG1 5′-GGATTTCAAGTTGTACTGGACCG-3′PAG2 5′-CTTGTGCATTTAATGCGATTGG-3′LEF1 5′-AACCCGTACTTGTAATCCAATCTTC-3′LEF2 5′-GACGGACTATCAATTAACCCTCCTG-3′CAP1 5′-GTGTTAGGGTTGCTACTCTTGG-3′CAP2 5′-TATCCAATGCACTGGCAACTGG-3′

a. The homology region to the coding sequence is marked in capital letters. The restriction sites used for cloning of the corresponding PCRfragments are underlined.

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For the complementation plasmid pMntA, the mntA com-plete gene was cloned as a BamHI-digest of PCR product(COM1 and COM2 primers), replacing the pagA gene in thepreviously described vector pASC-α (Cohen et al., 2000).

All plasmids transformed into the Vollum strain were firstpropagated in a methylation deficient E. coli strain GM2929(Table 1). B. anthracis cells were electrotransformed asdescribed (Cohen et al., 2000).

To disrupt the mntA gene by homologous recombination,an allelic exchange technique was performed as follows:plasmid pEO-mntA was introduced into competent cells ofthe Vollum strain and transformants were selected for KmR at30°C. Integrants were recovered by growing transformants inLB broth at 30°C for 1.5 h, shifting to 42°C (non-permissivetemperature) for 6 h, and then plating serial dilutions on LBplates containing Km, incubated at 42°C for 12–16 h. Singlecolonies were selected, resuspended in LB broth and spottedon LB plates containing Km or Em, then incubated at 42°Cfor 12–16 h. Deletion mutants were isolated as KmR EmS

clones. The deletion of the internal fragment of mntA andinsertion of the KmR cassette into the chromosome, as wellas the presence of pXO1 (pagA and lef genes) and pXO2(capA gene) native plasmids, were confirmed by PCR (prim-ers used: MNT1 and MNT2 for the mntA gene, PAG1 andPAG2 for pagA, LEF1 and LEF2 for lef and CAP1 and CAP2for capA).

Complementation was accomplished by transformingpMntA into the ΔmntA mutant strain. The expression of MntAwas verified by Western blot analysis. The presence of thechromosomal deleted mntA containing the KmR cassette;pXO1 (lef gene) and pXO2 (capA gene) were confirmed byPCR, as above.

Membrane-enriched proteinous fraction preparation, 2-DE separation and MALDI-TOF mass spectroscopic identification

Subcellular fractions enriched in membranal proteins wereprepared as previously described (Chitlaru et al., 2004).Briefly, 1010 cfu per millilitre of B. anthracis vegetative cells ofthe Vollum or ΔVollum strains were disrupted by sonication.The insoluble material was extracted in 8 M urea, 4% (w/v)CHAPS, 40 mM Tris, 2% DTT and 0.2% Bio-Lyte 3/10 (Bio-Rad). Following centrifugation, the supernatant represents amembranal proteinous enriched fraction (∼1.5 mg proteinml−1). These fractions (200 μg protein) were resolved first byisoelectric focusing (IEF) on ready-made 17 cm, immobilizedpH gradient (IPG) strips (Immobiline DryStrips, Pharmacia),applied to a Protean IEF cell (Bio-Rad). Electrophoretic sep-aration on the second dimension was performed on 12.5%SDS-PAGE on an Ettan DALT II System (Pharmacia). Gelswere stained with G-250 Coomassie blue (Bio-safe, Bio-Rad)and spots detected and analysed by scanning on a GS-800Calibrated Densitometer assisted by the PDQuest 2-D Soft-ware (Bio-Rad). Protein spots were cut from the 2-DE gelsand subjected to in-gel overnight digestion with 6.25 μg ml−1

trypsin (Promega). Peptides were eluted with 1% TFA fol-lowed by 50% CH3CN, dried and resuspended in 10 μl of 25%CH3CN, 0.1% TFA. Two microlitres were mixed with an equalvolume of a α-cyano matrix (Sigma) and applied to a MALDI-TOF target. Mass spectra were acquired on a TofSpec 2E

apparatus (Micromass) in positive ion reflectron mode. Spec-tra were compared with theoretical tryptic digest fragmentsof the genomic sequence of the B. anthracis Ames. Identifi-cation of proteins was based on a peptide-coverage of morethan 30% and peptide mass deviation between observed andcalculated values of less than 100 p.p.m.

Generation of sera and serological tests

A guinea pig anti-B. anthracis (Vollum strain) antiserum wasobtained following exposure of the animals to a lethal chal-lenge (200 LD50), treatment with antibiotics and re-challeng-ing, as described (Altboum et al., 2002). Enzyme-linkedimmunosorbant assay (ELISA) tests for detection of anti-PAand anti-LF antibodies were carried out as detailed previously(Cohen et al., 2000; Mendelson et al., 2005). The antiserumexhibited anti-PA and anti-LF titres of 1:128 000 and1:32 000, respectively, and it was used for evaluation of MntAantigenic potential and in vivo expression, by probing with the2-DE-derived Western blots.

For generation of specific anti-MntA antibodies by DNAimmunization, the pCI-mntA vector preparation was mixedwith gold particles (Bio-Rad), and gene gun bombardmentwas carried out in female outbred ICR mice (5–6 weeks old),using the Helios Gene Gun System (Bio-Rad), as describedbefore (Grosfeld et al., 2003). Following a schedule of fourbombardments at 2-week intervals, serum was withdrawn.The mice were handled according to the National ResearchCouncil 1996 Guide for the Care and Use of LaboratoryAnimals and the IIBR Animal Use Committee.

Measurement of oxidative stress sensitivity

To determine the sensitivity to H2O2, log-phase culturesgrown in BHI medium were treated with 0, 0.1, 0.5, 1, 2.5, 5,10 and 25 mM H2O2 and incubated at 37°C, with shaking of250 r.p.m., for 30 min. Per cent survival was determined byviable plate counting of serial dilutions. To determine thesensitivity to intracellular superoxide, log-phase culturesgrown in BHI medium, were treated with 75 mM methyl viol-ogen (paraquat, Sigma), and incubated at 37°C, 250 r.p.m.,in parallel to untreated cultures. At defined time points, sam-ples were removed from treated and untreated cultures andplated to obtain viable counts. The assays were performed intriplicates.

Infection of macrophages and macrophage toxin-dependent lysis assay

A tissue culture model of macrophages was used to assessthe characteristics of the ΔmntA mutant strain under condi-tions mimicking the early stages of infection. RAW264.7macrophage-like cells (ATCC TIB 71) were cultured andmaintained in the modified DMEM. The macrophages werediluted to 5 × 105 cells per millilitre in medium, seeded to 24-well tissue culture plates and allowed to adhere overnight at37°C, 7.5% CO2. Infection was carried out by addition of5 × 105 spores per millilitre (multiplicity of infection 1:1). Theplates were centrifuged and incubated for 1 h to allow phago-cytosis, then washed four times with medium containing

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2.5 μg ml−1 gentamycin, to kill extracellular bacteria, and incu-bated for additional 2.5 h in medium containing gentamycin.The wells were washed as before and re-incubated inmedium without antibiotics (t = 0) for up to 20 h. At selectedtime points, the culture supernatant was removed for subse-quent assessment of bacterial viable count and macrophagelysis, and the cells were fixed with 80% acetone (20 min,−20°C). The fixed cells were Gram-stained, for visualizationof bacterial infection, and the wells were then scanned bylight microscopy. The B. anthracis-dependent macrophagelysis was quantified by measuring lactic dehydrogenase(LDH) released from the infected RAW264.7 cells (by Cyto-Tox 96, Promega) to the culture supernatants.

Infection of guinea pigs

Female Hartley guinea pigs (Charles River Laboratories),weighing 220–250 g, were infected with spore preparationsof the mutant strains and compared with the parental Vollumstrain. Prior to infecting the animals, the spore preparationswere heat-shocked (70°C, 20 min) to synchronize germina-tion and kill residual vegetative bacteria and serially diluted10-fold in saline, to produce spore suspensions within therange 102−109 per millilitre. A 0.1 ml spore dose volume wasadministered subcutaneously (s.c.) to each guinea pig. Atotal of four guinea pigs per dose-strain were used. Theremaining spore dose suspensions were plated for total via-ble counts (cfu ml−1). The animals were observed daily for21 days or the indicated period. Upon observation of death,necropsies were performed and blood and spleen sampleswere taken for culture. At the end of the observation period,surviving animals were bled by cardiac puncture for serolog-ical studies or were challenged s.c. with the indicated lethaldose of the parental B. anthracis Vollum strain (LD50 = 100spores). The spore lethal dose required to kill 50% (LD50) ofthe animals was calculated by the method of Reed andMuench (1938).

The guinea pigs were cared for according to the NationalResearch Council 1996 Guide for the Care and Use of Lab-oratory Animals and the IIBR Animal Use Committee.

Assays for identification of the B. anthracis major virulence factors

The B. anthracis LT components, PA and LF, were detectedin the bacterial culture growth media (NBY-Bicarbonate broth)and the culture maintenance media (modified DMEM) ofinfected RAW264.7 macrophages. Supernatant were col-lected and filtered through 0.22 μm filters. For Western blotanalyses, polyclonal anti-PA and anti-LF antibodies, gener-ated in guinea pigs and rabbits injected with highly purifiednative PA or LF, respectively (Kobiler et al., 2002; Gat et al.,2003), were used. LT activity was determined by cytotoxicityassay of J774.1A macrophage-like cell-line, as described(Reuveny et al., 2001; Marcus et al., 2004). J774.1A mac-rophage cells were maintained as described above for theRAW264.7 macrophages. Specificity of the LT-dependentcytotoxicity was assessed by incubating the supernatantswith the specific guinea pigs polyclonal anti-PA antibodiesprior to incubation of the supernatants with the J774.1A cells.

Negative staining of capsule formation was done using Indiaink (Becton, Dickinson and Company).

Acknowledgements

We are grateful to Dr B. Velan and Dr A. Zvi for critical readingof the manuscript, and thank I. Inbar, G. Friedman and N.Zeliger for their excellent technical assistance and Y. Shlo-movitch for help in the animal studies.

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