FEMS Microbiology Letters 234 (2004) 289–295

www.fems-microbiology.org

Brucella abortus nicotinamidase (PncA) contributes to itsintracellular replication and infectivity in mice

Suk Kim a, Daisuke Kurokawa a, Kenta Watanabe a, Sou-ichi Makino b,Toshikazu Shirahata a, Masahisa Watarai a,*

a Department of Applied Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,

Obihiro-shi, Hokkaido 080-8555, Japanb Research Center for Animal Hygine and Food Safety, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,

Obihiro-shi, Hokkaido 080-8555, Japan

Received 4 February 2004; received in revised form 3 March 2004; accepted 24 March 2004

First published online 10 April 2004

Abstract

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-pro-

fessional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of vir-

ulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in

HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic

acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The

pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared

from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from

spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to

confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to

intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate

that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Brucella; Nicotiamidase; Macrophages

1. Introduction

Brucellosis is a major bacterial zoonosis that causes a

serious debilitating disease in humans and abortion and

sterility in domestic animals. The etiologic agents of

brucellosis are Brucella spp., small gram-negative and

facultative intracellular pathogens that can multiply

within professional and non-professional phagocytes[1,2]. In contrast to other intracellular pathogens, Bru-

cella species do not produce exotoxins, antiphagocytic

capsules or thick cell walls, resistant forms or fimbriae

* Corresponding author. Tel.: +81-155-49-5387; fax: +81-155-49-

5389.

E-mail address: watarai@obihiro.ac.jp (M. Watarai).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.03.038

and do not show antigenic variation [3]. A key aspect of

the virulence of Brucella is its ability to proliferate

within professional and non-professional phagocytic

host cells, thereby successfully bypasses the bactericidal

effects of phagocytes, and their virulence and chronic

infections are thought to be due to their ability to avoid

the killing mechanisms within host cells [4,5]. The mo-

lecular mechanisms and genetic basis for intracellularsurvival and replication, however, are not understood

completely. Some studies with non-professional phago-

cytes have shown that Brucella invades host cells and is

contained within early endosome-like vacuoles. These

vacuoles rapidly fuse with early autophagosomes that

acquire vacuolar Hþ-ATPase and lysosome-associated

membrane proteins (LAMP), mature into a late auto-

. Published by Elsevier B.V. All rights reserved.

290 S. Kim et al. / FEMS Microbiology Letters 234 (2004) 289–295

phagosome, inhibit fusion with lysosome and finally

become a replicating vacuole normally associated with

the endoplasmic reticulum [4,6–8]. The genetic basis of

Brucella virulence is still poorly understood. The VirB

type IV secretion system of Brucella has been identifiedrecently [9]. This operon is composed of 13 open reading

frames (ORFs) that share homology with other bacterial

type IV secretion systems in the intracellular trafficking

of pathogens. Bacteria with deletion or polar and non-

polar mutations of these ORFs were not able to repli-

cate and survive within phagocytes [10,11]. Thus, the

VirB proteins of B. abortus are thought to be constitu-

ents of the secretion apparatus.A preliminary characterization of Brucella abortus

pncA (nicotinamidase)mutant showed that it had reduced

intracellular growth within HeLa cells [12], suggesting

that this nicotinamidase might be a virulence-associated

factor. In this study, we examined the virulence of B.

abortus pncA mutant in mice and their intracellular rep-

lication in macrophages. The protection induced in mice

by a B. abortus pncAmutant against a challenge with thevirulent strain B. abortus 544 was also evaluated.

2. Materials and methods

2.1. Bacterial strains and media

All bacterial strains employed in this study are thederivatives from B. abortus 544 (ATCC23448), a smooth

virulent B. abortus biovar 1 strain. Isogenic mutants of B.

abortus 544 pncA::Tn 5Km2 and DvirB4 were described

previously [11,12]. All these strains were maintained as

frozen glycerol stocks and were cultured in Brucella

broth (Becton Dickinson, Sparks, MD) or Brucella broth

containing 1.5% agar. Kanamycin (30 lg/ml) and am-

picillin (100 lg/ml) were used when necessary.pSK pncA (pncAþ) was constructed by cloning a PCR

fragment intoKpnI/SacI-cleaved pBBR1MCS-4 [13]. The

1195-bp KpnI–SacI PCR fragment spanned a site located

393 nucleotides upstream of the 5‘ end of pncA to a po-

sition 48 nucleotides downstream from the 30-end [14] andwas amplified using the primers 50-GGTACCGCATC-

TGCGGCACCTGCAAGG-30 (KpnI site underlined)

and 50-GAGCTCAACCCGAACTGGCAGAACGAG-30 (SacI site underlined).

2.2. Cell culture

HeLa cells were grown at 37 �C in a 5% CO2 atmo-

sphere in eagle minimum essential medium (MEM)

(Sigma, St. Louis, MO) containing 10% fetal bovine

serum (FBS). Bone marrow-derived macrophages fromfemale BALB/c mice were prepared by the method de-

scribed previously [15]. After culture in L-cell condi-

tioned medium, macrophages were harvested and

resuspended in RPMI 1640 (Sigma) containing 10%

FBS. The HeLa cells or macrophages were seeded (2–

3� 105 per well) in 24-well tissue culture plates one day

before infection for all assays.

2.3. Comparative growth of the mutants in minimal

medium

Growth of the pncA mutant in minimal media, RPMI

1640, which contains no nicotinic acid and 1 lg/ml

nicotinamide, was assessed according to the modified

method described previously [16,17]. Briefly, 2–3� 1010

colony forming units (CFU) of B. abortus wild-type,pncA mutant and complemented strain were pelleted at

8000 rpm for 15 min and resuspended to 2–3� 107

CFU/ml in RPMI 1640 with 0.3 mM nicotinic acid or

0.3 mM nicotinamide, and then were incubated at 37 �Cfor 0, 6, 24 and 48 h with shaking. To measure the CFU

of brucellae, equal portions were withdrawn from each

culture, serially diluted into PBS, and spread on the

surface of brucella plates to determine the number ofviable cells at intervals of up to 48 h.

2.4. Intracellular growth of the mutants in cultured HeLa

cells and macrophages

Bacterial infection and intracellular survival assay

were done by using the modified method described

previously [12]. Briefly, HeLa cells or mouse bonemarrow-derived macrophages were grown on 24-well

microtiter plates in MEM or RPMI 1640 with 10% FBS.

Cells were infected with B. abortus strains at a multi-

plicity of infection (MOI) of 20 by centrifugation at 150g

for 10 min at room temperature. To determine the effect

of disruption of nicotinamidase in vivo, HeLa cells or

macrophages were preincubated for 30 min with the

nicotinic acid (0.1 mM) and nicotinamide (0.1 mM) [16],and infected with bacteria as above.

After centrifugation, infected cells were incubated at 37

�C in a 5% CO2 for 30 min, washed twice with 0.5 ml of

sterile PBS and cultured in the media containing genta-

micin (30 lg/ml) in the presence or absence of 0.1 mM

nicotinic acid or nicotinamide for 2, 24 and 48 h. Serial

dilutionsof the infected cells lysedwith distilledwaterwere

plated onto Brucella plates for determination of CFU.

2.5. LAMP-1 staining

LAMP-1 staining was performed as described previ-

ously [15]. Briefly, infected macrophages were fixed in 4%

periodate–lysine–paraformaldehyde (PLP)–sucrose for 1

h at 37 �C. All antibody-probing steps were for 1 h at 37

�C. Samples were washed three times in PBS for 5min andthen were permeabilized at )20 �C in methanol for 10 s.

After incubating three times for 5 min with a blocking

buffer (2% goat serum in PBS), the samples were stained

S. Kim et al. / FEMS Microbiology Letters 234 (2004) 289–295 291

withanti-LAMP-1 ratmonoclonal antibody1D4B,which

was obtained from the Department of Pharmacology and

Molecular Sciences, Johns Hopkins University School of

Medicine and the Department of Biology, University of

Iowa,diluted1:100 inblockingbuffer.Afterwashing threetimes for 5 min in blocking buffer, the samples were

stained with Texas red-goat anti-rat IgG (Molecular

Probes, Inc.). Then the samples were stained with anti-B.

abortus polyclonal rabbit serum [11] and fluorescein iso-

thiocyanate (FITC)-conjugated goat anti-rabbit IgG

(Molecular Probes, Inc.) in blocking buffer to identify the

bacteria, and then they were placed in mounting medium

and were visualized by fluorescence microscopy. Onehundred bacteria within macrophages were selected ran-

domly and the LAMP-1-positive bacteria were counted.

2.6. Virulence determination in mice

Six-week-old female BALB/c mice were infected in-

traperitoneally with approximately 104 CFU of brucel-

lae in 0.1 ml saline. Groups of five mice were infectedwith each strain. At each week post-infection, mice were

sacrificed by decapitation and their spleens were re-

moved, weighed and homogenized in saline. Tissue ho-

mogenates were serially diluted with PBS and were

plated on Brucella agar to count the number of CFU in

each spleen to assess the virulence of each strain.

2.7. Vaccination against challenge infection with a viru-

lent strain

Six-week-old BALB/c mice were vaccinated with 104

CFU of B. abortus S19 or pncA mutant. Each group

consisted of five mice. At 8 weeks postvaccination, mice

were challenged intraperitoneally with approximately

104 CFU of a virulent strain, B. abortus 544. Two weeks

after the challenge, the numbers of viable B. abortus 544in the spleens and the weights of the spleens were de-

termined as described [11].

Fig. 1. Bacterial growth of wild-type and pncAmutants under minimal

medium condition. Wild-type B. abortus, pncA mutant (pncA)) and

complemented strain (pncA+) were grown in RPMI 1640 (A), RPMI

1640with nicotinic acid (B) or nicotinamide (C) as described in Section 2.

The numbers of viable bacteria at each time point were determined by

making serial dilutions in PBS and plating on Brucella plates. Data

points and error bars represent themeanCFUof triplicate samples from

a typical experiment (performed at least four times) and their standard

deviation.

3. Results

3.1. Effect of disruption of pncA in vitro and in vivo

Brucella wild-type and complemented strain showed

almost the same growth yield but pncAmutant showed a

different growth comparing to that of wild-type in

RPMI 1640, RPMI 1640 with nicotinic acid or nico-

tinamide (Fig. 1A, B and C). In the media with nico-

tinamide, two strains with intact pncA showed almost

the same growth yield but pncA mutant did not grow

(Fig. 1A and C). In the media with nicotinic acid,growth of pncA mutant was restored (Fig. 1B).

As survival and multiplication in professional and

non-professional phagocytic host cells is an important

virulence mechanism of Brucella, we examined the in-

tracellular replication of Brucella strains in HeLa cells

and mouse bone marrow-derived macrophages. The

pncA mutant failed to replicate in HeLa cells, and

displayed a lower rate of intracellular replication in

292 S. Kim et al. / FEMS Microbiology Letters 234 (2004) 289–295

macrophages than the wild-type strain and comple-

mented strain (Fig. 2A and B). To further analyze the

effect of disruption of nicotinamidase, cells were prein-

cubated in the media supplemented with nicotinic acid or

nicotinamide and then infected with Brucella strains. Inthe presence of nicotinic acid, pncA mutant was able to

replicate inside the cells as well as wild-type (Fig. 2C and

Fig. 2. Intracellular replication of wild-type and pncA mutants within HeLa

macrophages (B), nicotinic acid (C and D) or nicotinamide (E and F) added H

mutant (pncA)) and complemented strain (pncA+) as described in Section 2. A

viable intracellular bacteria were determined. Data points and error bars rep

(performed at least four times) and their standard deviation.

D). In the medium with nicotinamide, the pncA mutant,

which was unable to convert nicotinamide to nicotinic

acid, showed a reduced ratio of intracellular replication

in HeLa cells and macrophages as compared to that in

other two strains (Fig. 2E and F). Nicotinamide deami-dase is known to be released into medium from HeLa

cells and macrophages and converts nicotinamide into

cells and mouse bone marrow-derived macrophages. HeLa cells (A),

eLa cells or macrophages were infected with wild-type B. abortus, pncA

t different times of incubation, the cells were lysed, and the numbers of

resent the mean CFU of triplicate samples from a typical experiment

Fig. 4. Kinetics of bacterial growth in infected mice. Mice were infected

intraperitoneally with wild-type B. abortus, pncA or virB4 mutant (104

CFU/0.1 ml). Recovery of viable bacteria from the spleen (A) and the

weights of spleens (B) of infected mice at 1–10 weeks postinfection are

shown. Error bars indicate standard deviation.

S. Kim et al. / FEMS Microbiology Letters 234 (2004) 289–295 293

nicotinic acid for NAD synthesis [18,19]. Therefore, it is

likely that by internalizing little amounts of nicotinic

acid, pncAmutant was able to replicate to some extent in

medium with nicotinamide.

Phagosomes containing virulent B. abortus arereluctant to fuse with lysosomes, whereas dead B.

abortus phagosomes co-localize with endocytic com-

partments in the early stage of infection in macrophages

[20]. To test the ability of B. abortus to target properly

within macrophages early in infection, interaction of the

mutants with the endocytic pathway was quantified by

immunofluorescence localization of LAMP-1, a mem-

brane protein of late endosomes and lysosomes [21]. Asexpected, most phagosomes containing the wild-type

and complemented strain did not co-localize with the

LAMP-1 (15.2� 2.3% or 13.8� 4.2% positive). Inter-

estingly, pncA mutation did not affect LAMP-1 acqui-

sition (17.2� 4.8% positive) (Fig. 3). It suggests that

both wild-type strain and pncA mutant are not co-lo-

calizing with either late endosomes or lysosomes.

3.2. B. abortus pncAmutant has reduced virulence in mice

To assess whether the reduced in vitro virulence of

pncA mutant as determined by the replication within

HeLa cells and macrophags correlates with a virulence in

the host, mice were infected with wild-type B. abortus,

pncA and virB4mutant. As bacterial growth in spleen and

liver are same level [22,23], only bacterial growth in spleen

Fig. 3. Localization of pncAmutant and late endosomal and lysosomal

marker LAMP-1 in bone marrow-derived macrophages by immuno-

fluorescence microscopy. Macrophages were infected with wild-type or

pncA mutant for 1 h, fixed and stained for LAMP-1 and intracellular

bacteria.

was checked in this study. Two weeks after infection,

many bacteria were recovered from the spleen of mice

infected with the wild-type strain (6.7� 105 CFU/spleen),

but fewer bacteria were recovered frommice infected with

pncA mutant (4.2� 104 CFU/spleen) (Fig. 4A). Bacteria

were still recovered from the spleen of mice infected with

wild-type strain at 10 weeks after infection (6.8� 103

CFU/spleen), while fewer bacteria were recovered frommice infected with pncA mutant (7.8� 102 CFU/spleen).

In contrast, virB4mutant was cleared to the undetectable

level in the spleen at four weeks after infection (Fig. 4A).

Wild-type strain induced splenomegaly as a conse-

quence of host inflammatory response, but pncA mutant

induced a reduced response compared to that of the

wild-type strain (Fig. 4B). Splenomegaly was not ob-

served at all in mice infected with virB4 mutant.

3.3. Effect of vaccination with B. abortus pncA mutant

One of the drawbacks of the vaccine strain B. abortus

S19 is that it displays some degree of virulence causing

abortion in pregnant cows [24]. In order to determine if

the less pathogenic pncA mutant remains immunogenic,

Fig. 5. Protection against B. abortus 544 in mice vaccinated with pncA

mutant or B. abortus S19. Mice were inoculated intraperitoneally with

104 CFU of pncA mutant or strain S19. Eight weeks postvaccination,

mice were challenged with B. abortus 544. Two weeks later, mice were

killed and the numbers of viable Brucella recovered from the spleens

(A) and the weights of the spleens (B) were determined as described in

Section 2. Error bars indicate standard deviation.

294 S. Kim et al. / FEMS Microbiology Letters 234 (2004) 289–295

experiment of protection was carried out in mice. It was

shown that vaccination with B. abortus pncA mutant

protected mice against a challenge with the virulent

strain B. abortus 544 to the same extent as B. abortus

S19 (Fig. 5). This indicated that pncA mutant with areduced virulence and an impaired ability for intracel-

lular multiplication completely retained the ability to

induce the protective immunity in mice.

4. Discussion

The pncA gene encoding nicotinamidase/pyrazinami-dase is an enzyme involved in the production of NAD.

Salmonella emterica serovar Typhimurium and Esche-

richia coli use PncA in the preferred exogenous pathway

for NAD synthesis [25]. In the less preferred endogenous

pathway, NadB, NadA, NadC catalyse the conversion

of aspartate to nicotinate DD-ribonucleotide. The genome

of Brucella melitensis and Brucella suis contains PncA

and PncB, NadD and NadE, but not NadB, NadA orNadC [14]. Likewise, B. abortus may rely upon a single

pathway for NAD synthesis. B. abortus lacking pncA

was able to survive and grow in vitro because Brucella

broth contains nicotinamide, nicotinic acid, NAD and

NADP. One or more of these compounds may permit

the growth of pncA mutant at the same rate as wild-type

strain. It is also possible that PncA participates in other

pathways required for survival and growth in vivo. As arecent study reported that plasmid-encoded PncA con-

tributed to infectivity of Borrelia burgdorferi in mice

[16], PncA may have important roles for virulence of

intracellular pathogens. Our results in this study indicate

that both wild-type strain and pncA mutant prevent

phagosome–lysosome fusion after uptake by macro-

phages. Thus, PncA does not contribute to intracellular

trafficking of B. abortus, but contribute to utilization ofnutrients required for intracellular growth.

PncA is involved in the conversion of pyrazinamide

to pyrazinoic acid. Pyrazinamide is an important antit-

uberculosis drug and mutation of pncA is a major

mechanism of pyrazinamide resistance in Mycobacte-

rium tuberculosis [26]. Unlike most antibacterial agents,

pyrazinamide, despite its remarkable in vivo activity,

has no activity against M. tuberculosis in in vitro exceptat an acidic pH [27]. Presumably, pyrazinamidase would

act under acidic environment. Vacuole acidification in

phagocytic cells to pHs between 4.0 and 4.5 has been

shown to be essential for intracellular survival during

early infection by B. suis [28]. Pyrazinamidase may

participate in the resistance of Brucella spp. to the acidic

condition of the phagosome.

B. abortus virB mutant was cleared from the infectedmice faster than wild-type strain. On the other hand,

pncAmutant did not replicate in mouse spleen, but it was

not cleared from the infected mice soon. These results

indicate that virB genes have important roles in resis-

tance to clearance by host immunity. Our results showed

that the pncA mutant protected mice against a challenge

with the pathogenic B. abortus strain 544 to the same

extent as attained by the B. abortus vaccine strain S19.The decreased virulence with retention of the capacity to

confer immunity suggests that detailed characterizations

of the pncA mutant may improve current live vaccines.

Acknowledgements

We thank Drs. A.R. Ghosh and Neeraj Rana forcritical reading of the manuscript. This study was sup-

ported, in part, by a grant from The 21st Century COE

Program (A-1), Ministry of Education, Culture, Sports,

Science, and grant from Uehara Memorial Foundation.

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