Peptidase N encoded by Salmonella enterica serovar Typhimurium modulates systemic infection in mice

R E S E A R C H A R T I C L E

PeptidaseNencodedbySalmonella enterica serovarTyphimuriummodulates systemic infection inmiceVeerupaxagouda Patil, Anujith Kumar, Sanjana Kuruppath & Dipankar Nandi

Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, India

Correspondence: Dipankar Nandi,

Department of Biochemistry, Indian Institute

of Science, Bangalore-560012, India. Tel.:

191 80 22933051; fax: 191 80 23600814;

e-mail: [email protected]

Received 29 December 2006; revised 17 July

2007; accepted 23 July 2007.

First published online October 2007.

DOI:10.1111/j.1574-695X.2007.00323.x

Editor: Willem van Eden

Keywords

cytosolic protein degradation; enzyme;

infection; Salmonella typhimurium .

Abstract

The cytosolic protein degradation pathway, involving ATP-dependent proteases

and ATP-independent peptidases, is important for modulating several cellular

responses. The involvement of pathogen-encoded ATP-dependent proteases is well

established during infection. However, the roles of ATP-independent peptidases in

this process are not well studied. The functional role of Peptidase N (PepN), an

ATP-independent enzyme belonging to the M1 family, during systemic infection of

mice by Salmonella enterica serovar Typhimurium (Salmonella typhimurium)

was investigated. In a systemic model of infection, the number of CFU of

S. typhimurium containing a targeted deletion in peptidase N (DpepN), compared

with wild type, was significantly higher in the lymph node and spleen. In addition,

S. typhimurium replicated in the thymus and greatly reduced the number of

immature CD41CD81 thymocytes in a dose- and time-dependent manner. Strains

lacking or overexpressing pepN were used to show that the reduction in the

number of thymocytes, but not lymph node cells, depends on a critical number of

CFU. These findings establish a role for PepN in reducing the in vivo CFU of

S. typhimurium during systemic infection. The implications of these results, in the

context of the roles of proteases and peptidases, during host–pathogen interactions

are discussed.

Introduction

Cytosolic protein degradation plays an important role in the

maintenance of cellular homeostasis and is of great signifi-

cance in various cellular processes. This pathway can be

categorized into two distinct stages based on the need for

ATP. The proximal ATP-dependent protein unfolding and

proteolytic events are followed by ATP-independent proces-

sing and degradation of peptides into free amino acids. La

endopeptidase (Lon) and Caseinolytic protease (ClpP) are

the major ATP-dependent proteases in bacteria, whereas the

ATP-independent events are performed by multiple pepti-

dases, which lead to the generation of free amino acids

(Chandu & Nandi, 2004; Nandi et al., 2006).

Salmonella typhimurium is a facultative intracellular

pathogen that exhibits a wide host range, infecting humans,

cattle, mice and chickens. Infection manifests as enterocoli-

tis in humans and cattle; however, in mice, the organism

crosses the intestinal barrier and infects systemic organs.

The roles of two Salmonella pathogenicity islands (SPIs),

each encoding a type III secretion system for virulence, are

well studied. SPI1 is involved in infecting intestinal cells

whereas SPI2 is responsible for systemic pathogenesis (van

Asten & van Dijk, 2005). Recently, there have been several

studies that implicate ATP-dependent proteases in the

determination of virulence. Salmonella typhimurium Dlon

survives poorly within macrophages, colonizes systemic

organs less efficiently and is highly attenuated (Takaya

et al., 2003). Furthermore, Lon degrades key proteins and

negatively regulates the expression of SPI1 (Boddicker &

Jones, 2004; Takaya et al., 2005). Also, S. typhimurium DclpP

exhibits reduced capacity to survive within macrophages

and is impaired in the colonization of the spleen and liver in

oral and systemic models of infection (Yamamoto et al.,

2001).

Although there is relatively better knowledge on the

effects of the ATP-dependent proteases, the roles of ATP-

independent peptidases during the infection process are not

well established. PepN belongs to the M1 family of metallo-

peptidases and is responsible for the majority of the cyto-

solic aminopeptidase activity in Escherichia coli. Unlike most

aminopeptidases, it cleaves both amino and endopeptidase

FEMS Immunol Med Microbiol 51 (2007) 431–442 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

substrates; in fact, it is the only known aminoendopeptidase

in eubacteria. Furthermore, E. coli lacking pepN is more

adept in resisting sodium salicylate-induced stress, demon-

strating a role for pepN in modulating selective stress

responses (Chandu & Nandi, 2003; Chandu et al., 2003).

Interestingly, PepN from Brucella melitensis is recognized

by sera from patients with acute and chronic brucellosis,

suggesting that it is an immunogenic aminopeptidase with

possible diagnostic significance (Contreras-Rodriguez et al.,

2003). Notably, pepN is downregulated during infection of

a macrophage cell line by S. typhimurium (Eriksson et al.,

2003). However, there are no functional data to validate this

observation in an infection model system. Therefore, this

study was initiated to investigate the functional role of pepN

during systemic infection of mice with S. typhimurium.

Materials and methods

Bacterial strains and culture conditions

Salmonella typhimurium NCTC 12023 (Chakravortty et al.,

2005) was used as the wild type (WT) strain for construction

of DpepN, using the following strategy (Chandu et al., 2003):

The E. coli pepN construct harboring the kanamycin resis-

tance cassette in place of conserved catalytic motifs was

transformed into S. typhimurium containing pKD46 encod-

ing lRed. Positive clones were selected on Luria–Bertani

(LB) agar plates containing 30mg mL�1 kanamycin at 30 1C

and later cured of pKD46 by overnight growth at 42 1C. The

pepN overexpression plasmid, #5423, containing the endo-

genous promoter, was a kind gift from Charles Miller,

University of Illinois, Urbana-Champagne. This plasmid

was used to generate pepN overexpressing transformants in

both WT and DpepN (Table 1) that were grown in LB

containing 100 mg mL�1 ampicillin. The WT strain STM8c

and DinvA were gifted by E. Charpentier, University of

Vienna, Austria, and have been characterized previously

(Galan & Curtiss, 1989). DinvA was grown in LB containing

50mg mL�1 tetracycline.

Strains were grown in LB broth in the presence or absence

of appropriate antibiotics. Different strains were streaked on

Salmonella Shigella agar (SSA) plates and incubated over-

night at 37 1C. A single colony was picked and inoculated

in LB broth for 12 h at 37 1C. This preinoculum was diluted

in LB broth and cultured for 3 h (log phase) or overnight

(static phase) and used for appropriate experiments. Bacter-

ia were centrifuged, washed, resuspended in Hank’s-buf-

fered salt solution (HBSS) or phosphate-buffered saline

(PBS) and used for infection.

Enzyme assays

Enzyme assays were performed as described previously

(Chandu & Nandi, 2003). Briefly, cytosolic lysates (5–25 mg

total protein) were incubated with 0.5 mM Arg-AMC,

0.5 mM Suc-LLVY-AMC or 0.5 mM Cbz-GGL-bNA (Sigma

Chemical Co., St Louis, MO). Free bNA or AMC released

post cleavage was measured and specific activity was calcu-

lated as the nanomoles of bNA or AMC released per

microgram of protein per hour at 37 1C.

Mice

BALB/c female mice (5–8-week age group) were procured

from the Central Animal facility, IISc, or the National

Institute of Nutrition, Hyderabad. Mice were housed in the

departmental facility according to institutional guidelines.

Isolation of thymocytes and lymph node cells

Mice were infected with indicated doses of CFU of S.

typhimurium via the intraperitoneal route. After appropriate

days, the mesenteric lymph node and thymus were dissected

and the CFU and cell numbers were determined from the

same organ. Cells were removed by teasing, washed and an

aliquot was used for fluorescence-activated cell sorting

(FACS) staining or determination of viable cell numbers

using Trypan Blue. Cells were stained with monoclonal

antibodies to CD4 and CD8 conjugated to fluorescein

isothiocyanate (FITC) and phycoerythrin (PE), respectively

(BD, San Diego, CA), and analyzed on a Becton Dickinson

FACScanTM flow cytometer. The CELLQUEST (Becton Dick-

inson) software was used for acquisition, and WINLIST (Verity,

Topsham, ME) software was used for further data analysis,

as described previously (Prasanna & Nandi, 2004).

Table 1. Salmonella typhimurium strains used in this study

Sl. No. Strain designations Strain characteristics

1 WT S. typhimurium NCTC 12023

2 DpepN WT containing a targeted deletion in pepN

3 WT/Vector WT transformed with pBR322

4 DpepN/Vector DpepN transformed with pBR322

5 WT/pepN WT transformed with #5423, the pepN overexpressing plasmid

6 DpepN/pepN DpepN transformed with #5423, the pepN overexpressing plasmid

FEMS Immunol Med Microbiol 51 (2007) 431–442c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

432 V. Patil et al.

Macrophage isolation and culture

Mice were injected via the intraperitoneal route with c. 4 mL

of 4% Brewer’s Thioglycollate broth (Himedia) and sacri-

ficed after 4–5 days. Thioglycollate-elicited macrophages

were harvested by peritoneal lavage with c. 5 mL of 0.32 M

ice-cold sucrose. Cells were washed, resuspended in appro-

priate volume of plain Roswell Park Memorial Institute

(RPMI) and plated in 96-well flatbottom cell culture plates

(Tarsons, India). Cells were incubated for 2 h at 37 1C in a

CO2 incubator to facilitate adherence to plastic. At the end

of the incubation period, nonadherent cells were removed

by washing with HBSS. Cells were detached from the plate

using ice cold 0.5 mM EDTA, washed and purity was

confirmed by FACS analysis. Viable cells were counted by

the Trypan blue exclusion method using a hemocytometer.

Macrophages were cultured at 37 1C, in the presence of 5%

CO2, in RPMI 1640 medium supplemented with 25 mM

HEPES (Sigma), 2 mM L-glutamine (Himedia, India), 5 mM

b-mercaptoethanol (E Merck, San Diego, CA), 100 mg mL�1

penicillin, 250mg mL�1 streptomycin, 50 mg mL�1 gentami-

cin (Himedia) and 10% heat-inactivated fetal bovine serum

(FBS) (Sigma).

Macrophage cytotoxicity assay

The crystal violet staining protocol was standardized to

study the macrophage cytotoxicity caused by S. typhimur-

ium (Lundberg et al., 1999). Briefly, adherent thioglycollate-

elicited macrophages were infected with log-phase-grown

bacteria at different multiplicities of infection (MOI) for a

period of 1 h in RPMI 10% FBS without antibiotics. Cells

were washed with PBS, fresh medium with antibiotics was

added and incubated further for 1 h. After washing with

PBS, cells were stained with 100 mL of 0.2% crystal violet in

20% methanol for 15 min at 37 1C. Extensive washings were

performed with water to remove excess dye and the plates

were dried completely. A solution comprising 0.1 M sodium

citrate (pH 5.4) and 20% methanol was added to the wells

and incubation was performed for 30 min at 37 1C. At the

end of the incubation period, the absorbance was measured

at 620 nm. The absorbance of untreated cells was taken as

100%. The percentage of cell survival was defined as the

relative absorbance of treated vs. untreated cells.

Intracellular bacterial survival assay

Thioglycollate-elicited peritoneal macrophages were plated

at c. 2–5� 105 cells per well in RPMI 10% FBS medium in

24-well plates and incubated overnight at 37 1C. The cells

were infected with static-phase bacteria with indicated MOI.

Synchronization and enhancement of infection was achieved

by centrifugation of plates at c. 50 g for 5 min. Phagocytosis

was facilitated by incubating cells for a period of 30 min. At

the end of the incubation period, extracellular bacteria were

removed by washing three times with PBS. Fresh medium

containing 100mg mL�1 gentamicin was added to the wells

and incubated further for a period of 1 h to kill the residual

extracellular bacteria. After washing with PBS, fresh med-

ium containing 20mg mL�1 gentamicin was added. The cells

were incubated for appropriate durations of time under this

condition. At the end of the incubation period, cells were

washed with PBS three times and lysis was achieved by

incubating the cells with 500 mL of 0.1% Triton X-100 for

10 min at room temperature. The cell lysates thus obtained

were appropriately diluted in PBS and were plated in

triplicate individually on SSA plates. The colonies were

counted after overnight incubation at 37 1C, and CFU per

2–5� 105 cells were calculated.

Nitrite assay

Thioglycollate-elicited macrophages (c. 2� 105 cells well�1)

were seeded in 96-well flatbottom plates. The cells were

treated with lipopolysaccharide (Sigma) or infected at

indicated MOI of static-phase cultures of live or heat-killed

S. typhimurium (WT or DpepN). The cells were incubated

for 1 h under an antibiotic free condition. At the end of the

incubation period, the cells were washed and a fresh

medium with antibiotics was added. The cells were incu-

bated for specified time periods at 37 1C in the CO2

incubator. Culture supernatants were harvested at different

time points and nitrite was quantified using the Griess

reagent (Malu et al., 2003).

Determination of burden of infection

Mice were infected via the intraperitoneal route with log-

phase cultures of indicated CFU of either WT, DpepN or

pepN overexpression strains. Postinfection, different organs

were weighed and lysates were prepared in PBS using the

Dounce homogenizer. The lysates were appropriately diluted

and plated on SSA. The data were represented as CFU per

organ and GRAPHPAD.PRISM version 4 was used to plot values.

Mice survival assays

Mice were infected via the intraperitoneal route with log-

phase cultures of WT or DpepN. The animals were mon-

itored for death every 12 h, and GRAPHPAD.PRISM version 4 was

used to plot survival curves.

Statistical analysis

The statistical significances of the CFU per organ values

obtained from infection burden experiments were tested by

the Mann–Whitney U-test. The statistical significance of the

survival curves was tested by a log rank test using GRAPH-

PAD.PRISM version 4.


433Salmonella typhimurium encoded Peptidase N and systemic infection

Results

Selected amino and endopeptidase substratesare not cleaved by DpepN

The endogenous pepN was replaced with a homologous

disrupted pepN harboring the kanamycin resistance cassette

to generate DpepN. After PCR amplification, genomic DNA

from WT amplified a band of c. 2.6 kb, whereas the identical

primers amplified a band of c. 3.8 kb from DpepN, consistent

with expected results (Fig. 1a). Cytosolic lysates from DpepN

and DpepN/vector were unable to cleave Suc-LLVY-AMC, an

endopeptidase substrate, and Arg-AMC, an aminopeptidase

substrate (Fig. 1b). However, overexpression of pepN en-

hanced the cleavage of these substrates. Notably, there was

no difference in the hydrolysis of Cbz-GGL-bNA, which is

not cleaved by pepN. These studies, using DpepN and pepN

overexpression, demonstrate that pepN from S. typhimur-

ium encodes an enzyme that is an aminoendopeptidase,

similar to its counterpart in E. coli (Chandu & Nandi, 2003;

Chandu et al., 2003).

Next, we investigated whether PepN modulated the in

vitro growth of S. typhimurium. There was no difference

in terms of growth and CFU (Fig. 1c) between WT, DpepN

and pepN overexpressing strains in LB at all the time

points studied. Thus, pepN does not play a role in the

growth and survival of S. typhimurium in a nutrient-rich

medium.

PepN does not modulate macrophagecytotoxicity, intracellular bacterial survival andnitrite production induced by S. typhimurium

After gaining entry into the gastrointestinal tract, S. typhi-

murium infects host macrophages and induces cell death.

This rapid death of macrophages is dependent on SPI1 and

host caspase-1, which leads to successful colonization of the

intestinal tract. To study the effect of pepN on the quick

death of macrophages induced by S. typhimurium, thiogly-

collate-elicited macrophages were infected at indicated MOI

and the extent of death was quantified. Consistent with

previous results, DinvA was unable to cause the rapid death

of macrophages due to an impairment of the inner mem-

brane component of the SPI1 secretion apparatus (Galan &

Curtiss, 1989). However, no significant difference was found

between WT and DpepN (Fig. 2a), which demonstrates that

pepN does not play a role in macrophage cytotoxicity caused

by S. typhimurium.

Salmonella typhimurium is a facultative intracellular

pathogen that resides and replicates within macrophages.

Intracellular survival was investigated at two different MOI

using thioglycollate-elicited macrophages. The extent of

intracellular survival exhibited by WT and DpepN was

similar (Fig. 2b). Thus, pepN does not modulate

0

0.4

0.8

1.2

1.6

2

2.4

2.8

3.2

0

50

100

150

200

250

300

350

WTpepN

WT/V

ector

pepN/V

ector

WT/pe

pN

pepN/pep

N

nmol

AM

C r

elea

sed

(µg

prot

ein)

−1

h−1

Cbz-GGL- NASuc-LLVY-AMC Arg-AMC

M 1 2

3.8 Kb

2.6 Kb

6 Kb

3 Kb

2 Kb

Log

CF

U

0

2

4

6

8

10

12

14

16

18

0 4 8 12 16 20 24

0 4 8 12 16 20 240

0.5

1

1.5

2

2.5

3

3.5

WT

pepN

WT/Vector

WT/pepN

(a)

(b)

(c)

Time (h)

OD

600

nm

pepN/pepN

pepN/Vector

β

(µg

prot

ein)

−1 h

−1nm

ol

NA

or

AM

C r

elea

sed

β

Fig. 1. PepN, an aminoendopeptidase, does not modulate in vitro

growth of Salmonella typhimurium. (a) Genomic DNA from S. typhimur-

ium WT (lane 1) and DpepN (lane 2) were amplified using gene-specific

primers. Lane M represents the 1 kb ladder. (b) Cytosolic lysates of WT,

DpepN and pepN overexpressing strains were assayed for their ability to

cleave Cbz-GGL-bNA, Suc-LLVY-AMC and Arg-AMC. The data represent

mean� SE, n = 3 experiments. (c) Indicated strains were grown in LB

media at 37 1C and cultures were aliquoted at different time points for

reading at OD600 nm or were plated on SS agar plates for CFU analysis.

The data are representative of two independent experiments.


434 V. Patil et al.

intracellular survival of S. typhimurium within thioglycol-

late-elicited macrophages.

Macrophages are capable of producing a repertoire

of reactive radicals that inhibit or kill intracellular

pathogens. Nitric oxide is an important effector molecule

and iNos�/� mice are susceptible to systemic salmonellosis

(Mastroeni et al., 2000). The ability of live and heat-killed

WT and DpepN to modulate nitrite production by thiogly-

collate-elicited macrophages was studied. The culture super-

natants were harvested every 24 h and amounts of nitrite

were quantified. Macrophages treated with lipopolysacchar-

ide as a positive control induced high amounts of nitrite

(Fig. 2c). However, both WT and DpepN elicited similar

amounts of nitrite production, which demonstrates that

pepN does not modulate nitrite production by macro-

phages.

Role of pepN during a systemic model ofinfection by S. typhimurium

Mice were infected with 103 CFU of WT or DpepN via the

intraperitoneal route of infection. After 3 days of infection,

the number of CFU for WT and DpepN was approximately

the same in the mesenteric lymph node and spleen. How-

ever, on day 5, DpepN exhibited significantly more CFU

(c. 18–58-fold) compared with WT (Fig. 3a), which clearly

demonstrates that pepN reduced the in vivo growth of

S. typhimurium during systemic infection in mice.

0

20

40

60

80

100

120

1 :

0.01

1 :

0.1

1 :

1

1 :

0.01

1 :

0.1

1 :

1

1 :

0.01

1 :

0.1

1 :

1

1 :

0.01

1 :

0.1

1 :

1

STM8c ∆ invA WT ∆ pepN

0

1

2

3

4

5

6 LPSWT (Live)

pepN(Live)WT (HK)

pepN(HK)

24 h 48 h 72 h

0

1

2

3

4

5

6

7

8

9 WT (MOI 10)

pepN (MOI 10)

WT (MOI 1)

pepN (MOI 1)

2 h 8 h 16 h

% V

iabi

lity

CF

U p

er 2

–5 ×

105

Cel

ls

Time

Nit

rite

(µM

)

Time

(a)

(b)

(c)

Fig. 2. Salmonella typhimurium-encoded pepN does not modulate macrophage cytoxicity, intracellular bacterial survival and nitrite production. (a)

Approximately 105 thioglycollate-elicited macrophages from BALB/c mice were infected with indicated MOI of the above-mentioned strains for 1 h, and

macrophage viability was determined. (b) Thioglycollate-elicited macrophages were infected at two different MOI with indicated strains. Macrophages

were lysed at indicated periods of time and the lysates were plated. (c) Thioglycollate-elicited macrophages were stimulated with lipopolysaccharide

(10 mg mL�1), live or heat-killed strains of S. typhimurium (MOI 1) and incubated for different periods of time. At the end of each incubation period,

nitrite production by macrophages was quantified. The data show mean� SD for triplicate determinations. The data are representative of two

independent experiments.



The above result led to the question of whether this burden

of infection increased the death of mice. At the highest dose

of CFU used (104), all mice died after 5 days of infection,

whereas at lower doses of CFU (102 and 103) all mice died by

day 7. However, there was no significant difference in the

death profile between WT and DpepN (Fig. 3b).

Effect of pepN overexpression during thesystemic model of infection by S. typhimurium

In order to confirm the role of pepN, studies using pepN

overexpressing strains (WT/pepN, DpepN/pepN) and their

corresponding vector controls (WT/vector and DpepN/vec-

tor) were designed. Mice were infected intraperitoneal with

103 CFU and the burden of infection in systemic organs was

determined on indicated days after infection. As observed in

Fig. 4, there was no significant difference in the number of

CFU between WT/vector and DpepN/vector after 3 days of

infection. However, overexpression of pepN reduced CFU

after 3 days of infection. After 5 days of infection, the

number of CFU of DpepN/vector was far greater compared

with WT/vector (40–169-fold); however, pepN overexpres-

sion greatly reduced the number of CFU. Together, data

from DpepN and pepN overexpressing strains confirmed that

the number of CFU by S. typhimurium during systemic

infection of mice is modulated by pepN.

Effect of S. typhimurium infection on lymphnode cells and thymocytes

Infection by several pathogens reduces the number of

thymocytes due to increased apoptosis (Savino, 2006);

however, the effect of S. typhimurium infection on thymo-

cytes has not been studied. Mice were injected intraperito-

neal with 10 or 103 CFU of S. typhimurium and the

intracellular bacterial proliferation and cell numbers in the

lymph node and thymus were determined. The number of

CFU from mesenteric lymph node cells was enhanced after 5

days of infection with 103 CFU compared with 10 CFU (Fig.

5a). Also, there was a slight reduction in the numbers of

lymph node cells isolated in mice injected with 103 but not

10 CFU (Fig. 5b). However, the numbers of CD41 and

CD81 mature T cell subpopulations in infected mice were

comparable to uninfected controls (Fig. 5c–e). Concur-

rently, the number of CFU from the thymus was increased

after 5 days of infection with 103 CFU compared with

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8

Day 3 Day 5

Lymph node

Spleen

CF

U p

er o

rgan

× 1

03

103 CFU per mouse

102 CFU per mouse

104 CFU per mouse

WT pepN WT pepN

3

4

5

6

7

8

9

0

500

1000

1500

2000

0

50

100

150

200

250

0

50 000

100 000

150 000

200 000

Per

cent

sur

viva

l

WTpepN

0 1 2 3 4 5 6 7 80

25

50

75

100

125

0

25

50

75

100

125

0

25

50

75

100

(b)(a) 125

Time (days)

Fig. 3. Salmonella typhimurium DpepN demonstrates increased number of CFU during systemic infection. (a) Groups of five BALB/c mice were

administered with 103 CFU of WTor DpepN via the intraperitoneal route of infection. The mesenteric lymph node and spleen were dissected at indicated

intervals of time and the infection burden was determined. Increased number of CFU of DpepN was found to be statistically significant at Po 0.05. The

data are representative of two independent experiments. (b) Groups of five BALB/c mice were administered intraperitoneal with 102, 103 and 104 CFU

of WT or DpepN and monitored for death. The data are representative of two independent experiments.


436 V. Patil et al.

10 CFU (Fig. 5f). Thymocyte numbers were not greatly

affected upon infection with 10 CFU after 3 or 5 days

postinfection. However, infection with 103 CFU greatly

reduced the number of thymocytes after 5 days of infection

but not earlier (day 3) (Fig. 5g). FACS analysis revealed that

this reduction in thymocyte numbers occurred in the

immature CD41CD81 subpopulation (Fig. 5j).

PepN modulates the number of thymocytesduring S. typhimurium infection

The modulation of CFU in lymph node and spleen by

DpepN and pepN overexpressing strains led to investigation

of the effect on the host response with respect to thymocyte

numbers. As observed in Fig. 6a, the number of CFU greatly

increased in the lymph node upon infection with the

DpepN/vector. This increase was greatly reduced upon over-

expression of pepN, which is consistent with previous results

(Fig. 4). There was a slight reduction in the number of

lymph node cells upon infection; however, there was no

major difference upon infection with DpepN/vector and

pepN overexpressing strains (Fig. 6b). Compared with the

WT/vector, infection with the DpepN/vector greatly in-

creased CFU in the thymus (Fig. 6c). However, this great

increase in CFU did not further reduce thymocyte numbers

compared with infection with WT/vector. Overexpression of

pepN reduced the number of CFU in the thymus, which led

to increased numbers of thymocytes (Fig. 6d). This relation-

ship is shown clearly in Fig. 6f where an increased load of

CFU (compare DpepN and WT) did not lead to further

reduction in thymocyte numbers; however, over expression

of pepN resulted in lower loads of CFU, which, in turn,

increased the number of thymocytes. Importantly, this

relationship between CFU and cell numbers was not ob-

served in case of mesenteric lymph node cells (Fig. 6e).

Discussion

An emerging area of host–pathogen interactions is the role

of enzymes involved in cytosolic protein degradation. There

are, at least, three main mechanisms by which pathogen-

encoded enzymes are involved during infection. First, these

enzymes may be directly involved in the breakdown of host

proteins into amino acids. For example, the enzymes

involved in the breakdown of hemoglobin by Plasmodium

falciparum play a crucial role (Liu et al., 2006). Second,

proteases may degrade regulatory proteins and modify the

expression of important proteins required for virulence

(Boddicker & Jones, 2004; Takaya et al., 2005). Finally, they

may be involved in resisting reactive radicals generated by

host defense mechanisms (Darwin et al., 2003). Although

the roles of proteases in modulating infection are well

known, there are few reports on the functional roles of

peptidases during infection. A periodontal pathogen, Por-

phyromonas gingivalis, lacking dipeptidyl aminopeptidase

IV is less virulent compared with WT probably due to

CF

U p

er o

rgan

× 1

03

A B C D A B C D0

50

100

150

200

250

A : WT/Vector

B : pepN/Vector

C : WT/pepN

D : pepN/pepN

0

5

10

15

20

0

500

1000

1500

2000

0

2500

5000

7500

10 000

Spleen

Day 3 Day 5

Lymph node

Fig. 4. Overexpression of pepN reduces the number of CFU of Salmonella typhimurium during systemic infection. Groups of five BALB/c mice were

infected with 103 CFU of indicated strains via the intraperitoneal route. The mesenteric lymph node and spleen were dissected at indicated intervals of

time and the infection burden was determined. The difference was found to be statistically significant at Po 0.05.



0

50

100

150

200

250

300

350

Day 3 Day 5

15.3 ± 2.3

45.3 ± 3.4 57.3 ± 9.2

Uninfected Infected with 10 CFU Infected with 1000 CFU

CD4CD4

Uninfected Infected with 10 CFU Infected with 1000 CFU

0

5

10

15Uninfected10 CFU

103 CFU

Day 3 Day 50

20

40

60

80

100

120

Day 3 Day 5

10 CFU

103 CFU

0

1

2

3

4

5

6

Cel

l No

per

orga

n ×

107

103CFU

Day 3 Day 5

10 CFU103 CFU

CF

U p

er o

rgan

× 1

03

10 CFU

Uninfected

103

CD

8

102

101

14.3 ± 2.0

4.2 ± 3.74

CD

8

103

102

101

14.6 ± 1.5

103

CD

8

103CD4

103 103

CF

U p

er o

rgan

× 1

03

Cel

l No

per

orga

n ×

107

82.2 ± 1.9

CD

8

103CD4

103CD4

103CD4

CD

8

77.5 ± 2.1

103

CD

8

37.1 ± 16.4

(a)

(c)

(f)

(h) (i) (j)

(g)

(d) (e)

(b)

Fig. 5. Salmonella typhimurium infection reduces CD41CD81 thymocytes in a systemic model of infection. BALB/c mice were injected intraperitoneal

with c.10 and c.103 CFU of WT, and the infection burden and total cell count of mesenteric lymph node (a, b) and thymus (f, g) were determined after 3

or 5 days postinfection. The data shown are mean� SE of n = 3 mice. The data are representative of two independent experiments. Lymph node cells

(c–e) and thymocytes (h–j) from uninfected mice or infected with c.10 and c.103 CFU for 5 days were stained with monoclonal antibodies to CD4 and

CD8, and FACS analysis was performed.


438 V. Patil et al.

reduced destruction of connective tissue (Yagishita et al.,

2001; Kumagai et al., 2003). Also, the intra-erythrocytic

development of the malaria parasite is blocked by Bestatin,

an aminopeptidase inhibitor. Overexpression of a leucyl

aminopeptidase by Plasmodium falciparum reduces the

efficacy of Bestatin in blocking growth, demonstrating that

this aminopeptidase may be one of the targets of Bestatin

(Gardiner et al., 2006). Some aminopeptidases from

S. typhimurium have been biochemically characterized

(Mathew et al., 2000; Larsen et al., 2001); however, their role

during infection has not been studied.

In this study, a genetic approach was used to evaluate the

functional contribution of S. typhimurium-encoded pepN

during systemic infection in mice. Salmonella typhimurium-

encoded pepN, similar to its counterpart in E. coli (Chandu

& Nandi, 2003), cleaved both aminopeptidase and endopep-

tidase substrates (Fig. 1b). Initially, WT, DpepN and pepN

overexpressing strains (WT/pepN and DpepN/pepN) were

Cell No per organ × 107

0

1

2

3

4

5

6

7

0 1 2 3 4 5

Uninfected

WT/Vector

pepN/Vector

WT/pepN

pepN/pepN

0

1

2

3

4

5

6

7

0 2 4 6 8 10

Lymph node

Thymus

ThymusLymph node

0123456789

10

0

1

1.5

2

2.5

3

3.5

4

4.5

pepN/V

ector

WT/V

ector

WT/pep

N

pepN/pep

N

Uninfected

pepN/V

ector

WT/V

ector

WT/pep

N

pepN/pep

N

Uninfected

0

200

400

600

800

1000

pepN/V

ector

WT/V

ector

WT/pep

N

pepN/pep

N

0

1000

2000

3000

4000

5000

pepN/V

ector

WT/V

ector

WT/pep

N

pepN/pep

N

CF

U p

er o

rgan

× 1

03

0.5Cel

l No

per

orga

n ×

107

CF

U p

er o

rgan

× 1

03

Cel

l No

per

orga

n ×

107

Log

CF

U

Cell No per organ × 107

Log

CF

U

(a) (b)

(c) (d)

(e) (f)

Fig. 6. Overexpression of pepN decreases the number of CFU and increases thymocyte numbers in mice infected with Salmonella typhimurium. BALB/c

mice were infected with c.103 CFU of indicated strains via the intraperitoneal route. The mesenteric lymph node (a, b) and thymus (c, d) were dissected 5

days postinjection and the infection burden and cell count per organ were determined. The data shown are mean� SE of n = 3 mice. The correlation

between cell number and CFU in the lymph node (e) and thymus (f) is shown.



characterized in vitro with respect to growth and survival in

LB broth. There was no significant difference among the

strains in the above-mentioned parameters (Fig. 1c). PepN

is not essential for in vitro growth of S. typhimurium,

probably due to the presence of redundant peptidases. In

fact, mutations in four peptidases (pepN, pepA, pepB and

pepD), but not single peptidase genes, in S. typhimurium

result in a significant decrease in cytosolic protein degrada-

tion (Yen et al., 1980). Also, pepN did not modulate nitrite

production by macrophages (Fig. 2c) or macrophage cyto-

toxicity caused by S. typhimurium (Fig. 2a). Salmonella

typhimurium lacking lon (Takaya et al., 2003) or clpP

(Yamamoto et al., 2001) are highly sensitive to oxidative

stress compared with the WT and survive less efficiently

within macrophages. However, no difference was observed

between the ability of WT and DpepN to survive within

thioglycollate-elicited macrophages (Fig. 2b). Thus, DpepN

was not compromised in its ability to interact with macro-

phages under in vitro conditions. Notably, these studies

clearly demonstrate that the phenotypes of ATP-dependent

proteases, lon and clpP, are distinct from that of pepN, an

ATP-independent peptidase involved in cytosolic protein

degradation.

There are several evidences to demonstrate an in vivo role

for S. typhimurium-encoded pepN during systemic infection

of mice. First, 5 days after infection, the number of CFU of

DpepN was far greater in comparison with WT in the

mesenteric lymph node and spleen (Fig. 3). Previous studies

have shown that transformation of S. typhimurium with

plasmids reduces virulence (Abromaitis et al., 2005; Knodler

et al., 2005). However, studies with untransformed or vector

transformed strains (Figs 3, 4 and 6) demonstrated the role

of PepN in reducing CFU in systemic organs. Overexpres-

sion with pepN reduced CFU in the spleen, lymph node and

thymus, demonstrating that this effect was pepN-dependent

(Figs 4 and 6). Together, the data from DpepN and pepN

overexpressing strains demonstrate a functional role of pepN

in reducing the number of CFU in spleen, lymph node and

thymus during systemic infection. Although the number of

CFU increased upon infection with DpepN compared with

WT, there was no difference in the numbers or kinetics of

mice survival (Fig. 3b). It is important to note that the

increase in the infection burden in case of DpepN was

observed at a later time point. Hence, it is possible that the

high infection burden at a later time point did not translate

into reduced survival of mice.

Infection with several pathogens, e.g. Trypanosoma cruzi,

Francisella tularensis, mouse hepatitis virus, etc., causes

thymic atrophy. The induction of pathogen-induced thy-

mocyte apoptosis is due to an excessive host response due

to the production of steroids, chemokines, cytokines, etc.

(Savino, 2006). However, there are no studies on the effect of

S. typhimurium infection on thymocytes. The important

aspects of this part of the study are as follows: first,

S. typhimurium replicated in the thymus and reduced the

number of thymocytes in a dose- and time-dependent

manner (Fig. 5). Therefore, infection with c.10 CFU after

5 days of infection does not lead to significant reduction

in thymocytes unlike that observed with c.103 CFU. This

reduction in the number of thymocytes occurs later during

infection (day 5 compared with day 3). Second, the number

of CFU in the thymus was less compared with that observed

in the lymph node (Figs 5 and 6). Despite this higher CFU

load in the lymph node, there was no major reduction

in cellular subpopulations. However, replication of S. typhi-

murium in the thymus greatly reduced the number of

immature CD41CD81 thymocytes, as shown previously

with other pathogens (Savino, 2006). Third, WT/vector,

DpepN/vector and pepN overexpressing strains were used to

closely study the relationship between the number of CFU

and thymocyte numbers during S. typhimurium infection

(Fig. 6). It appears that beyond a certain threshold limit,

increased CFU (compare WT/vector and DpepN/vector)

does not translate into an increased reduction in thymocyte

numbers. However, reduction in CFU (by overexpressing

pepN) below this threshold limit increased the number of

thymocytes during S. typhimurium infection. Importantly,

this relationship between the load of CFU and host immune

cell numbers was observed for immature CD41CD81

thymocytes but not mature CD41 or CD81 lymph node T

cells (Fig. 6). Additional studies are required to investigate

the mechanisms responsible for the reduction in thymocytes

during infection with S. typhimurium.

PepN and its orthologs possess specialized functions in

organisms from different kingdoms. These roles can be

broadly categorized into two distinct groups. In the first

group, pepN and its orthologs are required for some func-

tions. For example, Lactobacillis lactis lacking pepN displays

lowered growth in medium containing casein as a carbon

source (Mierau et al., 1996). Further, mice lacking puromy-

cin-sensitive aminopeptidase are sterile due to defective

reproductive processes (Osada et al., 2001a, b). Also, mice

lacking ERAAP1 display unstable MHC class I molecules on

the surface and lowered CD81 T cell responses (Hammer

et al., 2006). In the second group, the absence of pepN and its

orthologs enables an organism to resist and grow better

under some stress conditions. For example, when glucose is

exhausted during diauxic growth, glycogen accumulation

occurs in Saccharomyces cerevisiae. This accumulation of

glycogen is considered to be a marker for stress in yeast but

Saccharomyces cerevisiae lacking aap-1 accumulates less

glycogen (Caprioglio et al., 1993). Also, E. coli lacking pepN

are able to grow better, compared with WT, in the presence

of sodium salicylate and overexpression of pepN reverses this

phenotype (Chandu & Nandi, 2003). It appears that pepN

encoded by S. typhimurium belongs to this second group as


440 V. Patil et al.

it is important for restricting the number of CFU at a later

stage during systemic infection. There are at least two broad

reasons for this effect. First, pepN may reduce the prolifera-

tion of S. typhimurium during systemic infection so as to

minimize the damage to the host. It is important to point

out that some pathogen-encoded genes modulate the num-

ber of bacteria to allow for a successful infection that retains

the host niche (Tierrez & Garcia-del Portillo, 2005). Alter-

nately, S. typhimurium is exposed to different stress during

infection (Rychlik & Barrow, 2005) and pepN may modulate

this in vivo stress response. Consequently, DpepN may be

able to resist increased stress occurring during the later

phase of systemic infection, resulting in higher CFU com-

pared with WT. Further work is required to fully compre-

hend the mechanisms by which pepN modulates the

systemic infection of mice by S. typhimurium. In addition,

the role of pepN during the oral route of infection, which is

more physiologically important, by S. typhimurium needs to

be investigated.

This study demonstrates a specialized role for pepN

during systemic infection by S. typhimurium in mice. It is

important because it documents a functional role for an

ATP-independent enzyme involved in cytosolic protein

degradation. This study has made a beginning and further

investigations are required to understand the mechanisms

involved in the process. A comprehensive understanding of

the roles of ATP-dependent and ATP-independent enzymes

involved in cytosolic degradation will add a novel dimension

towards understanding host–pathogen interactions.

Acknowledgements

The authors appreciate the encouragement and suggestions

by Drs Dipshika Chakravortty, N.V. Joshi, P. Sadhale,

R. Manjunath, K. Balaji and members of the DpN labora-

tory. The authors thank Drs C. Miller and E. Charpentier

for the gift of plasmids and strains. The authors are

grateful to the staff of the Central Animal Facility and

the FACS facility in IISc for their cooperation. This study

was funded by grants from the Council of Scientific and

Industrial Research and the Department of Biotechnology,

Government of India.

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Peptidase N encoded by Salmonella enterica serovar Typhimurium modulates systemic infection in mice

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