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Immunobiology 217 (2012) 354– 362

Contents lists available at ScienceDirect

Immunobiology

jo u rn al homepage: www.elsev ier .de / imbio

oles of Salmonella enterica serovar Typhimurium encoded Peptidase N duringystemic infection of Ifn�−/− mice

anoj Bhosale, Jayachandra C. Kadthur, Dipankar Nandi ∗

epartment of Biochemistry, Indian Institute of Science, Bangalore 560012, India

r t i c l e i n f o

rticle history:eceived 6 April 2011ccepted 5 July 2011

eywords:ytokinesost response

mmunocompromisedFN�nfectioneptidase. typhimurium

a b s t r a c t

Pathogen encoded peptidases are known to be important during infection; however, their roles inmodulating host responses in immunocompromised individuals are not well studied. The roles of S.typhimurium (WT) encoded Peptidase N (PepN), a major aminopeptidase and sole M1 family member,was studied in mice lacking Interferon-� (IFN�), a cytokine important for immunity. S. typhimurium lack-ing pepN (�pepN) displays enhanced colony forming units (CFU) compared to WT in peripheral organsduring systemic infection in C57BL/6 mice. However, Ifn�−/− mice show higher CFU compared to C57BL/6mice, resulting in lower fold differences between WT and �pepN. Concomitantly, reintroduction of pepNin �pepN (�pepN/pepN) reduces CFU, demonstrating pepN-dependence. Interestingly, expression of acatalytically inactive PepN (�pepN/E298A) also lowers CFU, demonstrating that the decrease in CFU isindependent of the catalytic activity of PepN. In addition, three distinct differences are observed betweeninfection of C57BL/6 and Ifn�−/− mice: First, serum amounts of TNF� and IL1� post infection are signifi-

−/−

cantly lower in Ifn� mice. Second, histological analysis of C57BL/6 mice reveals that damage in spleenand liver upon infection with WT or �pepN is greater compared to �pepN/pepN or �pepN/E298A. Onthe other hand, Ifn�−/− mice are highly susceptible to organ damage by all strains of S. typhimuriumused in this study. Finally, greater survival of C57BL/6, but not Ifn�−/− mice, is observed upon infectionwith �pepN/pepN or �pepN/E298A. Overall, the roles of the host encoded IFN� during infection with S.typhimurium strains with varying degrees of virulence are highlighted.

ntroduction

An area of emerging interest is the role of pathogen encodedroteases and peptidases in modulating the infection process andhe host response. There are multiple mechanisms by which thesenzymes affect the virulence of pathogens: first, they may bemportant in destruction of host tissue (Kumagai et al. 2003;agishita et al. 2001) or degrading host proteins and recyclingmino acids for pathogen growth (Gardiner et al. 2006). Second,hey may degrade key regulatory proteins required for virulence

Boddicker and Jones 2004; Swenerton et al. 2011; Takaya et al.005). Third, they may regulate the production of free radicalsDarwin et al. 2003), suppress host immune effector components

Abbreviations: AMC, amino methyl coumarin; ATP, Adenosine tri phosphate;FU, colony forming units; ELISA, enzyme-linked immunosorbent assay; FCS, fetalalf serum; IFN�, interferon-gamma; IL1�, interleukin 1 beta; IL6, interleukin 6; i.p.,ntraperitoneal; PBS, phosphate buffered saline; PCR, polymerase chain reaction;epN, Peptidase N; SEM, standard error of mean; SPI, Salmonella pathogenesitysland; TNF�, tumor necrosis factor alpha; WT, wild type.∗ Corresponding author. Tel.: +91 80 22933051; fax: +91 80 23600814.

E-mail address: nandi@biochem.iisc.ernet.in (D. Nandi).

171-2985/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.imbio.2011.07.010

© 2011 Elsevier GmbH. All rights reserved.

(Gropp et al. 2009) or affect inflammatory responses (Naglik et al.2003). Indeed, immunization with leucine aminopeptidase showshigh level of protection with Fasciola hepatica demonstrating itsefficacy as a vaccine candidate (Acosta et al. 2008).

Enzymes belonging to the cytosolic protein degradation path-way consisting of ATP-dependent proteases and ATP-independentpeptidases are also known to play important roles in modulatingthe host response. ATP-dependent proteases are involved in unfold-ing and cleavage of substrate protein whereas ATP-independentpeptidases cleave peptides into amino acids. Our laboratory hasbeen studying an ATP-independent peptidase known as PeptidaseN, a member of the M1 family. It is a monomeric enzyme pos-sessing amino- and endo-peptidase catalytic abilities (Chandu andNandi 2003; Chandu et al. 2003). PepN plays important roles dur-ing sodium salicylate (Chandu and Nandi 2003) and nutritionaldownshift plus high temperature stress (Kumar et al. 2009; Kumarand Nandi 2007). Other microbial members of M1 family alsoplay diverse cellular roles: S. cerevisiae lacking alanine/arginineaminopeptidase (AAP1) shows greater glycogen accumulation, a

stress marker (Caprioglio et al. 1993). Tricorn interacting factorF2 and Tricorn interacting factor F3 from Thermoplasma aci-dophilum are involved in the recycling of amino acids togetherwith 20S proteasomes (Tamura et al. 1998). There are several

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M. Bhosale et al. / Immun

ammalian members of M1 family peptidases which play dis-inct physiological roles: Endoplasmic reticulum aminopeptidasealso known as adipocyte derived leucine aminopeptidase)-1 and

are involved in the final proteolytic processing of peptides pre-ented by major histocompatibility complex class I moleculesSaveanu et al. 2005; Yan et al. 2006) and regulating hyperten-ion (Watanabe et al. 2003). Another member, Leukotriene A4ydrolase (LTA4H) is important for the host response as it cat-lyzes the conversion of LTA4 to LTB4, which is an inflammatoryediator (Haeggström 2004). These studies clearly demonstrate

he specialized roles played by M1 family members in differentrganisms.

Salmonella has been used as a model to study the roles ofnzymes involved in protein degradation. S. typhi causes typhoidever in humans whereas S. typhimurium has a broad host rangend causes self limiting gastroenteritis in humans. Importantly,nfection of mice with S. typhimurium is commonly used as a

ouse model of typhoid. The roles of several effectors proteins,.g. SPI-I, SPI-II etc, that are important in virulence are wellnown (Haraga et al. 2008). S. typhimurium lacking the pro-ease Lon are highly sensitive to H2O2 and acid, are unable tourvive within macrophages and proliferate in systemic organso cause disease (Takaya et al. 2003). Lon has been shown tolay a central role in degrading HilC and HilD, which are pos-

tive regulators of SPI-I expression. Consequently, the amountsf SPI-I are elevated in S. typhimurium lacking Lon which pre-ents the induction of SPI-II and impairs the establishment ofnfection (Boddicker and Jones 2004; Takaya et al. 2005). Also, S.yphimurium lacking ClpP protease are not completely cleared byhe host leading to persistence (Yamamoto et al. 2001). Out of thehree paralogs of the periplasmic serine proteases present in S.yphimurum, DegQ has no role for virulence but DegP and DegS areequired (Mo et al. 2006).

Peptidase N, the sole M1 member in S. typhimurium, is respon-ible for majority of aminopeptidase activities in cytosolic extractsnd shows a broad substrate preference (Kumar and Nandi 2007).icroarray analysis of S. typhimurium encoded gene expression

pon infection of the RAW macrophage cell line shows pepN expres-ion to be reduced upon infection (Eriksson et al. 2003). Also,almonella encoded pepN expression is reduced upon infection of774, U937 and HeLa cells (Ge et al. 2010; Hautefort et al. 2008).nterestingly, PepN from Brucella melitensis is an immunogenicminopeptidase that may possess diagnostic value (Contreras-odriguez et al. 2003). PepN has also been shown to reduce colony

orming units (CFU) at a later stage in infection of BALB/c micePatil et al. 2007). There are at least two possible explanationsor the role of PepN during infection. First, PepN may belong to

group of pathogen encoded proteins that reduce CFU duringnfection (Tierrez and Garcia-del Portillo 2005). Second, pathogensncounter several stresses during infection and those that areetter able to resist stress show increased proliferation and sur-ival under these conditions (Rychlik and Barrow 2005). However,he role of catalytic activity of PepN during stress and infectionas not been studied. This aspect is important as recent studiesave shown the role of LTA4H, a M1 family member, in contribut-

ng to chronic obstructive pulmonary disease (Snelgrove et al.010). More importantly, LTA4H regulates the balance betweenro- and anti-inflammatory host responses and polymorphisms

n LTA4H influence susceptibility to tuberculosis and leprosy inumans (Tobin et al. 2010).

Cytokines, e.g. TNF�, IL1� and IFN�, are induced and playmportant roles during infection with S. typhimurium (Bao et al.

000; Everest et al. 1998; Mizuno et al. 2003; Pie et al. 1997;aupach et al. 2003, 2006; Rhee et al. 2005). IFN� is an impor-ant cytokine that modulates several host responses (Saha et al.010). The roles of IFN� and its receptor has been shown to be

gy 217 (2012) 354– 362 355

important during infection with several organisms, e.g. Mycobac-terium (Cooper et al. 1993; Dorman and Holland 1998; Flynnet al. 1993), Listeria (Harty and Bevan 1995), Chlamydia pneumo-niae (Rottenberg et al. 2000), Toxoplasma gondii (Scharton-Kerstenet al. 1996) etc. However, the roles of pathogen encoded pep-tidases during infection in immunocompromised mice have notbeen shown. Therefore, this study was performed in C57BL/6and Ifn�−/− mice, a model to study the host response underimmunocompromised conditions. Together this study sheds bet-ter light on the roles of endogenous IFN� in modulating thehost response to different S. typhimurium strains, i.e. WT, �pepN,�pepN/pepN and �pepN/E298A, which display varying degrees ofvirulence.

Materials and methods

Strains

S. typhimurium NCTC 12023 derived strains used in this studyare listed in Supplementary Table 1 (Kumar and Nandi 2007). WTand �pepN transformed pBR322 were used and plasmid p5423,a pBR322-based plasmid in which S. typhimurium pepN is clonedwith its endogenous promoter was introduced in �pepN for com-plementation studies (�pepN/pepN). Strains were grown in LB(Himedia Labs, India) in the presence of 100 �g/ml ampicillin(Himedia) at 37 ◦C.

Site directed mutagenesis

The pepN mutant E298A was generated (�pepN/E298A)based on multiple sequence alignment of M1 family members(Supplementary Fig. 1). pepN was PCR amplified using primers withE298A mutation, the amplified product was treated with DpnI andtransformed. The mutation was confirmed by restriction digestionusing MluI (Supplementary Fig. 2A) and sequencing. The expres-sion of E298A was checked by Western analysis using antisera thatdetects E. coli and S. typhimurium PepN (Chandu and Nandi 2003;Patil et al. 2007).

Growth analysis

Pre-inoculum (0.2%) of different strains were grown overnightat 37 ◦C and transferred to either M9 minimal or LB medium (Kumarand Nandi 2007). Bacterial strains were initially grown at 37 ◦C for3.5 h and then shifted to 42 ◦C. Cultures were aliquoted at differenttime intervals and dilutions were plated on Salmonella-Shigella (SS)agar (Himedia) plates for CFU analysis.

Enzyme purification and spectroscopic studies

Cultures of DH5��pepN/pepN or DH5��pepN/E298A weregrown for 10 h at 37 ◦C. PepN was purified by passing cytosolicextracts through a series of chromatography columns, e.g. DEAE-cellulose, Q-sepharose and Butyl toyopearl (Sigma, St Louis, USA), aspreviously reported (Kumar and Nandi 2007). Further, Sephacryl-200 (Pharmacia) FPLC gel filtration was performed to obtainapparently homogenous purified proteins. Enzyme purity waschecked by SDS-PAGE with Coomassie staining (Supplementary Fig.3A).

Enzyme assays

Total cell extracts of different strains (20 �g) or purifiedenzymes (10 ng) were assayed for hydrolysis of aminopeptidase(0.5 mM) substrates, in 20 mM phosphate buffer, pH 8.0, at 37 ◦C for1 h. The reactions were terminated with 80% ethanol and released

356 M. Bhosale et al. / Immunobiology 217 (2012) 354– 362

Fig. 1. Catalytic activity of PepN is required for growth of S typhimurium �pepN during minimal medium and high temperature stress. Total cell extracts were preparedfrom indicated strains of S. typhimurium and Western analysis (A) and enzymatic activities (B) were performed. The specific activity shown as mean ± S.E. is representativeo s werec SS agas

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f three independent experiments. (C) Overnight grown cultures of indicated strainultures were transferred to 42 ◦C, aliquoted at different time points and plated on

hown as mean ± SE.

MC was measured fluorimetrically with excitation wavelength70 nm and emission wavelength 430 nm (Kumar et al. 2009).

ntracellular replication assay

The RAW 264.7 macrophage cell line (2 × 105) was grown inMEM medium containing 10% FCS in 24 well plates. After 12 h,

he plates were washed thrice with PBS and adherent cells werenfected with WT or �pepN or �pepN/pepN or �pepN/E298A (MOI:10) in triplicates. Plates were centrifuged at 50 × g for 10 min and

ncubated for 30 min in 5% CO2. During the initial 1 h, plates werencubated in DMEM medium containing 100 �g/ml gentamycin,hen washed thrice with PBS and incubated in 10 �g/ml of gen-amycin till completion of experiment. At indicated time points,ulture supernatants were collected for cytokine analysis. Cellsere detached from surface by scraping, counted and lysed with

% Triton X-100. Appropriate dilutions were plated on SS agar forFU analysis (Eriksson et al. 2003).

ntraperitoneal infection of mice

Mice were procured and housed in the Central Animal Facil-ty, Indian Institute of Science as per institutional guidelines. Fornfection in mice (C57BL/6 and Ifn�−/−), 0.5–1 × 103 CFU of differ-nt strains of S. typhimurium (WT, �pepN and �pepN/pepN andpepN/E298A) in 0.5 ml of PBS were administered via intraperi-

oneal (i.p.) route (Patil et al. 2007). Mice survival was monitoredfter every 12 h. For CFU studies, mice were sacrificed on day two

nd day four after infection and organs were dissected. Organs wereeighed and homogenized in 1 ml PBS; the homogenized lysatesere centrifuged at 500 × g for 5 min and appropriate dilutions of

he supernatant were plated on SS agar plates for CFU analysis.

transferred to either LB or minimal medium. After initial growth for 3.5 h at 37 ◦C,r for CFU analysis. The data is representative of four independent experiments and

Cytokine analysis

Serum samples were collected from mice and cytokine amountswere quantified by ELISA according to manufacturer’s instructions(eBiosciences San Diego, USA).

Histopathology

Liver and spleen samples from sacrificed mice were collectedin 10% neutral buffered formalin and fixed for 48 h. After fixa-tion, tissue samples were processed by routine paraffin embeddingmethod, sectioned as 5 �m sections and stained with haematoxylinand eosin for histopathological examination (Gamble and Wilson2002).

Statistical analysis

Significant differences between CFU and cytokine were analyzedby Student’s unpaired t test and log-rank test was used for sur-vival analysis. Graph pad PRISM software (Version 5, San Diego,USA) was used for data plotting and analysis. P < 0.05 was consid-ered as statistically significant, *represents P < 0.05, **P < 0.01 and***P < 0.001.

Results

Generation of S. typhimurium encoded E298A PepN

Catalytically inactive PepN (E298A) was generated using site

directed mutagenesis and mutants were screened by restrictiondigestion with MluI which led to the release of 1 kb fragment(Supplementary Fig. 2). The E298A mutant expressed PepN asobserved by Western analysis of total cell lysates (Fig. 1A). Analysis

M. Bhosale et al. / Immunobiolo

Fig. 2. PepN expression in S. typhimurium does not affect TNF� production andintracellular proliferation in a macrophage cell line. RAW264.7 cells were infectedwith indicated strains of S. typhimurium (MOI 1:10). At different time points, super-nae

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atants were removed for cytokine analysis (A). Further, cells were harvested, lysednd CFU (B) analysis was performed. Data is representative of three independentxperiments and shown as mean ± S.E.

f total cell lysates of �pepN/pepN showed Ala-AMC or Arg-AMCleavage; however �pepN/E298A extracts did not show any activityith these substrates (Fig. 1B).

omparative biochemical analysis of WT and E298A PepN

In order to study the biochemical properties of catalyticallynactive PepN, WT and E298A were purified to near homogeneitySupplementary Fig. 3A). WT PepN showed cleavage of Ala-AMCnd Arg-AMC, however, purified E298A did not cleave these sub-trates (Supplementary Fig. 3B). No significant differences werebserved in WT and E298A PepN between far UV CD spectrumSupplementary Fig. 3C) and CD-Tm (Supplementary Fig. 3D). Them of both these proteins was ∼54 ± 0.8 ◦C. Overall, this suggestshat the loss in catalytic activity did not lead to any major structuralhanges.

atalytic activity of PepN is required for growth in nutritionalownshift and high temperature stress

Previous studies have shown that PepN is required for optimalrowth of S. typhimurium and E. coli during nutritional downshiftnd high temperature stress (Kumar et al. 2009; Kumar and Nandi007). To study the roles of catalytic activity of PepN under thesetress conditions, indicated strains were grown at 37 ◦C for 3.5 hnd then transferred to 42 ◦C in LB or minimal media (Fig. 1C). Noifference in growth was observed in LB among different strains.

onsistent with previous results (Kumar and Nandi 2007) �pepNhowed reduced growth, as compared to WT, after transfer toinimal medium at 42 ◦C. Increased growth was observed withpepN/pepN; however, reintroduction of E298A did not rescue the

gy 217 (2012) 354– 362 357

growth of �pepN. Thus, catalytic activity of PepN was required forgrowth of �pepN during nutritional downshift and high tempera-ture stress.

S. typhimurium encoded PepN does not play a role in RAW 264.7cell line infection

To address the role of PepN during infection, RAW 264.7 cellswere infected with different strains of S. typhimurium. Upon infec-tion, increase in TNF� amounts (Fig. 2A) and CFU (Fig. 2B) weremonitored and maximal amounts were observed by 18–24 h. How-ever, no significant differences were observed in TNF� productionand CFU in cells infected with different strains. The results demon-strated that PepN did not further modulate S. typhimurium inducedTNF� production or CFU during RAW 264.7 infection.

PepN reduces growth of S. typhimurium upon intraperitonealinfection of C57BL/6 and Ifn�−/− mice

Cytokine analysis upon S. typhimurium infection in mice clearlyshowed increased amounts of IFN�, which is known to play impor-tant roles in the host immune response (Saha et al. 2010). Therefore,we wished to understand the roles of endogenous IFN� in contextof the host response upon infection with different strains used inthis study. C57BL/6 and Ifn�−/− mice were infected with indicatedstrains of S. typhimurium and Ifn�−/− mice showed higher CFU inspleen (Fig. 3A) and liver (Fig. 3B) compared to C57BL/6 mice inall the strains. �pepN showed higher CFU in spleen and liver com-pared to WT in C57BL/6 mice (∼5–6 fold) but this difference wasreduced to ∼2–3 fold in Ifn�−/− mice. Infection with �pepN/pepNand �pepN/E298A showed further decrease in CFU (∼10–15 fold inC57BL/6 and ∼4–5 fold in Ifn�−/− mice) compared to �pepN.

Host encoded IFN� increases serum amounts of TNF ̨ and IL1ˇupon S. typhimurium infection

A comparative cytokine analysis was performed in C57BL/6and Ifn�−/− mice upon S. typhimurium infection. The amounts ofcytokines analyzed were increased upon infection. However, infec-tion with �pepN/pepN or �pepN/E298A reduced cytokine amountsin sera by ∼2–3 fold in C57BL/6 mice. Despite the higher CFU, theamounts of TNF� and IL1�, but not IL6, were reduced in Ifn�−/−

mice. Furthermore, no significant differences in cytokines amountswere observed in Ifn�−/− mice upon infection with various strainsof S. typhimurium used in the study (Fig. 4).

Endogenous IFN� decreases host tissue damage and increasessurvival upon infection with S. typhimurium

Severe damage was observed in the livers of WT and �pepNinfected C57BL/6 mice compared to infection with �pepN/pepNand �pepN/E298A (Fig. 5). In WT and �pepN infected mice, dif-fuse areas of necrosis were observed in hepatic parenchyma.On the other hand, in �pepN/pepN and �pepN/E298A infectedmice, multi-focal and smaller areas of necrotic and inflamma-tory foci were observed. However, liver of Ifn�−/− mice infectedwith S. typhimurium showed severe hepatic damage but no dif-ferences were observed between different strains. Also, spleensof Ifn�−/− infected mice showed diffused areas of inflammatoryreactions with presence of inflammatory cells, necrotic cell debrisand expansion of red pulp irrespective of S. typhimurium strains

used in the study (Supplementary Fig. 4). These results under-score the increased susceptibility to organ damage upon infectionof Ifn�−/−mice to different strains of S. typhimurium used in thestudy.

358 M. Bhosale et al. / Immunobiology 217 (2012) 354– 362

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nd on day four post-infection mice were sacrificed, CFU load in spleen (A) and liver (npaired t test was used for statistical analysis. P < 0.05 was considered as statistica

Infection in C57BL/6 mice showed increased survival comparedo Ifn�−/− mice (Fig. 6A and B). C57BL/6 mice survival analy-is showed no differences in survival between WT and �pepNnfected mice (50% survival ∼110 ± 12 h), whereas infection with

pepN/pepN and �pepN/E298A increased survival (50% survival140 ± 12 h). Ifn�−/− mice showed early onset of death compared

o C57BL/6 mice. However, no differences were observed in sur-ival of Ifn�−/− mice (50% survival ∼100 ± 12 h) upon infection withifferent strains of S. typhimurium.

iscussion

The cytokine IFN� plays key roles in host immunity (Sahat al. 2010) and serum amounts of IFN� and IL12 are increasedn patients with Salmonellosis (Mizuno et al. 2003). The expres-ion of IFN� in intestines increases with age, which correlatesith better anti-microbial responses in adults compared to youngups (Rhee et al. 2005). There are several mechanisms by which

FN� increases host resistance against Salmonella: increasing thenternalization efficacy of macrophages, early intracellular killingollowed by cytokine production, decreasing the availability of ironrom Salmonella, increasing the production of anti-microbial pep-ides, nitric oxide etc. (Bao et al. 2000; Gordon et al. 2005; Nairzt al. 2008; Raupach et al. 2003; Rhee et al. 2005). Ifn�−/− miceemonstrate increased susceptibility to Salmonella with greater

acterial dissemination and increase in tissue damage lesions (Baot al. 2000; Raupach et al. 2003; Rhee et al. 2005). Interestingly,he requirement of IFN� and TNF� varies depending on the typef Salmonella mutant and the route (systemic or oral) of infection

monitored. The data is representative of three independent experiments. Student’snificant, *indicates P < 0.05, **indicates P < 0.01 and ***indicates P < 0.001.

(Raupach et al. 2003). The roles of S. typhimurium PepN during infec-tion were investigated in the current study. In comparison withC57BL/6, high basal CFU were observed in Ifn�−/− mice; conse-quently, the fold modulation of CFU by �pepN was comparativelyless (Fig. 3). The serum amounts of four cytokines analyzed, e.g.IFN�, TNF�, IL6 and IL1�, were increased upon S. typhimuriuminfection. Although differences were observed with respect to CFUbetween WT and �pepN, only IL1� amounts correlated with CFUin C57BL/6 mice. The lowered serum amounts of IFN�, TNF�, IL6and IL1� upon infection with �pepN/pepN and �pepN/E298A weremost likely due to lower CFU (Fig. 4). This finding is consistent witha study that showed that C57BL/6 mice (low bacterial clearance)produce high amounts of IFN� and IL10 whereas CBA mice (highbacterial clearance) produce less amounts of these cytokines (Pieet al. 1997).

Enhanced infiltration of neutrophils and eosinophils at infectionsites and lower amounts of IL12p40 but higher IL4 and IL5 by spleniccells are observed in Ifn�−/− mice post infection with T. gondii(Scharton-Kersten et al. 1996). Also, IL12 amounts are reduced inIfn�−/− mice upon infection with C. pneumoniae (Rottenberg et al.2000). However, in a human patient with mutation in IFNGR2, PHA-induced IFN� was greatly reduced whereas TNF� was not affected(Dorman and Holland 1998). Our studies with Ifn�−/− mice haveshown the clear role of IFN� in enhancing the amounts of TNF� andIL1� (Fig. 4). Therefore, the increased amounts of TNF� and IL1� in

C57BL/6 mice appear to be indicative of a robust host response andare consistent with the roles of IFN� in enhancing host immunity.

Due to the lowered host response, attenuated strains of T. gondii(Scharton-Kersten et al. 1996) or S. typhimurium (Bao et al. 2000)

M. Bhosale et al. / Immunobiology 217 (2012) 354– 362 359

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Fig. 4. Reduction of serum amounts of TNF� and IL1� upon infection is observed in Ifn�−/− mice. Mice were infected with ∼0.5–1 × 103 CFU of indicated strains of S.t amoue P < 0.0a

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nd ***indicates P < 0.001.

re virulent in Ifn�−/− mice. However, attenuated Listeria generatesntigen-specific CD8+ T cell responses that are capable of protectingaïve hosts (Harty and Bevan 1995), demonstrating the genera-ion of protective T cell responses in the absence of IFN�. In thistudy, all strains irrespective of pepN expression caused tissue dam-ge lesions in Ifn�−/− mice (Fig. 5). Most likely, the lower immuneefense network due to lack of IFN� led to increase in CFU (Fig. 3)nd greater tissue damage (Fig. 5), leading to reduced mice survivalFig. 6A, B). Consequently, the differential pepN expression depen-ent survival pattern observed in C57BL/6 mice was not observed

n Ifn�−/− mice. These results clearly demonstrate the key role of

ndogenous IFN� in mediating host resistance to Salmonella strainsith lowered virulence, e.g. �pepN/pepN and �pepN/E298A.

Although the roles of several pathogen encoded proteases andeptidases in the infection process are well known, the roles of

nts were quantified using ELISA. Data shown is representative of three different5 was considered as statistically significant, *indicates P < 0.05, **indicates P < 0.01

their catalytic activity has not been properly investigated. Candidaalbicans encoded secreted aspartate proteases are well known viru-lence factors that play several roles (Naglik et al. 2003). Pepstatin A,the aspartate protease inhibitor, lowers the invasion of C. albicanswith intestinal epithelial cells (Naglik et al. 2008). However, NF�Bupregulation and production of cytokines occurs in the presence ofPepstatin A, demonstrating that the induction of the inflammatoryresponse is independent of Pepstatin A (Pietrella et al. 2010). Also,the catalytic activity of proteasomes is required for some microbialfunctions: First, culture of T. acidophilum together with proteasomeinhibitors reduces growth during stress, e.g. heat shock (Ruepp

et al. 1998). Second, the M. tuberculosis encoded proteasomal path-way is required for optimal growth and resistance to nitric oxidein vitro and during in vivo infection (Darwin et al. 2003; Gandotraet al. 2010; Lamichhane et al. 2006). Interestingly, the survival of M.

360 M. Bhosale et al. / Immunobiology 217 (2012) 354– 362

Fig. 5. Increase in organ damage is observed upon infection of Ifn�−/− mice. Mice (∼8) were infected with indicated strains of S. typhimurium with ∼0.5 × 103 CFU, livertissue from infected and uninfected mice were stained with haematoxylin and eosin. Black arrows indicate necrotic foci whereas black triangles indicate inflammatory foci,magnification is ×200.

B A C57BL/6 Ifnγ-/-

0 50 100 150 200 2500

20

40

60

80

100

Time (h)

% S

urvi

val

WT/pBR322

ΔpepN/pBR322ΔpepN /pepNΔpepN/E298A

% S

urvi

val

0 50 100 150 200 2500

20

40

60

80

100

Time (h)

Fig. 6. Ifn�−/− mice showed reduced survival upon infection with S. typhimurium. Mice (∼8) were infected with indicated strains of S. typhimurium with ∼0.5 × 103 CFU,survival C57BL/6 (A) and Ifn�−/− (B) upon infection was monitored for every 12 h. P < 0.05 was considered as statistically significant. Data is representative of two independentexperiments.

obiolo

tatntirbamaFstiHiSTmsw

rgbebci(2paatoimvre2priiicwi

A

hfsG

A

t

M. Bhosale et al. / Immun

uberculosis during stationary phase growth in Middlebrook mediand long term persistence in lungs of infected mice requires pro-easomal activity. However, catalytic activity of the proteasome isot required for optimal growth in Middlebrook media, resistanceo nitric oxide mediated stress and initial growth of M. tuberculosisn lungs of infected mice (Gandotra et al. 2010). To investigate theoles of catalytic activity of PepN, the E298A mutant was generatedased on the multiple sequence alignment (Supplementary Fig. 1)nd the crystal structure (Addlagatta et al. 2006; Ito et al. 2006). Thisutant demonstrated complete loss in catalytic activity (Fig. 1B)

lthough there were no major structural changes (Supplementaryig. 3C, Fig. 3D). During nutritional downshift and high temperaturetress, high amounts of denatured proteins need to be degraded andhe recycled amino acids are used for growth. Here, the role of PepNs manifested and catalytic activity of PepN was required (Fig. 1C).owever, PepN lowers growth of E. coli during sodium salicylate-

nduced stress (Chandu and Nandi 2003) and lowers CFU during. typhimurium infection of mice during the later stage (Fig. 3B).his study clearly shows that the lowered CFU upon infection isediated by PepN but is independent of catalytic ability. Further

tudies are in progress to better understand the mechanisms byhich PepN modulates systemic infection.

It is possible that proteases and peptidases may play specializedoles under some conditions that are required for infection androwth in some hosts. The ProA/MspA metalloprotease secretedy Legionella pneumophile, the causative agent of Legionnaire’s dis-ase, is required for infection of Hartmannella vermiformis amoebaut not for infection of human macrophages or Acanthamoebaastellanii amoeba (Rossier et al. 2008). PepN and other M1 fam-ly members play specialized roles during some stress conditionsCaprioglio et al. 1993; Chandu and Nandi 2003; Kumar et al.009; Kumar and Nandi 2007) and infection (Patil et al. 2007). Theresent study clearly demonstrates the requirements of catalyticctivity of PepN during nutritional downshift and high temper-ture stress but not during systemic infection of mice with S.yphimurium. The differences in the host response to various strainsf S. typhimurium expressing differential amounts of PepN aremportant and highlight the robust roles of endogenous IFN� in

odulating serum cytokine amounts, CFU, organ damage and sur-ival during infection. Although previous studies have shown theole of IFN� during Salmonella infection (Bao et al. 2000; Gordont al. 2005; Nairz et al. 2008; Raupach et al. 2003; Rhee et al.005), the current study is a comprehensive analysis of multi-le aspects of the host response that are regulated by IFN� inesponse to infection by different strains of S. typhimurium exhibit-ng varying degrees of virulence. This aspect is important especiallyn understanding host response to possible vaccine strains inmmunocompromised patients. Overall, further studies on the spe-ialized roles of proteases and peptidases in modulating infectionill enrich and add a novel dimension to the area of host-pathogen

nteractions.

cknowledgements

We thank Prof. R. Manjunath, Prof. P. Sadhale and Prof. R. Med-amurthy for encouragement and incisive suggestions. The support

rom all members of the DpN laboratory is greatly appreciated. Thistudy was funded by a grant from the Department of Biotechnology,overnment of India.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.imbio.2011.07.010.

gy 217 (2012) 354– 362 361

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