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Microbes and Infection xx (2008) 1e9www.elsevier.com/locate/micinf

Original article

Transcriptional analysis of diverse strains Mycobacterium aviumsubspecies paratuberculosis in primary bovine monocyte

derived macrophages

Xiaochun Zhu a, Zheng J. Tu b, Paul M. Coussens c, Vivek Kapur d,e, Harish Janagama e,Saleh Naser f, Srinand Sreevatsan a,e,*

a Veterinary Population Medicine, University of Minnesota, 1365 Gortner Avenue, 225 VMC, Saint Paul, MN 55108, USAb Minnesota Super Computing Institute, University of Minnesota, Minneapolis, 117 Pleasant St. SE, Minneapolis, MN 55455, USA

c Department of Animal Sciences, Michigan State University, East Lansing, MI 48824, USAd Department of Microbiology, University of Minnesota, 1971 Commonwealth Avenue, Rm 330, Saint Paul, MN 55108, USA

e Veterinary Biomedical Sciences Department, University of Minnesota, 1971 Commonwealth Avenue, Rm 330, Saint Paul, MN 55108, USAf Department of Molecular Biology and Microbiology, University of Central Florida, 4000 Central Florida Boulevard, Orlando, FL 32816, USA

Received 28 December 2007; accepted 11 July 2008

Abstract

In this study we analyzed the macrophage-induced gene expression of three diverse genotypes of Mycobacterium avium subsp. para-tuberculosis (MAP). Using selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human,and sheep exposed to primary bovine monocyte derived macrophages for 48 h and 120 h we created and sequenced six cDNA libraries. Sequenceannotations revealed that the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolateup-regulated 35 and 358 genes, at the two time points respectively. Thirteen to thirty-three percent of the genes identified did not have anyannotated function. Despite variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred bypathway analysis. For example, 10e12% of the genes expressed by all three strains at each time point were associated with cell-wallbiosynthesis. All three strains of MAP studied up-regulated genes in pathways that combat oxidative stress, metabolic and nutritional starvation,and cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes respond with similar modusoperandi for survival in the host.� 2008 Elsevier Masson SAS. All rights reserved.

Keywords: Mycobacterium avium subspecies paratuberculosis; SCOTS; Gene expression; macrophage; Virulence

1. Introduction

Mycobacterium avium subspecies paratuberculosis (MAP)is the causative organism of Johne’s disease (JD), a debili-tating chronic gastroenteritis in ruminants [1,2]. Crohn’sdisease (CD) is also a chronic inflammation of distal intes-tines and exhibits pathology similar to that of JD in ruminants.

* Corresponding author. Veterinary Population Medicine Department,

College of Veterinary Medicine, University of Minnesota, 1365 Gortner

Avenue, 225 VMC, St. Paul, MN 55108, USA. Tel.: þ1 612 625 3769.

E-mail address: sreev001@umn.edu (S. Sreevatsan).

1286-4579/$ - see front matter � 2008 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.micinf.2008.07.025

Please cite this article in press as: X. Zhu et al., Transcriptional analysis of diverse

monocyte derived macrophages, Microb Infect (2008), doi:10.1016/j.micinf.2008

Several studies have associated MAP with a proportion of CDcases [3,4]. Although strain sharing has been documented [5],the evidence for a link remains controversial as a causal roleof MAP has not been demonstrated [6,7]. In a recent study weshowed that MAP strains isolated from CD patients wereclustered with strains derived from animals with Johne’sdisease, suggesting possible inter- and intra-species trans-mission and the association of human disease and strainssharing with animals [5]. Viable MAP has also been isolatedfrom commercial pasteurized milk [8,9] and these carry thecommon 7G and 4 or 5 GGT short sequence repeats geno-types (Sreevatsan, unpublished)dan overlap in the strains

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present in the human milk supply and those identified in CDpatients.

JD is now recognized to be of serious economic and animalhealth consequences in domesticated ruminant species(primarily dairy and beef cattle, sheep and goats) throughoutthe world [2,10]. Thus there is a critical need to devisescience-based intervention strategies to control JD in domesticanimal populations.

Molecular subtyping studies on MAP [11,12] have shownthat MAP isolates are genetically diverse and an associationbetween MAP genotype and host species exists [13e15].Despite this level of understanding of MAP diversity,a comprehensive understanding of intracellular transcrip-tional profiles of MAP is still lacking. Understanding themodus operandi of MAP in a macrophage environment isexpected to reveal intracellular survival mechanisms of thisinsidious pathogen. Indeed, several studies on pathogen-genotype specific host response studies exist in the literature[16e21].

Selective capture of transcribed sequences (SCOTS) hasrecently led to the discovery and characterization of virulencefactors in bacterial pathogenesis. These include studies on M.tuberculosis [21,22], the two subspecies of Salmonella enter-ica: Salmonella typhimurium and S. typhi [23,24], M. avium[25], and Listeria monocytogenes [26]. While all these studiesaddressed questions related to gene expression among typestrains within a single bacterial species, no attempts have beenmade thus far to delineate transcriptional differences amongststrains within a subspecies. We here evaluated the bovinemonocyte derived macrophage associated gene expressionprofiles amongst genetically similar-epidemiologically diverseand genetically dissimilar strains of MAP derived from theUnited States.

2. Materials and methods

2.1. Bacterial isolates and bacterial cultures

Three MAP strains selected in this study have been wellcharacterized by short sequence repeats (SSR) typing usingtwo polymorphic (G and GGT) loci and by microarray anal-ysis for genome content differences [27]. The SSR genotypesand the hosts of origin for these isolates were: 1018 (SSRfinger-print: 7G, 4GGT; cattle isolate), MAP6 (SSR finger-print: 7G, 5GGT; human isolate) and 7565 (SSR finger-print:15G, 3GGT; sheep isolate). The cattle strain (7G, 4GGT)represents a common genotype amongst MAP in the UnitedStates. The human strain (7G, 5GGT) was isolated froma patient with CD and is genetically similar to cattle strain.Sheep isolate (15G, 3GGT) is distinct from cattle and humanstrains. The cattle and human strains studied fall under the C-type while the sheep strain represents the typical S-typeclassification identified by Whittington et al. [15].

All the MAP isolates were cultured as described[13,14,28]. A few MAP colonies of MAP were harvested fromMB7H9 agar plate and inoculated into MB7H9 broth sup-plemented with OADC enrichment medium and 2 mg/ml of

Please cite this article in press as: X. Zhu et al., Transcriptional analysis of diverse

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mycobactin J. Broth culture was incubated at 37 �C ona shaker set at 120 rpm for 4 days to achieve metabolicallyactively bacteria. Four-day-old cultures were vigorously vor-texed for 3 min and kept still to settle bacterial clumps for30 min at room temperature. Bacteria were collected fromsupernatants and re-suspended in 1� PBS (GIBCO, Invi-trogen Co., Carlsbad, CA) to obtain the optical density at600 nm (OD600) to determine the colony forming units(CFU) of bacteria using the formula: 0.3 at OD600 ¼ 109 CFU/ml based on a comparative analysis of OD600 to CFUs per-formed in our laboratory. Bacteria were used at a 20:1multiplicity of infection (MOI) in all infections. The choice ofMOI was based on a series of phagocytosis experiments usinga different bacterial load. The bacterial count that showed atleast 90% efficiency in phagocytosis in 2 h was selected for allmacrophage studies.

2.2. Macrophage cell lines

Monocyte derived macrophages (MDMs) were preparedfrom fresh peripheral blood mononuclear cells of a JD freecow as described [19,29,30].

2.3. Macrophage infections

Macrophage monolayers (w2 � 106 cells per 25-cm2 flask)were incubated with MAP bacilli (20 MOI) for 2 h at 37 �Cwith 5% CO2 in RPMI1640 with 2% autologous serum. Afterinfection the non-phagocytized bacteria were removed bywashing the monolayers with pre-warmed RPMI 1640 threetimes. The infected monolayers were maintained at 37 �C with5% CO2 in RPMI1640 medium containing 2% autologousserum until the cells were harvested for RNA extraction.Culture medium was changed every two days.

2.4. Genomic DNA extraction

Genomic DNA of MAP was isolated from Middlebrookbroth culture by mechanical lysis of mycobacterial cells asdescribed [31].

2.5. Extraction of RNA form MAP broth culture andMAP infected macrophage

Four milliliters of 4-day-old MAP culture was collected ina 2 ml sterile screw-cap microcentrifuge tube and 1 ml ofTRIzol reagent (Invitrogen, Life Technologies) and 0.6 ml ofsterile RNase-free 0.1 mm zirconium beads were added totube. The bacteria were homogenized in a Mini Bead-Beaterfor 4 min. Cell lysate was transferred to a 1.5 ml sterileRNase-free microcentrifuge tube. The following steps werefollowed the TRIzol reagent instructions. Similarly, infectedmacrophages were lysed in 1 ml TRIzol (25 cm2 culture flask).RNA was extracted as above. All total RNA samples weretreated with RNase-free DNase I (Ambion, Inc., Austin, TX)after extraction.

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2.6. cDNA synthesis

Three micrograms of total RNA isolated from broth cultureor infected macrophages was converted to first-strand cDNAand second strand cDNA as described [22]. Each cDNAmixture was amplified by the polymerase chain reaction (PCR)using the corresponding defined primers for each set of cDNAmixtures (Table 1.) with Hotmaster Taq DNA polymerase(Eppendorf, Westbury, NY) and the following thermal cyclerconditions: initial denaturation at 94 �C for 2 min followed by30 cycles of denaturation at 94 �C for15 s, annealing at 52 �Cfor 15 s, and extension at 65 �C for 40 s. A final extension wasperformed at 65 �C for 7 min.

2.7. Selective capture of transcribed sequences (SCOTS)

The SCOTS procedure used in the present study was per-formed as described [22,25] with some modifications.Genomic DNA of MAP was adjusted to concentration of1 mg/ml (1 mg/ml) in TE buffer (10 mM Tris, 1 mM EDTA, pH7.4). The DNA samples were denatured by boiling for 5 minfollowed by a quick chill on ice for 1 min. The Psoralen-PEO-Biotin (Pierce Chemical Co., Rockford, IL) stock solution(13.8 mg/ml) was added to DNA samples in dilution of 1:100.Mixtures were kept on ice and irradiated under a long wave-length UV source (365 nm) 1e10 cm for 15 min. The bio-tinylated DNA was purified from excess biotinylation reagentby precipitation with 0.1 volume of 4 M NaCl and 1 volume ofisopropanol. After centrifugation, the DNA pellet was washedwith 70% ethanol, air-dried and re-dissolved in TE buffer.Labeling was repeated three times.

Plasmid DNA, pYA1401 (kindly gifted by Dr. JosephineClark-Curtiss), containing the ribosomal operon of M. aviumand Biotinylated DNA were sheared to 1w2 kb by sonication.Sheared DNA samples were mixed in ratio of 0.6 mg of MAPDNA and 5 mg of pYA1401 DNA in 8 ml of 10 mM N-(2-hydroxyethyl) piperazine-N-(3-propanesulfonic acid) (EPPS)e1 mM EDTA.

Hybridizations were performed as described [22,25] using30 mg of Dynalbeads M270 streptavidin (Invitrogen) followingthe manufacturer’s instruction. The bacterial cDNA-genomicDNA hybrids were separated from hybridization mixture bybinding to 30 mg of Dynalbeads M270 streptavidin in 1 ml of1� Binding & Washing buffer (5 mM pH 7.5 TriseHCl,0.5 mM EDTA, 1 M NaCl). The beads were washed and re-suspended in 100 ml of sterile water. These beads were boiled

Table 1

List of primers used in selective capture of transcribed sequences

Primer identity Sequence (50e30) Pu

SCOT09 ATC CAC CTA TCC CAG TAG GAG NNN NNN NNN cD

SCOT0 ATC CAC CTA TCC CAG TAG GAG PC

SCOT189 GAC AGA TCC GCA CTT AAC CCT NNN NNN NNN cD

SCOT18 GAC AGA TCC GCA CTT AAC CCT PC

SCOT1109 ATG CGA ATC CAG ACT GTA AGA NNN NNN NNN cD

SCOT110 ATG CGA ATC CAG ACT GTA AGA PC

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at 100 �C for 5 min and chilled on ice and the supernatant wasamplified by PCR as described above.

In the first iteration of SCOTS, five separate hybridizationreactions were set up for each growth condition in order toenhance the possibility of capture of cDNA molecules corre-sponding to a more complete diversity of transcripts present atthe time of RNA preparation for each growth condition. Thenthese five cDNA preparations were combined after separationby Dynalbeads M270 and applied to next round of SCOTS.Three iterations of SCOTS were performed for cDNA mixturefrom each growth condition.

2.8. Enrichment for cDNA of M. paratuberculosis frominfected macrophages and creation of cDNA libraries

This step was used to enrich the bacterial genes preferen-tially transcribed or up-regulated in macrophages. Thiscompetitive hybridization enrichment strategy was performedas described [22,25].

2.9. Synthesis of cDNA probes

Digoxigenin-labeled cDNA probes were generated fromcDNA mixtures represented each growth condition after threerounds of SCOTS or enrichment when PCR amplification wasdone using digoxigenin-11-dUTP (Roche Applied Science)/dNTP mixture (digoxigenin-11-dUTP:dTTP ¼ 7:13) and cor-responding primers (Table 1).

2.10. Dot-blot hybridization to screen for up-regulatedgenes

Five microliters of PCR amplicon of each white colony wasadded to 50 ml of 6� SSC in a 96-well PCR plate, denatured at98 �C for 5 min and quickly chilled on ice. Samples weretransferred to Nytran SPC 0.45 Nylon Transfer Membrane(Whatman, Inc., Sanford, ME) using Minifold Spot Blotsystem (Whatman Inc.). Each membrane was cross hybridizedwith digoxigenin-probe generated from broth culture cDNAafter three rounds of SCOTS or cDNA of corresponding timepoint of infected macrophages after three rounds of enrich-ment. The membranes were prehybridized with DIG Easy Hyb(Roche, Indianapolis, IN) at 45 �C for 30 min and thendenatured probes were added. Hybridization was continued inDIG Easy Hyb (Roche, Indianapolis, IN) at 45 �C for 12e16 h. The membranes were washed twice with 2� SSCe0.1%

rpose

NA synthesis for total RNA from MAP broth culture

R amplification of cDNA of broth culture

NA synthesis for total RNA from infected macrophages 48 h post infection

R amplification of cDNA of 48-h time point

NA synthesis for total RNA from infected macrophages 120 h post infection

R amplification of cDNA of 120-h time point

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SDS at room temperature for 5 min and then twice with 0.1�SSCe0.1% SDS at 55 �C for 15 min each time. Themembranes were washed in 1� PBSeTween 20 and thenblocked in a casein blocking buffer (Blocker in Casein in PBS,Pierce Biotechnology, Rockford, IL) at 37 �C for 1 h. Anti-digoxigenin-POD conjugate were added in blocking buffer andincubated at 37 �C for 1 h. Then the membranes were washedin 1� PBSeTween 20 twice at room temperature for 15 minand three times for 5 min. Chemiluminescent signals weredetected after applying 1:1 mixture of Stable Peroxide Solu-tion and Luminol/Enhancer Solution (Pierce Inc.) according tothe manufacturer’s instructions. Clones showing strong signalwhen hybridized with probes of infected macrophages and notthe probes from broth culture were considered as up-regulatedgenes and selected for sequencing.

2.11. Estimation of coverage of cDNA libraries

All sequences were analyzed against M. paratuberculosisK-10 genome to obtain their gene identity. The redundancy ofeach gene was counted cumulatively and the library coveragewas calculated using Analytic Rarefaction 1.3 (http://www.uga.edu/strata/software/Software.html) [32,33]. Coveragevalues were calculated by equation C ¼ [1 � (n/N )] � 100,where n is the estimated species of genes and N is the numberof colony counts.

2.12. Sequence analysis and annotation of gene function

Fig. 1. (A, B) Rarefaction analysis curves demonstrating coverage of cDNA

libraries of 1018, MAP6 and 7565 isolates at 48 h time point (A) and 120 h

post infection (B). Shown are library coverage for the bovine (1018) isolate

All sequences were annotated against genomes of M. par-atuberculosis K-10, M. avium 104, M. bovis AF2122/97, M.leprae TN, M. tuberculosis CDC1551 and M. tuberculosisH37Rv available in NCBI database.

(blue lines); the human (MAP6) isolate (Pink lines); and the sheep (7565)

isolate (red lines).

2.13. Real-time RTePCR analysis to evaluatedconsistency of gene expression

MDMs derived from a second JD-free cow were infectedby MAP isolates 1018, MAP6 and 7565. RNA was extracted at48 h and 120 h post infection using TRIzol reagent asdescribed above. Eight common up-regulated genes identifiedin SCOTS were confirmed by real-time PCR in ABI 7500system. Bacterial broth cultures were used as base line andhsp65 was used as a house-keeping gene. The expression levelwas calculated by the 2�DDCt method [34].

3. Results

3.1. Estimates of diversity and coverage of cDNAlibraries

Six cDNA libraries from three isolates representing two timepoints (48 h and 120 h post infection) were established. Rare-faction analysis indicated that saturation was achieved for allthree 48-h cDNA libraries showing 82% coverage for the bovineisolate, 83% for MAP6 and 78% for 7565 (Fig. 1A); at 120 hMAP6 showed 82% coverage, while 62% coverage was

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achieved for 1018 and 7565 cDNA libraries (Fig. 1B), indicatingthat most of representative genes in each library were identified.

3.2. Analysis of overall gene expression profiles

The number of genes identified in the six cDNA librariesand the numbers of commonly expressed genes across isolatesor the two time points are shown in Fig. 2. We identified 27, 22,and 35 genes in bovine, human, and sheep isolates, respectivelyat 48 h post infection. At 120 h post infection, a total of 241,53, and 358 genes were up-regulated in the bovine, human, andsheep isolates, respectively (Fig. 2A and B). Genes wereclassified into the following major categories adapted from thegenome annotation of M. tuberculosis H37Rv [35]: small-molecule degradation, energy metabolism, amino acidbiosynthesis, lipid biosynthesis, broad regulatory functions,synthesis and modification of macromolecules, cell envelope,transport/binding proteins, virulence, IS (insertion sequence)element/repeat sequences/phage, antibiotic production andresistance, conserved hypothetical proteins, unknown unique toMAP, and other miscellaneous genes. Functional classifications

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Fig. 2. Shown are the numbers of unique and commonly up-regulated genes in by each isolate at 48 h (A) and 120 h (B) post infection. Also shown in lower panes

C and D are genes sharing either the cattle or the sheep isolate across the two post-infection time points. The human isolate did not show a shared expression

pattern over time.

Table 2

Up regulated genes shared by three isolates

Name Putative function

Group 1: 48 h 1018/M6/7565 common genes

MAP0065 Probable conserved transmembrane protein

MAP1352 Translation initiation factor InfC

MAP2838c FadE20_2: acyl-CoA dehydrogenase

MAP4041c low-affinity inorganic phosphate transporter PitA

MAP4270 Mpr protein:DNA interaction

MAP4281 Transposase

Group 2: 120 h 1018/M6/7565 common genes

MAP0854 Hypothetical protein

MAP0855 Hypothetical protein similar to DNA repair

protein RADA

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in each cDNA library are summarized in pie charts (Supple-mentary figures S1eS6). The detailed functional classificationand the pathways in which they are involved are presented insupplemental data (Supplementary table S1).

The human isolate (MAP6) up-regulated fewer genes andshowed no common genes across time points or in comparisonto profiles identified in the other two organisms. In the cattleisolate (1018) four up-regulated genes were shared between48 h and 120 h post infection while six genes were sharedacross the two time points in the sheep strain. The numbersand identities of up-regulated genes, by 1018 and MAP6 at48 h post infection were similar.

MAP4041c and MAP4281 were up-regulated by all threeMAP strains and participated in transport/binding protein andIS element/repetitive sequences/phage during early infection(Table 2). At 120 h post infection relatively large number ofgenes were up-regulated by the cattle and sheep strains. Ashared expression pattern was identified amongst the threegenetically distinct isolates at 48 h post infection and theseincluded members involved in translation (infC ), lipid degra-dation ( fadE20_2), membrane transportation ( pitA), DNArepair (radA, MAP3760c (probable methyltransferase gene)),virulence (mpr), transposase (MAP4281). At 120 h post infec-tion most of shared genes were unknown function (Table 2).

MAP1064 Hypothetical protein

MAP1065 Hypothetical protein

MAP1718c Hypothetical protein

MAP1821c Hypothetical protein

MAP3167c Hypothetical protein

MAP3467c Hypothetical protein

MAP3759c Probable transposase

MAP3760c Probable methyltransferase

3.3. Macrophage induced up-regulated genes classifiedby function

3.3.1. Small-molecule degradationWe identified 4 and 13 genes that participated in small-

molecule degradation to be up-regulated in 1018 at 48 and

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120 h post infection; three genes in both MAP6-48/120hcDNA libraries; 3 and 16 genes in 7565-48h and 7565-120hcDNA libraries, respectively were also identified in this cate-gory. Most of these shared genes were predicted to play a rolein fatty acid degradation except one that was involved incarbon compound degradation (1018-48, MAP2696c); onegene (1018-48, MAP3022) relevant to phosphorous compounddegradation; one gene (MAP0744c) involved in amino acidand amine degradation was identified both in cattle and sheep

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isolate derived cDNA libraries at 120 h post infection. Whilethere were more genes up-regulated at 120 h than 48 h postinfection in cattle and sheep isolates, only one gene(MAP2838c) in this category was shared by the isolates at48 h post infection.

3.3.2. Energy metabolism, oxidative stress, iron metabolismThe tricarboxylic acid cycle (TCA) is a common pathway

in oxidation of carbohydrate, fatty acids and proteins ineukaryotes and prokaryotes. Most of the 13 up-regulated TCAgenes were identified exclusively in the sheep isolate at 120 hpost infection and one at 48 h post infection. Three genesinvolved in electron transport were identified in the sheepisolate at 120 h post infection. We identified several oxidore-ductases in the sheep isolate at 120 h post infection. Oneoxidoreductase gene at the same time post infection wasidentified in the human isolate. Superoxide dismutase (sodA)was up-regulated at 48 h post infection by sheep and humanisolates. Other genes involved in reactive oxygen detoxifica-tion were also up-regulated and included: MAP2329 (bacter-ioferritin comigratory protein Bcp, 1018-120), MAP1589c(alkyl hydroperoxide reductase AhpC, 7565-120) andMAP2667c (epoxide hydrolase EphC, 7565-120). Althoughthere were no oxidoreductase genes up-regulated in the cattleisolate, this isolate expressed monooxygenase (MAP0180,MAP1620) and dioxygenase (MAP0720c, MAP3141c) familyof proteins that serve the same function.

Two iron acquisition genes, MAP3742 (probable mbtB) andMAP1872c (mbtH_2) were up-regulated in the cattle andsheep isolates at 120 h post infection. However, no ironregulatory genes were identified as up-regulated by the humanisolate (MAP6).

3.3.3. Cell envelope proteinsA high proportion of genes associated with biosynthesis of

the components of cell envelope were identified among threeMAP isolates across time points; up to 9% of the genesidentified for all strains and all time points (Figs. S1eS6). Weobserved that 7 mmpL (major membrane protein Large) familygenes were up-regulated in the 120-h post infection libraries ofcattle and sheep isolates; one mmpL gene was identified in thehuman isolate at 120 h. One mmpS gene (MAP1241c) wasidentified in the 120-h post infection libraries of cattle andsheep isolates. A different gene in the mmpS family(MAP2238) was up-regulated in the human isolate at 48 h postinfection. Other conserved transmembrane proteins were alsoidentified to be up-regulated in each library.

3.3.4. Amino acid metabolismSCOTS identified some genes involved in amino acid

biosynthesis, such as glutamate family, aspartate family, serinefamily, aromatic amino acid family, and pyruvate family. Mostgenes were identified in the late time post infection points ofcattle and sheep isolates. MAP0414c was also identified at48 h post infection in the cattle isolate. No genes in thiscategory were identified in the human isolate at both timepoints post infection.

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3.3.5. DNA damage repair and replicationThe gene (radA) encoding DNA repair protein RadA was

up-regulated in the 120-h post infection libraries of cattle andsheep isolates. Several genes including dnaB (MAP0071) anddnaE1 (MAP1257) were identified in the 120-h post infectionlibraries of cattle and sheep isolates. MAP1257 (a probableATP dependent DNA ligase) (MAP0855) and MAP1335(uvrB) were identified in the human and sheep isolates at 120 hpost infection.

3.3.6. Immunological and virulence related genesImmunological and virulence related genes include mce

(mammalian cell entry) families, PE/PPE families, and othergenes, such as mpr and yrbE1B. In the present investigation,the human isolate revealed MAP1507 (PE_4) and MAP1506(probable PPE family) at 48 h post infection while PE/PPEgenes are up-regulated in cattle and sheep isolates at late timepoints post infection. These genes included: MAP1003c (PE),MAP1507 (PE_4), MAP1506 (PPE), MAP3725 (PPE),MAP3782 (PPE) and MAP3765 (PPE). MAP3604 (mce1_2)was up-regulated in the sheep isolate at 120 h post infection.Other probable mce family proteins are identified in cattle andsheep isolates post infection. No mce gene is observed to beup-regulated in the human isolate derived libraries.

3.4. Confirmation and consistency of up-regulated genesacross hosts using real time RTePCR

In order to confirm the results of SCOTS eight genesidentified in all three strains at 48 h or 120 h post infectionwere evaluated using quantitative real time RTePCR. Theresults showed that all eight genes were over-expressed by allthree isolates within bovine MDMs at both post infection timepoints. We noted that MAP0853, MAP0854, MAP1236c andMAP3167 were highly up-regulated by 1018 in both MDMsfrom two different cows at 48 h and 120 h post infection.

4. Discussion

MAP is an intracellular pathogen, which survives andpersists within macrophages. While it is well established thatMAP are a diverse group of organisms exhibiting a variety ofgenotypes and microbiological phenotypes, a comprehensiveanalysis that provides a functional attribution of this variationdoes not exist to date. In the absence of an adequate animalmodel, identification of MAP genes expressed within primarymonocyte derived macrophages is expected to provide a thor-ough understanding of the mechanisms and pathways used byMAP to survive within its hosts.

Previous studies have classified MAP strains into S strain,including predominantly sheep isolates, and C strains,including cattle, goat, deer and human isolates [11]. The truegenetic diversity of MAP isolates by short sequence repeat(SSR) [13,14] and strain dependent variation in host responseto MAP strains [19,27] have led to the consideration that theremay be strain dependent variation in functional gene expres-sion of MAP during interaction with their hosts.

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Transcriptional signatures of diverse MAP strains in thebovine macrophage environment suggest common themes inpathogenesis within the subspecies.

Gene expression profiles of M. tuberculosis within murinebone marrow-derived macrophages have revealed that theenvironment within macrophages is fatty acid rich, DNA andcell wall damaging, and iron deficient [36]. cDNA subtractivehybridization on MAA infected human macrophages revealedthat acyl coenzyme A (acyl-CoA) synthetase was up-regulated[37,38]. SCOTS analysis of MAA did not detect any fatty acidmetabolism related genes. One possible reason may be that thecDNA libraries were not screened to saturation [25]. Regard-less, direct comparisons of the gene expression profiles reportedby Hou et al. [23] and our data set identified several similaritiesin that we also report detection of transcripts belonging tomycobactin synthesis (mbtF), polyketide synthases (Rv1665,Rv1660), cobalamin biosynthesis (rv2062c), lipoprotein(Rv1970), PPE family (Rv1802, Rv0453), mce family(Rv3496c), Isocitrate dehydrogenase (Rv0066c), transcriptionalregulators (Rv0474), transposases (IS1311) and metal-lopeptidase (Rv3610c) as those reported for MAA inside humanmacrophages using SCOTS [23]. Furthermore our study iden-tified several genes involved in fatty acid degradation up-regulated in all three MAP isolates which is consistent with thecontention that regardless of the infecting genotypes, cell-walllipid synthesis and maintenance of cellular integrity is critical totheir survival and persistence inside the macrophages. Takentogether, fatty acid metabolism pathway may be one of commonthemes used by pathogenic mycobacteria during effacement,entry and growth within a macrophage environment.

Akin to other pathogenic mycobacteria MAP resist phag-osome-lysosome fusion [39,40]. Thus the ability to surviveunder oxidative stress is an important aspect of pathogenesisutilized by mycobacteria is considered as a virulence factor.The up-regulation of genes involved in combating oxidativestress and DNA damage and repair, at early times postinfection are consistent with this notion.

M. tuberculosis undergoes an iron-limiting environmentduring infection leading to up-regulation of genes required forsiderophore biosynthesis [41,42]. In our study, MAP3742(probable mbtB) and MAP1872c (mbtH_2) were up-regulatedin the cow and sheep isolates and polyketide synthase geneswere identified in all three strains. These findings suggest thatiron metabolism via the mycobactin biosynthesis pathway iscommonly used by MAP similar to several other mycobacteriawhen inside macrophages.

Since a larger proportion (9%) of envelope biosynthesisassociated genes were up-regulated, we infer that MAPundergoes changes of cell envelope composition duringgrowth in the macrophage. Most mmpL genes found in M.tuberculosis and other mycobacteria are related to the resis-tance-nodulation-division (RND) family involved in trans-portation of drugs, fatty acids, detergents, and dyes [43e45]and are thought to play a role in cell wall biogenesis, virulenceand other phenotypic characteristics. The identification ofmmpL family and other membrane protein genes expressed bythe three MAP strains suggested that up-regulation of envelope

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monocyte derived macrophages, Microb Infect (2008), doi:10.1016/j.micinf.2008

biosynthesis genes is a common theme for MAP survival inbovine macrophages.

Up-regulation of amino acid biosynthesis and degradationgenes suggests that the macrophage environment is alsonutrient deficient. Interestingly, all these amino acid metabo-lism genes were identified in 1018 and 7565 libraries and mostof these genes were up-regulated during late infection, sug-gesting that MAP responds aggressively to nutrient starvationin vivo.

We identified several putative virulence factors includingPE/PPE family and mce family genes in all MAP strainssuggesting that pathogenic mycobacteria may share commonvirulence pathways. PE/PPE proteins are thought to play animportant role in mycobacterial infection due to their specificdomains (Pro-Pro-Glu and Pro-Glu, respectively), whichcontain polymorphic repetitive sequences [46].

One paradigm in microbiology is that virulence factors areessential for pathogenesis and thus their use in vaccines wouldconfer protection against the infectious agent. Indeed, a recent[47] DNA vaccine study that included a cocktail of genesclassified as transport/binding protein, membrane proteins,virulence proteins and mycobactin/polyketide synthaseprovided protection in mice against MAP. Our results concurredin that all these genes were identified in response to bovinemacrophages and included membrane proteins (MAP1239c,MAP3049c and MAP3131), virulence proteins (MAP1003c),and mycobactin/polyketide synthase (MAP1796c, MAP1871c,MAP2230c, MAP3742 and MAP3764c) in 1018-120h and7565-120h libraries.

Genomic comparison of MAP and MAA reveals signaturesof a pathogen. Comparative genomics between MAP K10and MAA reveals that 136 MAP genes are absent from theMAA genome (http://cmr.tigr.org/tigr-scripts/CMR/shared/MakeFrontPages.cgi?page¼circular_display). Fifty of these136 genes were identified in our cDNA libraries among which,47 genes encode hypothetical proteins. Comparison of K-10genome against MAA, M. bovis AF2122/97, M. tuberculosisCDC1551, M. tuberculosis H37Rv, M. leprae TN, M. smeg-matis MC2 155 and Mycobacterium sp. MCS, 79 genes wereidentified as unique to MAP. Thirty-one of 79 MAP uniquegenes were up-regulated by all three strains studied. Thirty ofthese genes did not have any assigned functions. These MAP-unique genes may play an important role in MAP virulenceand pathogenesis. Further studies to understand the function ofthese unique genes up-regulated in the macrophage environ-ment are expected to lay strong scientific foundations inunderstanding JD pathogenesis.

Genomic comparison of sheep and cattle MAP strains hasrevealed three large deletions in the sheep strain [48]. One ofthese (‘‘deletion 2’’) deletions includes genes MAP1728ceMAP1744. Four of these genes (MAP1728 (YfnB), a predictedhydrolase; MAP1738 encoding MmpL5; and MAP1729c andMAP1730 classified as hypothetical proteins with unknownfunction) were up-regulated exclusively in our cattle strain at120 h post infection. Thus, these four genes could serve aspotential unique markers in the pathogenesis of JD caused bycattle strains.

strains Mycobacterium avium subspecies paratuberculosis in primary bovine

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During early infection (48 h post infection) all three MAPstrains expressed very few genes in bovine macrophages,consistent with the idea that MAP enter quietly and establishinfection in macrophages and become metabolically activewhen they begin replication. While the genes expressed byMAP isolates were not identical, the metabolic pathways thatthese sets of genes participated were common.

In summary, our study identified that several hypotheticalgenes unique to MAP were up-regulated by all three genotypesin a macrophage environment. While there were majorcommonalities between MAA and MAP genes expressed inmacrophages, identification of several genes unique to MAPsuggest a possible pathway(s) to the specialization of MAPinto a pathogen. We also showed that divergent strains ofMAP used common metabolic pathways for survival in bovinemacrophages. Further studies using specific deletion mutantsof genes/pathways identified in the current study and theircomplemented strains are required to start deciphering thefunctional genome of this agriculturally important pathogen.

Acknowledgments

This work was supported by a USDA-NRI grant (2005-35204-16106). We thank Dr. Josephine Clark-Curtiss forproviding pYA1401 plasmid used in these studies.

Appendix A. Supplementary material

Supplementary information for this manuscript can bedownloaded at doi: 10.1016/j.micinf.2008.07.025.

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