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Infection and Immunity, November 2006, p. 6046-6056, Vol. 74, No. 11
0019-9567/06/$08.00+0     doi:10.1128/IAI.00326-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Comparative Transcriptional Analysis of Human Macrophages Exposed to Animal and Human Isolates of Mycobacterium avium Subspecies paratuberculosis with Diverse Genotypes{dagger}

Alifiya S. Motiwala,1,{ddagger} Harish K. Janagama,2,{ddagger} Michael L. Paustian,4 Xiaochun Zhu,2 John P. Bannantine,4 Vivek Kapur,3 and Srinand Sreevatsan2*

Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey,1 Veterinary Population Medicine Department,2 Department of Microbiology and Center for Genomics, University of Minnesota, St. Paul, Minnesota,3 National Animal Disease Center, Agricultural Research Service, United State Department of Agriculture, Ames, Iowa4

Received 27 February 2006/ Returned for modification 11 April 2006/ Accepted 16 July 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in animals and has been hypothesized to be associated with Crohn's disease in humans. Recently, M. avium subsp. paratuberculosis isolates recovered from Crohn's disease patients were shown to have limited diversity, implying the existence of human disease-associated genotypes and strain sharing with animals (A. H. Ghadiali et al., J. Clin. Microbiol. 42:5345-5348, 2004). To explore whether these genotypic differences or similarities among human and animal isolates translated to functionally significant attributes such as variance in host preference and/or difference in magnitude of infections, we performed a global scale analysis of M. avium subsp. paratuberculosis isolates that were representative of different genotypes and host species using DNA microarrays. Genome-wide characterization of the transcriptional changes was carried out using a human monocytic cell line (THP-1 cells) in response to different genotypes of M. avium subsp. paratuberculosis isolates recovered from various hosts. We identified several differentially expressed genes during early intracellular infection, including those involved in common canonical pathways such as NF-{kappa}B, interleukin-6 (IL-6), mitogen-activated protein kinase/extracellular signal-regulated kinase, and Jun N-terminal protein kinase signaling, as well as genes involved in T helper type 1 (Th1) responses (such as CCL5 ligand) and those that encode several proinflammatory cytokines and chemokine receptors. The cattle and human isolates of M. avium subsp. paratuberculosis, regardless of their short sequence repeat (SSR) genotype, induced similar global gene expression patterns in THP-1 cells. They differentially regulated genes necessary for cell survival without causing major alterations in proinflammatory genes. In contrast, the sheep isolates representing diverse SSR genotypes closely resembled the global gene expression pattern of an M. avium subsp. avium isolate, and they significantly up-regulated proinflammatory genes related to IL-6, T-cell receptor, B-cell receptor, and death receptor signaling within THP-1 cells. Additionally, we demonstrated consistency among infecting genotypes of M. avium subsp. paratuberculosis isolated from diverse hosts [cattle (n = 2), human (n = 3), sheep (n = 2), and bison (n = 1)] in quantitative reverse transcription-PCR analysis of seven differentially expressed genes. While the levels of expression induced by the bison isolate were different compared with cattle or human isolates, they followed the common anti-inflammatory, antiapoptotic trend. Our data suggest that the macrophage responses to M. avium subsp. paratuberculosis isolates from cattle and human sources, regardless of genotype, follow a common theme of anti-inflammatory responses, an attribute likely associated with successful infection and persistence. However, these expression patterns differ significantly from those in THP-1 cells infected with sheep isolates of M. avium subsp. paratuberculosis or the M. avium subsp. avium isolate. These data provide a transcriptional basis for a variety of pathophysiological changes observed during early stages of infection by different strains of M. avium subsp. paratuberculosis, a first step in understanding trait-allele association in this economically important disease.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Johne's disease is a chronic intestinal inflammation caused by the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis. Johne's disease (JD) mainly affects ruminants such as cattle, sheep, and wildlife species including deer, bison, and elk (29). Thus, JD has serious economic and animal health consequences in domesticated ruminant and wild-life species worldwide. M. avium subsp. paratuberculosis is also of interest because of its possible involvement in human Crohn's disease, a chronic debilitating disease of unknown etiology that exhibits a pathology similar to JD (15, 18, 19, 45). Although several studies support the presence of M. avium subsp. paratuberculosis in the tissues of some Crohn's disease patients (5, 15, 21, 37, 41, 48-52), the evidence for a link remains inconclusive. Analysis of host cellular and molecular signaling in response to diverse genotypes of M. avium subsp. paratuberculosis is expected to provide key missing information in the understanding of the role of this pathogen in Crohn's disease.

The M. avium complex, which includes M. avium subsp. avium, M. avium subsp. paratuberculosis, Mycobacterium intracellulare, and Mycobacterium silvaticum, is characterized by over 90% similarity at the nucleotide level, but the organisms differ widely in terms of their host tropisms, microbiological and disease phenotypes, and pathogenicity. It is widely acknowledged that there are at least two phenotypically and genotypically distinct M. avium subsp. paratuberculosis strains associated with different disease characteristics (59). Most investigations have identified limited variation in M. avium subsp. paratuberculosis isolates from cattle and other ruminants (not including sheep strains) as well as in isolates from human Crohn's disease cases (6, 17).

However, recent analyses (1, 33, 35) of highly polymorphic repetitive loci (short sequence repeats, or SSRs) indicated that M. avium subsp. paratuberculosis is genotypically diverse. In the background of this diversity among animal isolates, the studies demonstrated that M. avium subsp. paratuberculosis strains from Crohn's disease patients had restricted allelic variation (17). The presence of only two alleles within the human isolates was interpreted to indicate that a few animal genotypes are associated with the pathobiology of Crohn's disease (17). The interpretation was based on the fact that JD in sheep is mostly caused by a distinct group of M. avium subsp. paratuberculosis strains which show minimal genome-wide variation. These studies suggested that specific genotypes may be associated with subclinical disease while others may lead to clinically overt JD in diverse hosts. In vitro analysis of M. avium subsp. paratuberculosis survival within primary bovine macrophage cells also showed clear differences in entry, survival, and persistence as a function of genotype (22). Thus, we hypothesized that host responses to divergent genotypes of M. avium subsp. paratuberculosis may be strongly associated with disease outcomes and persistence of specific genotypes within animal or human populations.

Host-pathogen interactions are a complex interplay between a host's defense mechanisms and the microorganism's attempts to circumvent these defenses (9-11). Recent studies using animal models and macrophages (primary cultures and cell lines) have demonstrated significant differences among clinical Mycobacterium tuberculosis isolates which were previously thought to exhibit little interstrain genotypic or phenotypic variation (3, 27, 28, 46, 47, 54, 55). Differential gene expression patterns have been demonstrated in mononuclear phagocytes infected with pathogenic and nonpathogenic mycobacteria (32). Inferring from these results and our own previous studies on M. avium subsp. paratuberculosis, we hypothesized that host responses to distinct genotypes of M. avium subsp. paratuberculosis may be strongly associated with disease outcomes and the persistence of specific genotypes within animal or human populations. To investigate our contention that differences in genome content and SSR fingerprint profiles would translate to functional and biomedically significant attributes (such as variance in host preference and/or difference in magnitude of infections), we performed a global-scale analysis of the expression profiles induced in a human monocytic cell line (THP-1 cells) in response to diverse M. avium subsp. paratuberculosis isolates using transcriptional analysis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. M. avium subsp. paratuberculosis isolates selected for the analysis have been previously characterized using several molecular markers (1, 33, 34) and were chosen to represent genotypes recovered from diverse host species and geographic locations (Table 1). One isolate each from a cow (1018), sheep (7565), and human (MAP4) source was used in transcriptional profiling. A well-characterized isolate of M. avium subsp. avium was included in the microarray analysis to enable comparison of the macrophage expression profiles in response to M. avium subsp. paratuberculosis and M. avium subsp. avium strains. Additionally one cattle (K-10), two human (MAP5 and MAP6), one sheep (5001), and one bison isolate (7560) were chosen to represent the genotypic diversity within each host species and applied in quantitative reverse transcription-PCR (RT-PCR) analysis of selected host genes to evaluate consistency in induced gene expression analysis. M. avium subsp. paratuberculosis strain K-10, a recently sequenced isolate recovered from a clinical case of JD was included in this analysis (25). The bison isolate was identical to the cattle isolate by SSR genotyping but was polymorphic in the IS1311 fingerprint.


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  		TABLE 1. Characteristics of isolates used in THP-1 cell stimulation and gene expression analysis

 
Whole-genome comparisons of M. avium subsp. paratuberculosis isolates. cDNA microarrays designed from the genome sequence of M. avium subsp. paratuberculosis K-10 were used to further define the gene content of the M. avium subsp. paratuberculosis isolates selected for host transcriptional analysis, which was performed as previously described (43). Briefly, purified genomic DNA was randomly sheared by nebulization to an average fragment size of 800 bp, and 4 µg was indirectly labeled with Alexa Fluor 555 or Alexa Fluor 647 succinimidyl ester dyes (Molecular Probes, Eugene, OR) using Klenow enzyme (USB, Cleveland, OH). Labeled cDNA from experimental mycobacterial isolates was mixed with alternately labeled M. avium subsp. paratuberculosis K-10 cDNA, applied to the M. avium subsp. paratuberculosis K-10 microarray, and allowed to hybridize overnight at 65°C. The arrays were washed sequentially for 3 min at room temperature and scanned with a ScanArray 4000 confocal laser scanner (Perkin Elmer, Fremont, CA). Each mycobacterial isolate was hybridized against M. avium subsp. paratuberculosis K-10 at least twice in a dye-flip experimental design.

Data transformation and analyses were performed as described previously (43) with minor modifications described here. Briefly, raw intensity measurements for each spot on the microarray were extracted from scanned images, background corrected, and LOWESS normalized. Poorly detected spots were removed, and any open reading frames (ORFs) not represented by at least two acceptable spots on a hybridized microarray were discarded from further analyses. Ratios of the spot intensities for experimental and control M. avium subsp. paratuberculosis K-10 DNA samples were calculated and log transformed for analysis. The software program Genomotyping Analysis (24) was utilized to grade the ORFs on a linear scale from –0.5 (0% estimated probability of being present) to 0.5 (100% estimated probability of being present). Grades from duplicated experiments were averaged, and ORFs were classified as divergent (average grade of <–0.325) or present (average grade of >0.325), with the remaining ORFs listed as having intermediate divergence. Results for these automated analyses were reported only if data were obtained from at least two hybridizations.

Macrophage cell line. The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (ATCC; Rockville, MD). Cell cultures were maintained in modified RPMI 1640 medium (ATCC, Rockville, MD) supplemented with 10% fetal bovine serum (ATCC, Rockville, MD) and antibiotic-antifungal mixture containing penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml) (Gibco-BRL, a division of Invitrogen Corp., Carlsbad, CA); cultures were incubated at 37°C in 5% CO2 in a humidified incubator. Cells were subcultured every third day, at an approximate density of 2 x 105 cells/ml. Cell culture medium without antibiotic-antifungal supplementation was used 24 h prior to infection to relieve the THP-1 cells of the antibiotics and antifungal agents. The macrophage cell density was determined prior to infection using a hemacytometer chamber.

Cell infections. All isolates were cultured, in duplicate, on MB7H9 solid medium (Becton Dickinson, Sparks, MD) supplemented with OADC (oleic acid-albumin-dextrose-catalase) enrichment medium and mycobactin J. After 4 weeks of incubation at 37°C, several loopfuls of growth were inoculated in MB7H9 broth (Becton Dickinson, Sparks, MD) supplemented with OADC enrichment medium and mycobactin J. The broth cultures were placed at 37°C on a shaker to allow uniform growth. Optical density at 600 nm was determined for 5-day-old cultures to obtain the CFU of the mycobacteria (a value of 0.3 for the optical density at 600 nm is the equivalent of 109 CFU/ml [C. C. Wu, personal communication]). A bacterium-to-macrophage cell ratio of 5:1 was used for infecting approximately 107 THP-1 cells/ml. All cell stimulations were performed for 2 h. Macrophages stimulated with sterile MB7H9 broth alone were used as negative controls to determine the basal gene expression profiles. Duplicate infections were carried out using two sets of broth cultures for each of the cattle (K-10 and 1018), human (MAP5 and MAP6), and sheep (5001 and 7565) M. avium subsp. paratuberculosis isolates. Control stimulations were also performed in duplicate with the exception of infection with M. avium subsp. avium (7337), for which only one infection was performed. In addition to the controls, one isolate each from sheep (7565), cattle (1018), and human (MAP6) source was used in the microarray analysis. In addition to controls, two human isolates (MAP5 and MAP6), two cattle isolates (K-10 and 1018), two sheep isolates (5001 and 7565), and one bison isolate (7560) of M. avium subsp. paratuberculosis were used in cell stimulations to confirm gene expression profiles by RT-PCR analyses.

cRNA target preparation and array hybridization. Cells were recovered from the culture suspension by centrifugation. RNA was harvested from the cells at 2 h postinfection by using 2 ml of TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instructions and was further purified using RNeasy Midi columns (QIAGEN, Valencia, CA). RNA quality and purity were examined using a Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto, CA). For each RNA sample, total RNA was processed and hybridized to the Human Genome U133 Plus 2.0 oligonucleotide GeneChip array according to standard Affymetrix protocols (Santa Clara, CA). Scanned output files were visually inspected for hybridization artifacts. A total of nine microarray hybridizations representing the biological duplicates of THP-1 cells infected with the cattle (n = 2), human (n = 2), and sheep (n = 2) M. avium subsp. paratuberculosis strains and with M. avium subsp. avium (n = 1) and noninfected control (n = 2) were analyzed.

Microarray data analysis. Array normalization, raw expression value calculation, relative change (n-fold) determination, and statistical analysis were performed using Genedata Expressionist Pro, version 1.0 (San Francisco, CA). Duplicate samples were treated as one group, data were normalized by using a LOWESS algorithm against noninfected negative controls, and an average expression value for each gene was calculated. The differentially expressed genes were then imported to the Ingenuity pathways database (Mountain View, CA [www.ingenuity.com]). Using the Ingenuity pathways knowledge base data set as a reference, in silico comparison was performed to reveal genes associated with biological functions and canonical pathways (8).

Analysis of altered gene expression by RT-PCR assays. To confirm the array data, expression profiles of seven genes that showed the most significant differences in various stimulations were assessed using real-time RT-PCR. These genes included tumor necrosis factor (TNF), NF-{kappa}BIA, neutrophil cytosolic factor 1 (NCF-1), CSNK2A1, MAP3K8, HLA-DQ1, and LRDD. Briefly, 1 µg of RNA was converted to cDNA using a SuperScipt First-Strand Synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) and amplified using a gene expression assay kit (SuperArray Bioscience Corp., Frederick, MD) per manufacturer recommendations. All real-time RT-PCRs were performed in an ABI 7500 (Applied Biosystems, Foster City, CA) instrument. Control reactions were done without RT mix to ensure that there was no contaminating DNA in the RNA samples being assayed. The 2–{Delta}{Delta}CT method was used to analyze the relative changes in gene expression from the real-time quantitative PCR data normalized against ß-actin expression levels (26). Mean changes in transcriptional profiles were analyzed for statistical significance using the Tukey's correction in the Proc GLM operation of SAS (Statistical Analysis System, Carey, North Carolina).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genome comparisons of M. avium subsp. paratuberculosis isolates. In order to define the gene content of the isolates used in the current study, genomic DNA from each of the M. avium subsp. paratuberculosis isolates (n = 7) examined was fluorescently labeled and competitively hybridized with M. avium subsp. paratuberculosis K-10 on a cDNA microarray designed from the M. avium subsp. paratuberculosis K-10 genome sequence. No ORFs were identified as significantly divergent from the M. avium subsp. paratuberculosis K-10 genome in the cattle, bison, and human isolates. Notably, 14 ORFs were identified as divergent in an M. avium subsp. paratuberculosis sheep isolate. These ORFs were grouped into three clusters on the genome. A manual reexamination of the microarray results for the regions surrounding these clusters resulted in the inclusion of an additional 11 ORFs and also indicated that these regions are deletions rather than sequence divergence due to the complete absence of fluorescently labeled sheep cDNA. Two of these regions correspond to deletions recently identified in a sheep isolate of M. avium subsp. paratuberculosis obtained from Australia (31).

The ORFs identified as divergent in the sheep isolate of M. avium subsp. paratuberculosis encode proteins with a variety of predicted functions, including four dehydrogenases, three putative regulatory proteins, as well as a PPE (Pro-Pro-Glu) family protein. Two putative membrane proteins and six hypothetical proteins with unknown functions were also encoded by ORFs located within the divergent regions. This sheep isolate conforms to the type I category genotype described elsewhere.

Comparative genomic hybridizations using oligonucleotide microarrays based on the genomes of M. avium subsp. paratuberculosis K-10 and M. avium subsp. avium 104 have revealed that sheep M. avium subsp. paratuberculosis isolate 7565 lacks approximately 33 ORFs that are present in the M. avium subsp. paratuberculosis K-10 genome and has acquired approximately 74 ORFs that are present in the M. avium subsp. avium 104 genome. Additionally, the genome of M. avium subsp. paratuberculosis sheep isolate 5001 appears to have lost approximately 144 ORFs that are traditionally present only in M. avium subsp. paratuberculosis isolates and 797 ORFs that are traditionally present only in M. avium subsp. avium isolates. Thus, this second sheep isolate represents a distinct M. avium subsp. paratuberculosis genotype that has not been described previously (M. Paustian et al., unpublished data).

Analysis of differentially expressed host genes. Raw data from each of the nine microarrays representing probe sets for a total of 57,675 genes were filtered using the Genedata Expressionist software (San Francisco, CA) yielding 23,793 genes. Compared to the negative (broth alone) stimulation control macrophage expression levels, 37 and 27 genes were up- or down-regulated 2.5-fold in the cattle and human M. avium subsp. paratuberculosis isolates, respectively (Fig. 1). Sheep isolate and M. avium subsp. avium-stimulated cells differentially regulated 4,780 and 1,800 genes, respectively.


Figure 1
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  		FIG. 1. Scatter plots representing differentially regulated genes in cells stimulated with cattle, human, and sheep M. avium subsp. paratuberculosis isolates and M. avium subsp. avium. Each panel compares gene expression of specific genotypes of M. subsp. paratuberculosis or M. avium subsp. avium relative to unstimulated cells. These are shown on a log scale on both the x and y axes. Inner line (blue), middle line (red), and outer lines (green) indicate 2.5-, 5-, and 10-fold cutoff values, respectively. Genes highlighted blue are more than 10-fold up- or down-regulated in the cells stimulated with either the M. avium subsp. avium or sheep M. avium subsp. paratuberculosis isolates. None of the genes was more than 10-fold differentially regulated in cells stimulated with human and cattle M. avium subsp. paratuberculosis.

 
To further distinguish the gene sets that belong to a known network of genes involved in biologically significant pathways, an in silico comparison was made using Ingenuity pathways (Mountain View, CA [www.ingenuity.com]). Ingenuity pathways with its continuously growing knowledge base offered an easy way to integrate our database into their database and identify genes that could be potentially involved in various cell signaling pathways (8). Thus, we identified genes that were a part of defined gene networks and were classified as being involved in critical biological functions (see supplemental material).

Several functionally significant pathways are differentially regulated by M. avium subsp. paratuberculosis isolates. Comparative analysis of the established canonical pathways induced specifically by M. avium subsp. paratuberculosis isolates in THP-1 cells identified 51 differentially regulated genes by all three M. avium subsp. paratuberculosis isolates analyzed (Fig. 2) (see supplemental material). Notably, this shared expression consisted of up-regulation of genes involved in apoptosis signaling by cells stimulated with a sheep M. avium subsp. paratuberculosis isolate. We also identified a gene expression pattern that was shared by the three M. avium subsp. paratuberculosis isolates and M. avium subsp. avium. Functional analysis determined that the shared genes were primarily associated with cytokine/chemokine signaling, NF-{kappa}B and apoptosis or cell death, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, and growth factor-associated signaling pathways (Fig. 3). These also included genes involved in oxidative burst and peroxisome proliferator-activated receptor signaling pathways. We also identified a subset of 28 genes that were associated with chemokine and cytokine signaling in cells stimulated with the three M. avium subsp. paratuberculosis isolates and the M. avium subsp. avium strain (see Table S10 in the supplemental material). Twenty-one genes were differentially expressed by cells stimulated with the human and sheep M. avium subsp. paratuberculosis isolates (see Table S2 in the supplemental material). Of these, all the transcription regulators including TIAF1 (transforming growth factor ß 1-induced antiapoptotic factor 1) and the T-cell receptor ß locus were up-regulated in cells stimulated with the sheep isolate.


Figure 2
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  		FIG. 2. Venn diagram depicting the genes that have shared induced expression profiles by the cattle, human, and sheep M. avium subsp. paratuberculosis isolates in THP-1 cells.

 

Figure 3
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  		FIG. 3. Shown are representative tile plots (hierarchical plots) in a color-coded display of expression values generated by the software Genedata Expressionist Pro, version 1.0 (as described in Materials and Methods), for cytokine and chemokine signaling (A), apoptosis and cell death signaling (B), genes regulated by MAPKs (C), and genes regulated via growth factor signaling (D) pathways. Each column corresponds to one individual experiment. Differences in intensity and color shades (expression pattern) represent levels of expression for each gene and are interpreted as differentially regulated relative to the color intensity in the "blank" or "nil" stimulated controls. Note that there are differences in expression patterns in gene families in cells stimulated with sheep M. avium subsp. paratuberculosis relative to cell stimulations with human and/or cattle M. avium subsp. paratuberculosis. Gene symbols are shown on the left side.

 
Cattle and human M. avium subsp. paratuberculosis isolates share a common pattern in the regulation of gene expression profiles within THP-1 cells. Cells stimulated with cattle and human M. avium subsp. paratuberculosis isolates differentially regulated genes (P < 0.05) involved in NF-{kappa}B, interleukin-2 (IL-2) signaling, IL-6 signaling, and vasculo-endothelial growth factor pathways. Cells stimulated with the cattle M. avium subsp. paratuberculosis isolate up-regulated HLA-DQB1 which is involved in the major histocompatibility complex class II antigen presentation pathway. The cattle isolate also down regulated genes involved in apoptosis signaling. Two genes (LRDD and PDCD8) related to death receptor signaling were down-regulated by 2.5-fold by the cattle M. avium subsp. paratuberculosis isolate compared to cell stimulations by the sheep M. avium subsp. paratuberculosis isolate (see Table S4 in the supplemental material). The core promoter element binding protein gene speculated to be involved in overall gene regulation and apoptosis was significantly down-regulated by the cattle isolate. The human isolate up-regulated TNF-{alpha}, which may also be involved in death receptor signaling (see Table S10 in the supplemental material).

Sheep M. avium subsp. paratuberculosis and M. avium subsp. avium share a common pattern in the regulation of gene expression profiles within THP-1 cells. Differentially expressed genes (P < 0.05) that were shared between cells stimulated with the sheep M. avium subsp. paratuberculosis and M. avium subsp. avium included those involved in proinflammatory pathways such as T-cell receptor signaling, B-cell receptor signaling, death receptor signaling, chemokine signaling, MAPK signaling, Jun N-terminal protein kinase signaling and integrin signaling pathways (Fig. 3) (see supplemental material). Differentially repressed gene sets that were shared between cells stimulated with sheep M. avium subsp. paratuberculosis and M. avium subsp. avium included genes that function as apoptosis inhibitors and genes involved in major histocompatibility complex class II expression. Twenty-one genes were differentially regulated by the sheep (type I) isolate. Notably, NCF-1, involved in oxidative burst within phagocytes, was up-regulated by more than 20-fold by the sheep M. avium subsp. paratuberculosis isolate. A tumor necrosis factor superfamily gene (TNFSF15) that acts as a decoy receptor and functions as an antiapoptotic gene was up-regulated (more than 2.5-fold) only in cells stimulated with the sheep M. avium subsp. paratuberculosis isolate. Gene expression unique to M. avium subsp. avium stimulations included GUCY1B3 (guanylate cyclase 1), a gene involved in oxidative burst within the phagocytes.

RT-PCR analysis of select targets to evaluate consistency of altered gene expression identified by microarray. We chose seven genes that were identified as being differentially regulated and expected to play a critical role in host-pathogen interactions to confirm the microarray data by real-time RT-PCR. The genes included TNF-{alpha}, NF-{kappa}BIA, CSNK2A1, LRDD, MAP3K8, NCF-1, and HLA-DQB1. RT-PCR data confirmed a similar trend in gene expression as suggested by microarray analysis for all the genes except HLA-DQB1. Microarray data suggested that HLA-DQB1 was up-regulated 2.5-fold by cells stimulated with cattle M. avium subsp. paratuberculosis alone. However, RT-PCR data showed that this gene was also up-regulated in cells stimulated with the sheep and the human isolates relative to the controls.

Consistency of altered gene expression across identical and variant genotypes of M. avium subsp. paratuberculosis isolates by RT-PCR. To evaluate the consistency of microarray data, RT-PCR analysis of the same set of seven genes was performed on cells stimulated with five additional isolates of M. avium subsp. paratuberculosis: cattle K-10 strain, two human isolates (MAP4 and MAP5), one divergent sheep isolate (5001), and a bison isolate (7560) (see Fig. S1 to S7 in the supplemental material). Bison and the type I sheep isolate triggered significant (P < 0.05) up-regulation of TNF transcripts. Cells stimulated with all three human and two cattle isolates representing three SSR genotypes showed similar trends of significantly (P < 0.05) lower TNF, NF-{kappa}BIA, and CSNK2A1 transcripts. Cells stimulated with the type I sheep isolate had significantly (P < 0.05) higher amounts of MAP3K8 gene transcripts relative to stimulations with cattle and human isolates. Transcript profiles of MAP3K8, LRDD, NCF, and CSNK2A1 were not significantly (P = 0.1) different in cells stimulated with the genotypically divergent or identical cattle and human isolates analyzed. Cells stimulated with the type I sheep isolate significantly (P < 0.05) up-regulated the HLA-DQB1 gene relative to cell stimulations with the three human isolates. While the expression trends were identical, the cells stimulated with the type I sheep isolate had significantly (P < 0.05) higher amounts of TNF, NF-{kappa}BIA, CSNK2A1, MAP3K8, LRDD, HLA-DQB1, and NCF-1 transcripts relative to cells stimulated with an atypical sheep isolate (no. 5001).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
M. avium subsp. paratuberculosis is an intracellular pathogen that infects, and persists within, host tissues. In the contest for survival between the host and pathogen, complex cell-mediated immune responses are elicited. Among the first steps in mycobacterial pathogenesis is phagocytosis of the bacteria by the macrophages. A key determinant of virulence is the bacterium's ability to enter and replicate within the phagosome in the early stages of infection, thereby evading the host's defense mechanisms (6, 27). A common theme that has emerged from molecular and population genetic analysis of pathogenic bacteria is that biomedically relevant traits, such as host range and virulence, are nonrandomly distributed among phylogenetic lineages (36). To evaluate this concept, we undertook a comprehensive characterization of genome content and functional phenotypic attributes of M. avium subsp. paratuberculosis isolates derived from cattle, sheep, human, and bison.

Whole-genome comparisons between M. avium subsp. paratuberculosis isolates confirm minimal large sequence polymorphisms. Typically, polymorphisms in mycobacteria are due to large insertions and/or deletions (indels) or to nonsynonymous single nucleotide polymorphisms. These polymorphisms can interrupt or abrogate the efficient transcription or translation to proteins, resulting in variation in biological function and/or host adaptation. Until recently, it was generally accepted that M. avium subsp. paratuberculosis isolates from different hosts and locations were restricted in diversity. To identify any possible large sequence polymorphisms, we performed whole-genome comparisons of well-characterized M. avium subsp. paratuberculosis strains from a variety of hosts. Competitive hybridization with M. avium subsp. paratuberculosis K-10 on a cDNA microarray indicated that no ORFs were significantly divergent from the M. avium subsp. paratuberculosis K-10 genome in the cattle and human M. avium subsp. paratuberculosis isolates while the sheep isolate had 14 divergent ORFs.

In JD, only two host-specific subgroups of M. avium subsp. paratuberculosis isolates have been consistently reported across all geographic origins: isolates from cattle are of the C type (type II), as are most isolates from goats and deer. In contrast, isolates from sheep have been of C, S (type I), or I (intermediate) type, with most countries tending to report only one type in their sheep population. Our whole-genome analysis of the seven isolates used in this study was, therefore, consistent with these previous reports of restricted variability (43). Although our comparative genomic microarray analyses of M. avium subsp. paratuberculosis isolates did not show large sequence polymorphisms or even indels within these phenotypically and SSR-based genotypically diverse groups of organisms, the possibility still exists that real differences that define specific microbiological or pathogenic characteristics might exist in short indels, single nucleotide polymorphisms, and/or in the regulatory machinery of M. avium subsp. paratuberculosis. Additionally, it is quite possible that plasticity may exist in acquired novel DNA segments not present in K-10, a finding supported by oligonucleotide array analyses.

The possibility that a particular group of M. avium subsp. paratuberculosis isolates may be involved in Crohn's disease has been investigated. However, in these studies only a limited number of M. avium subsp. paratuberculosis isolates from humans were analyzed, and given this limitation, it is not surprising that only a small number of genotypes were identified (6, 16, 17, 42, 44), even though there is clear diversity of isolates circulating in animal populations, as defined by short sequence repeats (17, 33). The absence of large sequence polymorphisms in the human isolates used in the present study also confirms this finding. Clustering of the human isolates with isolates derived from animal species is suggestive of inter- and intraspecies transmission and an association of a few animal M. avium subsp. paratuberculosis isolates with the pathobiology of Crohn's disease (16, 17, 59). These speculations are, however, based on genomic polymorphism identified at distinct sites within the genome. Thus, we analyzed the transcriptomes of isolates derived from human and animal hosts to obtain additional insight into a variation in biological function and/or host adaptation.

Transcriptional analysis of a human monocytic cell line (THP-1 cells) stimulated with diverse M. avium subsp. paratuberculosis isolates reveals shared as well as distinct expression profiles. Several studies have investigated gene expression profiles in which proapoptotic and proinflammatory pathways were induced by individual type strain(s) of M. avium subsp. paratuberculosis on bovine macrophages obtained from infected or uninfected cattle (9-14). However, the role of the bacterial genotype on the host's gene expression has yet to be clearly defined. In this study we examined the transcriptional changes that occur within infected macrophages and identified genes that are induced or repressed in response to different genotypes of M. avium subsp. paratuberculosis. The use of a transformed cell line (THP-1; ATCC) provides several benefits over the use of primary human cells including a control over the host genotype, reproducibility, economy of cost and time, practical simplicity, and availability. Several studies have indicated that THP-1, a human myelomonocytic cell line, is a representative model to evaluate differences in signaling pathways triggered by diverse species of mycobacterial species (40, 53). In addition, the transcriptional profiles identified in our study were consistent with those identified in bovine monocyte-derived macrophages (10, 12, 57) with one exception. The HLA-DQB1 gene, which has been shown to be down regulated by M. avium subsp. paratuberculosis, was consistently up-regulated by all isolates in our studies. This difference may be due to differences in either the genotypes of the infecting clinical isolates used in our study or in the types of host cells. Regardless, consistent transcriptional profiles identified between other studies and ours suggested that the THP-1 human macrophage cell line provided a convenient cell culture model system for these experiments.

Based on acid-fast staining and microscopy of THP-1 cells at 0, 0.5, 1, 2, and 4 h postinfection, at 2 h postinfection (95% infection rate) all strains of M. avium subsp. paratuberculosis used in this analysis were phagocytosed or in the process of being phagocytosed by the cells (data not shown). We chose this time point to represent early infection across all M. avium subsp. paratuberculosis isolates for the host gene expression analysis. We along with others have shown that M. avium subsp. paratuberculosis induced expression in host macrophages up to 110 h postinfection (10-12, 22). Using real-time PCR, our analysis in bovine macrophages has shown that M. avium subsp. paratuberculosis isolates persist and replicate until 120 h postinfection (22). A correlation between fluorescent staining and PCR has also been established (N. S. Gollnick et al., Abstr. 8th Int. Colloq. Paratuberculosis, abstr. 103, 2005). Thus, we believe that the bacteria were viable and active at 2 h postinfection in the THP-1 cells.

Macrophages and dendritic cells share a common gene expression pattern and a pathogen-specific gene expression pattern when infected with various intracellular pathogens (20, 38). When stimulated with pathogenic and nonpathogenic mycobacteria, U937 cells (a human histiocytic cell line used as an in vitro model for monocytes/macrophages) exhibited differential patterns of expression for genes related to cytokines, protein kinases, transcription factors, and genes involved in apoptosis (32). Human macrophages have been shown to differentially respond to infections with well-characterized clinical isolates of M. avium subsp. paratuberculosis (55), and the patterns of expression correlated with virulence attributes of the isolates. More recently, gene expression profiling of M. avium subsp. avium isolates with different growth characteristics exhibited both a shared gene expression profile and a pathogen-specific gene expression profile specifically in genes or gene families of cytokines and chemokines and genes involved in antiapoptosis, signal transduction, and chemokine receptors (4). We identified similar gene expression profiles for the M. avium subsp. avium isolate analyzed. The M. avium subsp. paratuberculosis isolates with unique SSR genotype and belonging to type I classification also paralleled the M. avium subsp. avium gene expression profiles, suggesting common themes in mycobacterium-host cell interactions. We acknowledge that our studies were not designed to evaluate differences or similarities in induced gene expression profiles between M. avium subsp. avium and M. avium subsp. paratuberculosis isolates. Also, because our study used only a single M. avium subsp. avium isolate and because there exists a great diversity among M. avium subsp. avium isolates (60), the parallels in gene expression patterns induced by sheep M. avium subsp. paratuberculosis and the M. avium subsp. avium isolate in the current study has to be interpreted with caution. Nevertheless, comparisons of gene expression profiles induced by the M. avium subsp. avium isolate in the present study corroborated those recently reported by Blumenthal et al. (4). Comparisons of our data revealed common themes in up-regulation of genes related to cytokines (TNF), chemokines (IL-8), receptors (CCR2 and CXCR4), signal transduction (TRAF1 and PDE4B), enzymes (ADA, PTGS2, and GCH1), antiapoptosis (TNFAIP3), and adhesion (TNFAIP6) in macrophages stimulated with four divergent M. avium subsp. avium isolates.

Host gene expression studies with M. tuberculosis using cellular models also show genotype-specific transcriptional profiles (27, 55). In addition, experimental infection in mouse models using clinical isolates of M. tuberculosis revealed that survival of mice correlated with the virulence traits of the isolate (3). If true, it is possible that the infecting genotype of the pathogen may play a significant role in downstream host responses.

In our studies the cattle M. avium subsp. paratuberculosis isolate, specifically, appeared to enter the macrophages without triggering any major proinflammatory cascades, an observation consistent with previous studies in bovine macrophages stimulated with a type strain of M. avium subsp. paratuberculosis and M. avium subsp. avium (12, 57). Cattle isolates also trigger anti-inflammatory pathways including the Flt-1 gene that modulates downstream events in the vasculo-endothelial growth factor pathway. The Flt-1 gene is associated with anti-inflammatory responses such as the production of nitric oxide and inhibition of chemotaxis (7, 23). The human isolate down-regulated MMP14, suggesting an anti-invasive pathway. These attributes are likely necessary for successful invasion and persistence of infection in the host (Fig. 4). Taken together, these findings suggest a parallel in host responses by the cattle and human isolates of M. avium subsp. paratuberculosis, irrespective of the pathogen genotype or host macrophage.


Figure 4
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  		FIG. 4. Depiction of the major pathways induced by M. avium subsp. avium and M. avium subsp. paratuberculosis (MAP) isolates from diverse host species. The diagram represents major pathways and potential outcomes of cellular infection. Dotted arrows represent pathways differentially regulated by the cattle and human isolates, while the solid arrows show those regulated by the sheep isolate.

 
Inasmuch as our previous studies suggested that SSR analysis provided epidemiologically meaningful diversity information, we studied the functional significance of M. avium subsp. paratuberculosis isolates from various hosts carrying diverse SSR genotypes. We identified common and genotype-specific cell signaling pathways. Regardless of the host of origin, identical SSR types showed similar gene expression patterns. While the magnitude of transcriptional profiles varied by genotype, our observations suggest that the cattle and the human M. avium subsp. paratuberculosis isolates, regardless of SSR genotypes, manipulate the host macrophages in similar ways: both down-regulated genes involved in death receptor signaling and several proinflammatory genes while up-regulating genes necessary for cell survival and cell differentiation. This suggests that the genotype of these isolates may provoke anti-inflammatory and anti-invasive pathways within the host cells and, hence, favor bacterial survival and suggest convergence to a common function.

Common themes in transcriptional profiles of the type I M. avium subsp. paratuberculosis isolate and M. avium subsp. avium are indicative of a less successful pathogen. The M. avium subsp. avium strain and (type I and atypical) sheep isolates of M. avium subsp. paratuberculosis elicited common signaling pathways in THP-1 cells. These observations corroborate our earlier findings in bovine monocyte-derived macrophages (22). Sheep isolates up-regulated some antiapoptotic genes and genes necessary for oxidative burst. Notably, the NCF-1 gene was up-regulated more than 10-fold relative to other isolates studied. NCF-1 is intricately involved in peroxisome proliferator-activated receptor signaling (56). IL-6 signaling receptor was down-regulated by the sheep M. avium subsp. paratuberculosis isolate. Blockade of IL-6 trans-signaling suppressed T-cell resistance against apoptosis in Crohn's disease (2). Oki et al. have recently shown that there was increased infiltration of Th1-type chemokines and chemokine receptors (CCL5 and CXCR3) in noncaseating granulomas which are characteristic of Crohn's disease (39). Our findings in THP-1 cells stimulated with M. avium subsp. paratuberculosis isolates also suggest a role for CCL5 and related chemokine receptors in the pathogenesis of JD.

This significant proinflammatory signaling induced by the sheep isolates (regardless of genotype; type I or atypical) may explain host adaptation and evolution and be associated with the relative rarity of these strains in other hosts. This proinflammatory phenotype parallels the response induced by a nonpathogen, M. avium subsp. avium, a likely reason for its rapid elimination from immunocompetent hosts. Since the type I strains and M. avium subsp. avium are similar in both gene content and in the ways in which they manipulate THP-1 cells, we speculate that S or type I strains of M. avium subsp. paratuberculosis represent an evolutionarily ancient clade during the divergence and specialization of M. avium subsp. avium into a pathogen.

The underrepresentation of S or type I isolates in cattle, deer, and goats has been assumed to reflect the difficulty of culturing of such isolates in the laboratory. However, the fact that they have been identified in sheep from a similar range of countries suggested that cultivability alone does not explain the apparent segregation observed among the host species. This raises the possibility that the M. avium subsp. paratuberculosis isolates display a degree of host specificity or at least host preference. Our findings that the sheep isolates induced proinflammatory pathways in a bovine (22) and a human monocytic cell line (THP-1 cells in the present study) suggest that these strains may be efficiently controlled and rapidly eliminated by some hosts and not others. This supports the hypothesis that M. avium subsp. paratuberculosis may show a degree of host preference. While some atypical sheep-adapted isolates that do not strongly induce proinflammatory pathways may exist and infect cattle in a given epidemiologic setting, their occurrence appears to be rare. Thus, typical sheep isolates may represent an evolutionary clade of pathogens that diverged from a common ancestor of the nonpathogenic M. avium subsp. avium.

The success of these organisms in sheep populations may be indicative of their ability to manipulate the sheep macrophages/immune responses efficiently to enable their persistence. Studies on these topics are under way in our laboratory and will provide insights into the concept of host specificity or host specialization in M. avium subsp. paratuberculosis infection.

In summary, we identified a global gene expression pattern that was shared and distinct between different genotypes of M. avium subsp. paratuberculosis in a human monocytic cell line (Fig. 2). Our observations with diverse genotypes of M. avium subsp. paratuberculosis correlated with similar studies utilizing clinical isolates of M. tuberculosis that suggested that the genotype carried by the pathogen may play a significant role in the "host response." We determined that cattle, human, and bison isolates of diverse genotypes belonging to the type II classification induced an anti-inflammatory and antiapoptotic gene expression pattern, a likely reason for the success and widespread occurrence of these organisms as pathogens. We also showed that sheep isolates, regardless of genotype, that were classified as type I differed in that they induced proinflammatory gene expression profiles and may represent a host-specialized clade associated almost always with disease in sheep. Further studies on host specialization and the role of host susceptibility-associated mutations in M. avium subsp. paratuberculosis pathogenesis will provide insights into the role of specific genotypes in human and/or animal infections.


   ACKNOWLEDGMENTS
 
This study was supported in part by a USDA-NRI (Animal Protection) grant awarded to S.S.

We thank the laboratory of Larry Schlesinger for helping optimize cell culture protocols used in this study. We also thank Zheng Jin Tu at the University of Minnesota Supercomputing Institute for technical support.


   FOOTNOTES
 
* Corresponding author. Mailing address: Veterinary Population Medicine Department, University of Minnesota, 136G Andrew Boss Laboratory-Meat Hygiene, 1354 Eckles Avenue, St. Paul, MN 55108. Phone: (612) 625-3769. Fax: (612) 624-4906. E-mail: sreev001{at}umn.edu. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: J. F. Urban, Jr.

{ddagger} A.S.M. and H.K.J. contributed equally to the work. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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