Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Ehrlichia ruminantium, a tick-transmitted pathogen, is the causative agent of heartwater in ruminants. In this study, a proteomic approach was used to identify host cell-specific E. ruminantium proteins encoded by the map1 multigene family, expressed in vitro in bovine endothelial and tick cell cultures. Two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to establish the identities of immunodominant proteins. Proteins extracted from E. ruminantium-infected endothelial cells were shown to be products of the map1 gene, whereas tick cell-derived E. ruminantium proteins were products of a different gene, map1-1. The expressed proteins were found to be glycosylated. Differential expression of MAP1 family proteins in vitro in mammalian and tick cell cultures indicates that the map1 multigene family might be involved in the adaptation of E. ruminantium to the mammalian host and vector tick.     

Mining the genetic diversity of Ehrlichia ruminantium using map genes family

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host.In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster.We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands).These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host–vector–pathogen interactions that could be crucial for developing broad-spectrum vaccines.     

Quantification of Ehrlichia ruminantium by real time PCR

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.     

Mycoplasma gallisepticum hemagglutinin V1hA, pyruvate dehydrogenase PdhA, lactate dehydrogenase, and elongation factor Tu share epitopes with Mycoplasma imitans homologues

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.     

Ligand-independent activation of steroid receptors

In several transformed cell lines, the growth factors IGF-I and epidermal growth factor (EGF) activate second messenger systems that cause the phosphorylation of the estrogen receptor (ER). One kinase catalysing receptor phosphorylation is mitogen activated protein (MAP) kinase, and the result of phosphorylation is an increase in receptor transactivation function. EGF and IGF-I, secreted locally and systemically, are involved in uterine-conceptus interactions in early pregnancy, and therefore it is of interest to determine whether these growth factors affect ER function in the uterus. An estrogen response element, chloramphenicol acetyl transferase reporter gene construct (CATERE) was transfected into bovine endometrial epithelial and stromal cells in vitro, and CAT measured during transient expression. Growth factors were added at various times following transfection, and MAP kinase phosphorylation was monitored by western blotting of p42 and p44. The MEK inhibitor U 0126 was used to determine whether the effect of IGF-I on CATERE expression was mediated through MAP kinase, and the anti-estrogen ICI 182780 was used to identify effects involving the ER. In stromal cells, reporter gene activity was increased in a dose dependent manner by IGF-I or hEGF in the presence or absence of estradiol-17beta. In the absence of estradiol the effect of IGF-I was not inhibited by ICI 182780. The effect of IGF-I occurred within an hour, before any detectable increase in cell proliferation, and the activation of CAT expression in response to IGF-I or EGF was blocked by U 0126. In contrast to their effects in stromal cells, neither IGF-I nor EGF affected CAT expression in bovine endometrial epithelial cells. Measurement of phosphorylated MAP kinases p42/p44 by western blotting showed that EGF but not IGF-I activated MAP kinase phosphorylation in both epithelial and stromal cells. In stromal cells, the fact that U 0126 blocked the CAT responses to IGF-I and EGF indicates the involvement of a MAP kinase. But since IGF-I did not activate p42/p44, a different MAP kinase, not detected by the antibody used here, is implicated. As the response was not blocked by ICI 182780, we conclude this effect is independent of ER activation. Therefore in bovine uterine cells in culture effects on MAP kinases p42/p44 can be dissociated from those on ERE-dependent gene expression, and reporter gene expression may be independent of ER activation.     

Proteomic analysis of plasma from Holstein cows testing positive for mycobacterium avium subsp. Paratuberculosis (MAP)

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

Identification of candidate host proteins that interact with LipL32, the major outer membrane protein of pathogenic Leptospira, by random phage display peptide library

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp. Rodent species are the major reservoir hosts that can excrete leptospires in their urine leading to environmental contamination. After gaining entry into the host via skin breaks and mucosa, leptospires disseminate through the bloodstream to target organs causing a wide range of disease manifestations in susceptible mammalian hosts. The crucial step of infection requires host-pathogen interactions. LipL32, the major outer membrane protein (OMP) of pathogenic Leptospira, is conserved among pathogenic leptospires, immunogenic, and expressed in target organs during acute infection in animal models. Therefore, it may play a key role in host-microbe interactions. To identify host proteins that interact with LipL32, phage display technology was employed in our study. Recombinant LipL32 was used as a target molecule for biopanning with a random heptapeptide phage library to enrich for phages expressing peptides with high affinity to LipL32. After three rounds of panning, 44 plaques of eluted phages were subjected to pyrosequencing. Six different peptide sequences were identified and used to search for matching proteins in the database. Putative proteins with potential binding to LipL32 are proteins known to be expressed on the surface of target cells of pathogenic Leptospira such as chloride channel accessory 2, glycoprotein VI, scavenger receptor expressed by endothelial cell isoform I (SREC-I), coronin 2A, laminin alpha 5, collagen XX, and prostaglandin receptor EP1. However, interactions of LipL32 with these host proteins and their role in the pathogenesis of leptospirosis requires experimental confirmation.     

Alteration in the Expression of Proteins in Unexplained Recurrent Pregnancy Loss Compared with in the Normal Placenta

The placenta is a unique pregnancy-related tissue and plays a key role in occurrence of unexplained recurrent pregnancy loss (URPL). Abnormal placentation might play a key role in occurrence of URPL. Therefore, the purpose of this study was to compare the human placental proteome between URPL placentas and normal placental matched for gestational week. Total placental proteins were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used for separation of the placental proteomes. Protein spots differentially expressed between URPL and normal placentas were selected and identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after being digested in the gel. Moreover, quantitative real-time PCR and Western blot techniques were used to confirm the differential expression mass results for some differentially expressed proteins. The results indicated that at least 19 protein spots were differentially expressed between URPL and normal placentas (P < 0.05), and twelve of them were successfully identified. While only two proteins were downregulated (calumenin and enolase 1), the remaining ten spots (actin gamma 1 propeptide, cathepsin D prepropeptide, heat shock protein gp96, tubulin beta, tubulin alpha 1, glutathione S-transferase, vitamin D binding protein, prohibitin, actin beta, apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. Real-time PCR also confirmed the downregulation of calumenin and upregulation of prohibitin and apolipoprotein A-I at the mRNA levels. In conclusion, the results of the present study showed that alteration in the expression of proteins involved in proliferation and migration of endothelial cells as well as control of coagulation by these cells might play an important role in the pathogenesis of URPL.     

MAP3K5基因在畜禽剩余采食量表型调控中的研究进展

随着分子遗传学的发展,已经鉴定出了影响剩余采食量(RFI)的大量数量性状位点和候选基因。有丝分裂原活化蛋白3激酶5(MAP3K5),也称凋亡信号调节激酶1(ASK1),属于MAPK超家族基因之一。目前,已有细胞外信号调节蛋白激酶(ERK)、c-Jun N-末端激酶(JNK)和p38丝裂原激活蛋白激酶(p38-MAPK)这3个MAPK家族成员在哺乳动物细胞中被克隆和鉴定,其主要作用机制是介导3条MAPKs信号通路,从而影响家畜的生长、体型及产奶性状等。课题组在前期对RFI的研究中筛选出与牛剩余采食量相关的MAP3K5基因,但其功能作用尚不明确,笔者在此基础上回顾了该基因的结构、生物学功能,概述了该基因在主要畜禽采食量变异及人类肥胖表型中的功能及作用,并从遗传学的角度重点分析了MAP3K5基因在畜禽RFI表型调控中的可能机制。通过对MAP3K5基因在畜禽RFI表型调控中的研究进展进行综述,期望为后期深入开展MAP3K5基因在畜禽采食量性状调控中的分子机制研究提供思路;对于其他可能通过MAP3K5基因影响畜禽表型的因素(如肠道菌群)有待进一步探讨。     

梅花鹿朊蛋白核心片段的基因克隆与高效表达

本试验根据梅花鹿朊蛋白基因序列设计引物,利用PCR的方法从梅花鹿基因组DNA中扩增朊病毒蛋白酶抵抗区域PrP^res,将该片段分别与表达载体pET-Trx和pET-His连接,构建重组表达载体pET-Trx-PrP^res和pET-His-PrP^res。分别将两个重组表达载体转入E.coli BL21(DE3) plys宿主菌中,37 ℃诱导4 h,经SDS-PAGE分析,Trx-PrP^res和His-PrP^res表达量分别为38.2%和30.1%。     

Proteomic survey of the pathogenic Mycoplasma hyopneumoniae strain 7448 and identification of novel post-translationally modified and antigenic proteins

Mycoplasma hyopneumoniae is an important pathogen for pigs, being the causative agent of enzootic pneumonia. Recently, the genome sequences of three strains, J, 7448 and 232 have been reported. Here, we describe the results of a proteomic analysis, based on two-dimensional gel electrophoresis of soluble protein extracts, immunoblot and mass spectrometry, which was carried out aiming the identification of gene products and antigenic proteins from the M. hyopneumoniae pathogenic strain 7448. A preliminary M. hyopneumoniae proteome map in two pH ranges (3-10 and 4-7) was produced. A total of 31 different coding DNA sequences (CDSs), including three hypothetical ones, were experimentally verified with the identification of the corresponding protein products by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. According to the Clusters of Orthologous Groups (COG) functional classification, the identified proteins were assigned to the groups of metabolism (13), cellular processes (5) and information and storage processing (4). Nine of the identified proteins were not classifiable by COG, including some related to cytoadherence and possibly involved in pathogenicity. Moreover, at least five highly antigenic proteins of M. hyopneumoniae were identified by immunoblots, including four novel ones (a heat shock protein 70, an elongation factor Tu, a pyruvate dehydrogenase E1-beta subunit and the P76 membrane protein). The now available proteome map is expected to serve as a reference for comparative analyses between M. hyopneumoniae pathogenic and non-pathogenic strains, and for methabolic studies based on cells cultured under modified conditions.     

Evaluation of the immunogenicity of Mycobacterium avium subsp. paratuberculosis (MAP) stress-associated recombinant proteins

The aim of this study was to assess the humoral immune responses in MAP-infected and uninfected sheep against 27 MAP stress-associated recombinant proteins that were regulated in in vitro models of physiological stress. These include evaluation of 5 proteins, which were previously reported by Gumber et al. (2009b), using serum samples from sheep with a wide range of disease stages. For purification of recombinant his-tagged proteins expressed as an insoluble protein, on-column refolding purification was applied as well as one-step denaturing purification. All purifications together resulted in a total of 48 recombinant antigen preparations. In antibody ELISA tests, 23 of these, representing 18 MAP proteins, showed significant differences in responses between infected and uninfected sheep. Recombinant antigen preparations MAP2281c, MAP3555 (refolded form), and MAP0711c (refolded form) when incorporated in an ELISA, had similar sensitivity to a commercial antibody ELISA test at the cutpoint of 90% specificity, and showed relatively high values in receiver operating characteristic (ROC) curve analysis. However, as some of the sera from uninfected sheep also reacted to recombinant antigens, further development of the assays is necessary prior to practical application. Compared to the commercial antibody ELISA, MAP0593c, MAP2281c, MAP2411, MAP3555, and MAP3200 detected more infected sheep with a lower grade of lesion, suggesting that these proteins identified in the in vitro models of stress were also expressed in vivo in MAP-infected sheep at an early stage of infection. This is consistent with the hypothesis of latency or dormancy in subclinical mycobacterial infection.     

Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.     

Adherence of Pasteurella multocida to fibronectin

For many pathogens, adherence and/or invasion involve association with host extracellular matrix molecules, such as fibronectin (Fn). Pasteurella multocida was found to bind significantly to Fn and collagen type IX but not to laminin and collagen types IV and X. The binding of P. multocida to Fn was dose-dependent and was inhibited by heparin (Hep). Removal of polysaccharide capsule enhanced the binding capacity of the bacterium to Fn and inhibition by Hep. Protease treatment of bacteria decreased binding, implicating surface protein(s) as adhesive components. Investigation of the binding domain(s) of P. multocida on the Fn molecule revealed preferential binding to the N-terminal Hep-binding domain of Fn but not to the carboxyl-terminal Hep-binding domain. Furthermore, Fn, and anti-Fn antibodies inhibited P. multocida adherence to Madin-Darby bovine kidney cells, suggesting the involvement of Fn in the bacterium adherence to host cells. Ligand blotting, batch affinity purification and MALDI-TOF mass spectrometry implicated several proteins as putative adhesins of P. multocida in the Fn-mediated adherence. Taken together, the data suggest that P. multocida-Fn interaction may play a role in the bacterium adherence to host cells, and this may be mediated by bacterial surface proteins with preferential affinity for the Hep-1 binding domain of Fn.     

Analysis of differential strategies to enhance detection of low‐abundance proteins in the bovine serum proteome

Serum‐based biomarkers hold propitious applications for addressing livestock health, and management. However, discovery of protein biomarkers in complex biological fluids like serum is wholly intractable due to the large dynamic range of protein concentrations; that is, ?10–12 high abundance proteins constitute >90% of the total protein content and effectively mask proteomic detection of low‐abundance biomarkers. Toward addressing this limitation, we test a continuous elution size‐based fractionation method, and two approaches that use affinity interaction‐based separation of proteins in preparing bovine serum, and compare liquid chromatography tandem mass spectrometry protein identification to neat serum. Our results identify the high‐abundance proteins in bovine serum, and demonstrate dynamic range compression and improved protein identification with the different enrichment methods. Although these findings indicate the highest protein number identified in bovine serum (445 proteins, all methods combined), and by any single sample processing method (312 proteins) to date, they still remain lower than levels deemed necessary for biomarker discovery. As such, this investigation revealed limitations to resolving the bovine serum proteome, and the need for species‐specific tools for immunodepleting high‐abundance proteins. In concert, this study represents a step toward advancing sample preparation methods for bovine serum biomarker identification.     

Establishment of a proteomic reference map for the gastrocnemius muscle in the rabbit (Oryctolagus cuniculus)

In several laboratory and production species, the establishment of a proteome reference map of a specific tissue has been accomplished. The rabbit is widely used as both a production and experimental animal. A lot of physiology research involving the gastrocnemius muscle of rabbit is described, although no reference proteome map is available. In this work, the first reference map of the rabbit’s gastrocnemius muscle using 2D gel electrophoresis and the identification of proteins through peptide mass fingerprinting (PMF) was established. A total of 45 proteins were localized and identified with three major roles: cell structure and contractile apparatus; metabolic and cell defense proteins. A reference map of major proteins expressed is described enabling possible comparisons with other physiological studies.     

Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus

Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis.     

猪丁型冠状病毒S1-CTD相互作用宿主蛋白的筛选和鉴定

猪丁型冠状病毒纤突蛋白S1亚基C端结合域(S1-CTD)是诱导中和抗体产生的主要区域,为研究与其相互作用的宿主蛋白,采用HEK-293T真核表达系统表达并纯化了S1-CTD,提取了猪回肠上皮细胞膜蛋白,通过免疫共沉淀筛选了可能与S1-CTD相互作用的蛋白并进行质谱分析,发现32个疑似相互作用的宿主蛋白。构建其中可能与S1-CTD互作的蛋白KIF1 binding protein (KIFBP)的真核表达质粒,通过免疫共沉淀和激光共聚焦验证上述宿主蛋白与S1-CTD是否存在相互作用,结果表明KIFBP和S1-CTD之间存在相互作用,共同转染时在细胞质共定位。进一步研究表明,过表达KIFBP能够有效降低病毒RNA水平和病毒滴度,其中mRNA水平降低了约70%,病毒滴度降低了10^1.6TCID₅₀。综上,本研究筛选并鉴定出一种与PDCoV S1-CTD相互作用的宿主蛋白KIFBP,为了解PDCoV的致病机制提供了理论基础。     

Relationship between Mycobacterium avium subspecies paratuberculosis, IL-1alpha, and TRAF1 in primary bovine monocyte-derived macrophages

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.