SAP Gene Research Articles

Molecular Detection of Secreted Aspartyl Proteinases (Saps) From Dental Isolates of Candida albicans and Targeting With Psidium guajava Biocompounds: An In Vitro and In Silico Analysis.

Introduction Candida albicans(C. albicans) is an opportunistic yeast-like fungus and is considered a functional biome of the oral and gut microbiomes. Thesapgene and its types play a vital role in the pathogenesis of C. albicans. The emergence of resistance traits is a major problem, and targeting the same with alternative medicines has sparked renewed interest in recent years. Objectives This study is thus aimed at detecting the frequency of sap gene types in the clinical isolates of C. albicans and evaluating the antifungal effect of the crude methanolic extract of Psidium guajava (P. guajava). Further in silico assessments will assess the inhibitory effect of six compounds of P. guajava against the Sap protein. Materials and methods C. albicans was characterized phenotypically in 20 patients with root caries, and the sap gene was detected by PCR. The crude methanolic extract was prepared, and its antifungal efficacy was evaluated by the agar well diffusion method. Auto-docking was performed to assess the best compound based on the docking and overall interactions. Results Six isolates were identified as C. albicans and sap genetypes 1-3 were detected in the four strains. P. guajava methanolic extracts showed a promising antifungal effect at varying concentrations. In silico analysis showed myricetin possessing the maximum number of hydrogen bonds and high docking energy with one violation. Conclusion The study concludes that P. guajava has a promising inhibitory effect againstC. albicans with myricetin as the best compound to target the sapgene of C. albicans. However, further experimental studies are to be considered for its effectiveness in treating the infections caused by C. albicans.

Genetic evidence for the interaction between Bacillus anthracis-encoded phage receptors and their cognate phage-encoded receptor binding proteins.

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein-protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between sap and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBPλBa03 binding to B. anthracis cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by B. anthracis phages, enabling future assay development.

Competitiveness and symbiotic efficiency in alfalfa of Rhizobium favelukesii ORY1 strain in which homologous genes of peptidases HrrP and SapA that negatively affect symbiosis were identified

In acid soils, Rhizobium favelukesii strains, known as Oregon-like strains, are a potential risk for alfalfa production given their parasitic behaviour. In this study, we isolated five parasitic strains (ORY1 to ORY5) from alfalfa nodules grown in Uruguayan acid soils, with a 99.7% and a 100% 16S rRNA gene sequence identity to R. favelukesii type strain of LUP83. The BOX profiles of the five isolates showed two different patterns, suggesting some diversity among these acid-tolerant isolates. The genome sequence analysis of R. favelukesii strains ORY1, LPU83, and Or191 showed that they have around 87.5% of common coding genes, including the symbiotic genes. Moreover, the phylogenetic analysis of ORY1 symbiotic genes nifH, nifD, nifK, nodA, nodB, and nodD were related to the symbiotic genes of E. meliloti. We teste ORY1 competitiveness by inoculating seeds with 99:1 and 1:99 ratios of ORY1::gusA/E. meliloti U143. In both treatments, ORY1::gusA occupied more than 50% of nodules, evidencing its high competitiveness. However, the aerial biomass in these treatments was remarkably different, suggesting that the nodules induced by the efficient strain are essential to provide enough N for optimal plant growth. These findings support the needing of inoculating in areas where inefficient strains are likely to be present. Finally, we found three genes that encode amino acid sequences for domains of M16 peptidases (with homology to bacterial hrrP and sapA genes), two of them were contiguous and located in an accessory plasmid, whereas the other one was a chromosomal gene. These genes are likely to be involved in the parasitic behaviour of ORY1 strain.

Differentiated sap (4–6) gene expression of Candida albicans isolates from HIV-positive patients with oral candidiasis and commensals in healthy individuals

Gene expression of SAP 4–6 based on the detection of mRNA was observed in Candida albicans isolates from HIV-positive patients with oral candidiasis and commensal from healthy individuals. The species of C. albicans strains were selectively isolated from both sources using CHROMagar Chromogenic Media. The obtained isolates were then induced to express SAP 4–6 using SAP 4–6 gene inducer media. Analysis of gene expression was performed on a molecular basis using the RT-PCR method. Molecular analysis of gene expression showed that the isolates CH3 from HIV-positive patients with oral candidiasis could express SAP 4–6 gene, while commensal isolates from healthy people could not. Based on the results of this study, it could be concluded that, in terms of molecular detection, only isolates from HIV-positive patients (CH3) could express their SAP 4–6 gene.

A conserved rhizobial peptidase that interacts with host-derived symbiotic peptides

In the Medicago truncatula-Sinorhizobium meliloti symbiosis, chemical signaling initiates rhizobial infection of root nodule tissue, where a large portion of the bacteria are endocytosed into root nodule cells to function in nitrogen-fixing organelles. These intracellular bacteria are subjected to an arsenal of plant-derived nodule-specific cysteine-rich (NCR) peptides, which induce the physiological changes that accompany nitrogen fixation. NCR peptides drive these intracellular bacteria toward terminal differentiation. The bacterial peptidase HrrP was previously shown to degrade host-derived NCR peptides and give the bacterial symbionts greater fitness at the expense of host fitness. The hrrP gene is found in roughly 10% of Sinorhizobium isolates, as it is carried on an accessory plasmid. The objective of the present study is to identify peptidase genes in the core genome of S. meliloti that modulate symbiotic outcome in a manner similar to the accessory hrrP gene. In an overexpression screen of annotated peptidase genes, we identified one such symbiosis-associated peptidase (sap) gene, sapA (SMc00451). When overexpressed, sapA leads to a significant decrease in plant fitness. Its promoter is active in root nodules, with only weak expression evident under free-living conditions. The SapA enzyme can degrade a broad range of NCR peptides in vitro.

Upregulation of secreted aspartyl proteinase genes of fluconazole-sensitive Candida albicans isolates.

Candida albicans infection development depends on several factors associated with this etiological agent, including secreted aspartyl protease (Sap) production. Sap expression commonly occurs under selective pressure caused by the presence of antifungals. Fluconazole is the main antifungal drug used for treatment or prophylaxis. This study investigated the influence of inhibitory and sub-inhibitory fluconazole concentrations on Sap activity and their gene transcription for three clinical C. albicans isolates. Two isolates presented significant increases in Sap activity and transcription of SAP 1-8 genes in the presence of 1 MIC80 of fluconazole compared to the absence of the antifungal agent. The results suggest that the increase in Sap activity occurs due to an upregulation of the SAP gene transcription influenced by fluconazole. This suggests the importance of all SAP genes in the progression of bloodstream infections compared to primary tissue infection. However, this phenomenon does not occur everywhere, and it is multifactorial. This may be related to the selective pressure effect on transcription modulators. Although preliminary, these results open a new perspective for the study of virulence factors.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive

A thermostable extracellular alkaline protease (called SAPA) was produced (4600U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70°C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Molecular detection of Bacillus anthracis: evaluation of the efficiency of DNA extraction and a novel dry PCR

Abstract Background Due to recent increase in mailings of anthrax spores, the detection of bioweapons has gained a great deal of interest. This study aimed to investigate the yield and purity of DNA obtained from spores and vegetative forms of Bacillus anthracis for detection by conventional (wet) and dry (lyophilized) PCR methods. Materials and methods Biosamples from stock solution were reconstituted to a concentration 108 cfu/mL followed by the spectrophotometric measurement of the yield and purity of acquired DNA. Twelve wet and 12 dry PCR studies of four various DNA dilution samples were performed for each three target gene (cap, pag, sap) of B. anthracis. Results Significant differences for both DNA yields and purity were found between liquid-agar and liquid-spore samples. No significant difference was observed between wet and dry PCR in concentration of 2.5 ng/μL DNA for all gene regions. PCR results of sap gene region with DNA concentrations of 1.5 ng/μL and 0.9 ng/μL was found statistically significant in favor of conventional wet PCR method (p = 0.047 and p ≤ 0.001, respectively). Conclusion It is concluded that B. anthracis cultivated in liquid medium is more usable than vegetative or spore form obtained on plate agar for proper DNA extraction.

Inhibitory Effect of Taurine on Biofilm Formation During Alkane Degradation in Acinetobacter oleivorans DR1.

Taurine, 2-aminoethanesulfonate, is known to function as an antioxidant or membrane stabilizer in eukaryotic cells, but its role in bacteria has been poorly characterized. Biofilm formation of Acinetobacter oleivorans DR1 was significantly reduced by taurine only during alkane degradation, suggesting that taurine affects alkane-induced cell surface. Structurally similar compounds harboring an amine group such as hypotaurine or ethylenediamine have a similar effect, which was not observed with sulfonate-containing chemicals such as ethanesulfonic acid, hexanesulfonic acid. Our biochemical assays and physiological tests demonstrate that taurine reduced cell surface hydrophobicity, which resulted in interruption of the interactions between cells and oily substrate surfaces, such that cells utilized alkanes less effectively. Interestingly, taurine-mediated reduction of quorum sensing (QS) signal production and QS-control sapA gene expression indicated that membrane permeability of quorum signals was also interfered by taurine. Composition and biomass of extracellular polymeric saccharides were changed in taurine-amended conditions. Taken together, our data provide evidence that amine-containing taurine can inhibit biofilm formation of DR1 cells during alkane degradation by (i) changing cell surface charge and (ii) reducing membrane hydrophobicity and QS sensing.

The SapA Protein Is Involved in Resistance to Antimicrobial Peptide PR-39 and Virulence of Actinobacillus pleuropneumoniae.

Antimicrobial peptides are essential to the innate immune defense of the mammal against bacterial infection. However, pathogenic bacteria have evolved multiple strategies to resist and evade antimicrobial peptides, which is vital to bacterial survival and colonization in hosts. PR-39 is a linear porcine antimicrobial peptide containing 39 amino acid residues with a high proline content. Resistance to antimicrobial peptide PR-39 has been observed in Actinobacillus pleuropneumoniae. However, little is known about the factors required for this resistance. In the present study, PR-39 exposure increased the expression of the sapA gene in A. pleuropneumoniae. The sapA gene, which encodes a putative peptide transport periplasmic protein, was deleted from this bacterium. The ΔsapA mutant showed increased sensitivity to PR-39 compared to the wild-type MD12 and complemented PΔsapA strains. However, the ΔsapA mutant did not exhibit any alterations in outer membrane integrity. Scanning electron microscopy showed that the ΔsapA mutant displayed morphological defects, as indicated by a deformed and sunken shape after PR-39 treatment. In addition, disruption of the SapA protein led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. Collectively, these data suggest that SapA acts as one mechanism for A. pleuropneumoniae to counteract PR-39-mediated killing. To the best of our knowledge, this is the first study to show a mechanism underlying antimicrobial peptide resistance in A. pleuropneumoniae.

The Slam/SAP signaling pathway regulates γδ T cell effector functions

Abstract The Slam/SAP (SLAM-associated protein) signaling pathway plays a central role in the host immune response to infections. Males that inherit an inactivated SAP gene develop X-linked lymphoproliferative syndrome (XLP), a serious immunodeficiency that often results in enhanced susceptibility to a variety of pathogens. SAP is a cytosolic adapter protein that transduces signals from Slam family receptors, a family of nine (SLAMf1 to SLAMf9) cell surface receptors that are expressed only on hematopoietic cells. Although the Slam/SAP axis is known to regulate the development and function of several lymphocyte subsets, their role in γδ T cell biology is less well understood. We analyzed simultaneously the expression of multiple Slam receptors on γδ T cells and observed significant heterogeneity among γδ T cell subsets. Interestingly, Slam expression patterns marked functional γδ T cell subsets in both naïve and influenza-infected mice. SLAMf6 expression on lung γδ T cells correlated with IFNγ-producing CD27+ cells while SLAMf1 expression characterized IL-17-producing CD27− RORγt+ γδ T cells. The heterogeneous Slam receptor expression patterns of γδ T cell subsets can already be seen during embryonic thymic development, suggesting a possible role in the developmental programming of γδ T cell effector function. Disruption of Slam signaling through deletion of SAP affected the development of RORγt+ CD44hi Vγ4 T cells in the embryonic thymus, and the frequency of RORγt+ Vγ4 T cells in the periphery was still diminished in the adult. These results suggest that the Slam/SAP signaling pathway is an important regulator of the functional programming γδ T cells undergo during thymic development which affects mucosal immune responses later in life.

Isolation and characterization of LcSAP, a Leymus chinensis gene which enhances the salinity tolerance of Saccharomyces cerevisiae.

A number of members of the SAP ("stress-associated protein") gene family have been implicated in the plant stress response. Here, a SAP gene has been isolated using PCR RACE from the perennial grass Leymus chinensis, a species which has reputation for ecological adaptability. The 17.6kDa LcSAP product comprised 161 residues, including both an A20 domain and an AN1 domain, a feature of type I SAPs. Using a semi-quantitative RT-PCR assay to profile its transcription, it was shown that LcSAP was more strongly transcribed in the leaf than in the root under control conditions. The level of LcSAP transcription began to rise 6h after the plant's exposure to 400mM NaCl, and the abundance of transcript remained stable for at least 24h. Exposing the plant to 100mM Na2CO3 also induced LcSAP transcription, but the abundance of SAP transcript faded after 6h. When LcSAP was introduced into yeast cells, the transgenic cells grew better than wild type ones when the medium contained 1.4M NaCl. The ability of LcSAP to respond to salinity stress in yeast suggests that it also makes a contribution to the stress tolerance shown by L. chinensis.

The Synergistic Effect of Azoles and Fluoxetine against Resistant Candida albicans Strains Is Attributed to Attenuating Fungal Virulence.

This study evaluated the synergistic effects of the selective serotonin reuptake inhibitor, fluoxetine, in combination with azoles against Candida albicans both in vitro and in vivo and explored the underlying mechanism. MICs, sessile MICs, and time-kill curves were determined for resistant C. albicans Galleria mellonella was used as a nonvertebrate model for determining the efficacy of the drug combinations against C. albicans in vivo For the mechanism study, gene expression levels of the SAP gene family were determined by reverse transcription (RT)-PCR, and extracellular phospholipase activities were detected in vitro by the egg yolk agar method. The combinations resulted in synergistic activity against C. albicans strains, but the same effect was not found for the non-albicans Candida strains. For the biofilms formed over 4, 8, and 12 h, synergism was seen for the combination of fluconazole and fluoxetine. In addition, the time-kill curves confirmed the synergism dynamically. The results of the G. mellonella studies agreed with the in vitro analysis. In the mechanism study, we observed that fluconazole plus fluoxetine caused downregulation of the gene expression levels of SAP1 to SAP4 and weakened the extracellular phospholipase activities of resistant C. albicans The combinations of azoles and fluoxetine showed synergistic effects against resistant C. albicans may diminish the virulence properties of C. albicans.

RT-PCR detection of SAP1 gene in Candida albicans isolated from Meningitis cases

Objective : The main objective of the sampling is to isolate fungi associated with cases of meningitis ,study of some Virulence factors of fungi and sensitivity to antifungal agents.Methods: 100 CSF sample were suspected of meningitis culture ,biochemical test and API-System for diagnosis of fungi in sample ,Real-time PCR based on SYBR Green I fluorescent dye for detection of sap gene in Candida albicans.Results: Depending on agricultures characteristics ,Biochemical tests and Api System the results showed that 32 samples, 32% were given the positive result for yeast growth, Candida albicans found in these samples by 87.5% and Cryptococcus neoformans 12.5% and 25(89.28%) of C.albicans isolates had Sap gene. C. albicans showed the lowest MICs (1 μg/ml) for amphotericin B. and higher MICs for fluconazole (4 - 32 μg/ml) and Nystatin (8-32 μg/ml) While C.neoformans showed low MICs for all antifungal agents used in this study ranged from (0.06-0.5 μg/ml) for amphotericin B and ( 0.25 - 1 μg/ml) for fluconazole and ( 1-4 μg/ml) for nystatinConclusion: Fungal Meningitis which caused by Candida albicans which have SAP gene that consider as virulence factors and Resistant to antifungal agents and Cryptococcus neoformans is a debilitating and potentially deadly disease that affects patients with both intact and impaired immune systems. early diagnosis and treatment is essential for optimal outcomes. Patients with Fungal Meningitis have multisystem disturbances and require a well-organized and executed plan of care.

Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins

The association of ESBLs (extended-spectrum beta-lactamases)/pAmpCs (plasmid-mediated AmpC β-lactamases) with PMQR (plasmid mediated quinolone resistance) in gram-negative bacteria has been of great concern. The present study was performed to characterize the diversity, gene location, genetic context, and evolution of ampC and qnrB alleles in isolates of Citrobacter freundii. Fifteen isolates of C. freundii were identified from a total of 788 isolates of Enterobacteriaceae derived from humans, animals, animal food products, and the environment between 2010 and 2012. Co-existence of qnrB/ΔqnrB with ampC was detected in all C. freundii isolates. Both ampC and qnrB genes were found to be located on the chromosome, but were distantly separated on the chromosome. Seven and six novel alleles were discovered for the 10 ampC and qnrB variants detected in this study, respectively. Phylogenetic analysis showed that the new alleles differed a little from the variants of ampC/qnrB previously described in this genus. The genetic context surrounding ampC genes was AmpR-AmpC-Blc-SugE. However, five different genetic contexts surrounding qnrB/ΔqnrB genes were observed, but they occurred in all cases between the pspF and sapA genes. Additionally, cloning experiments showed that the regions containing different qnrB alleles, even with different genetic contexts, contributed to the reduction of quinolone susceptibility. Our results showed that the chromosomal ampC and qnrB alleles are closely related to C. freundii. However, unlike ampC, qnrB alleles seemed to be related to the genetic contexts surrounding them. The evolution of these two genes in C. freundii isolates might be through different pathways.

Expression ofSAP5andSAP9inCandida albicansbiofilms: comparison of bloodstream isolates with isolates from other sources

Secreted aspartic proteases (Sap), encoded by a family of 10 SAP genes, are key virulence determinants in Candida albicans. Although biofilm-associated bloodstream infections (BSIs) are frequently caused by C. albicans, SAP gene expression in C. albicans biofilms formed by BSI isolates has not been evaluated. We compared the expression of two SAP genes, SAP5 and SAP9, in C. albicans biofilms formed by BSI isolates with those formed by isolates from other body sites. Sixty-three C. albicans isolates were analyzed, comprising 35 BSI isolates and 28 from other sites. A denture-strip biofilm model was used, and expression of the two SAP genes was quantified by real-time RT-PCR during planktonic or biofilm growth. Mean SAP5 expression levels of the BSI isolates were 3.59-fold and 3.86-fold higher in 24-h and 48-h biofilms, respectively, than in planktonic cells. These results did not differ from those for isolates from other sites (2.71-fold and 2.8-fold for 24-h and 48-h biofilms, respectively). By contrast, mean SAP9 expression during biofilm formation was higher in BSI isolates (2.89-fold and 3.29-fold at 24 and 48 h, respectively) than in isolates from other sites (1.27-fold and 1.32-fold at 24 and 48 h, respectively; both, P < 0.001). These results show, for the first time, that both SAP5 and SAP9 are upregulated in C. albicans biofilms formed by BSI isolates, and that BSI isolates may have a greater capacity to express SAP9 under biofilm conditions than isolates from other sites.

Campylobacter fetus subspecies: Comparative genomics and prediction of potential virulence targets

The genus Campylobacter contains pathogens causing a wide range of diseases, targeting both humans and animals. Among them, the Campylobacter fetus subspecies fetus and venerealis deserve special attention, as they are the etiological agents of human bacterial gastroenteritis and bovine genital campylobacteriosis, respectively. We compare the whole genomes of both subspecies to get insights into genomic architecture, phylogenetic relationships, genome conservation and core virulence factors. Pan-genomic approach was applied to identify the core- and pan-genome for both C. fetus subspecies and members of the genus. The C. fetus subspecies conserved (76%) proteome were then analyzed for their subcellular localization and protein functions in biological processes. Furthermore, with pathogenomic strategies, unique candidate regions in the genomes and several potential core-virulence factors were identified. The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes in an island specific for C. fetus subsp. venerealis. The genomic variations and potential core and unique virulence factors characterized in this study would lead to better insight into the species virulence and to more efficient use of the candidates for antibiotic, drug and vaccine development.

Game theoretical modelling of survival strategies of Candida albicans inside macrophages

The polymorphic fungus Candida albicans can live as an aggressive pathogen that causes a wide variety of diseases in humans. Host resistance against these infections is mediated predominantly by phagocytes, namely neutrophils and macrophages. This report provides two game theoretical models of ingested C. albicans cells in macrophages. Two strategies are available for each pathogenic yeast cell: avoiding lysis transiently (called silencing) or forming hyphae and escaping (called piercing because the macrophage is pierced from inside). In dependence on parameter values, two different outcomes can be derived from the model: when the difference of the costs of the two strategies is low, all fungal cells inside a macrophage will play the piercing strategy, while in the high-cost case, a mixed population of piercing and silencing cells is the only stable solution. Further, the role of the SAP gene family encoding secreted proteinases and the Sap proteins is investigated with the help of known studies and is put in relation to the costs of the strategies, the most important parameter of this model. Our results are in agreement with wet-lab results presented by other groups and the model parameters can be estimated from experimental data.

Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398bp) and SapA-C (1422bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P<0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P0.45: positive; S/P<0.4: negative; 0.45>S/P0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.

The proapoptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease

Deletion or mutation of the SAP gene is associated with the X-linked lymphoproliferative disease (XLP) that is characterized by extreme sensitivity to Epstein-Barr virus (EBV). Primary infection of the affected individuals leads to serious, sometimes fatal infectious mononucleosis (IM) and proneness to lymphoma. Our present results revealed a proapoptotic function of SAP by which it contributes to the maintenance of T-cell homeostasis and to the elimination of potentially dangerous DNA-damaged cells. Therefore, the loss of this function could be responsible for the uncontrolled T-cell proliferation in fatal IM and for the generation of lymphomas. We show now the role of SAP in apoptosis in T and B lymphocyte-derived lines. Among the clones of T-ALL line, the ones with higher SAP levels succumbed more promptly to activation induced cell death (AICD). Importantly, introduction of SAP expression into lymphoblastoid cell lines (LCL) established from XLP patients led to elevated apoptotic response to DNA damage. Similar results were obtained in the osteosarcoma line, Saos-2. We have shown that the anti-apoptotic protein VCP (valosin-containing protein) binds to SAP, suggesting that it could be instrumental in the enhanced apoptotic response modulated by SAP.