Houhui Song

Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay

Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 × 10(9) L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y = -0.6076x + 0.2441, R(2) = 0.9923) with the working range from 2.4 to 23.6 μg/kg, IC50 at 7.6 μg/kg and lower limit of detection at 1.4 μg/kg. The recovery rates in spiked samples were 91.2-110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R(2) = 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA.

Listeria monocytogenes varies among strains to maintain intracellular pH homeostasis under stresses by different acids as analyzed by a high-throughput microplate-based fluorometry

Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to determine pHi of listerial cells that were prelabeled with the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester and subjected to acid stress. We found that L. monocytogenes responds differently among strains toward organic and inorganic acids to maintain pHi homeostasis. The capacity of L. monocytogenes to maintain pHi at extracellular pH 4.5 (pHex) was compromised in the presence of acetic acid and lactic acid, but not by hydrochloric acid and citric acid. Organic acids exhibited more inhibitory effects than hydrochloric acid at certain pH conditions. Furthermore, the virulent stains L. monocytogenes EGDe, 850658 and 10403S was more resistant to acidic stress than the avirulent M7 which showed a defect in maintaining pHi homeostasis. Deletion of sigB, a stress-responsive alternative sigma factor from 10403S, markedly altered intracellular pHi homeostasis, and showed a significant growth and survival defect under acidic conditions. Thus, this work provides new insights into bacterial survival mechanism to acidic stresses.

A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA) was developed for rapid and sensitive detection of zearalenone (ZEN). The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904). The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283). We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

Listeria monocytogenes ArcA contributes to acid tolerance

The foodborne pathogen Listeria monocytogenes is able to colonize the human and animal intestinal tracts and subsequently crosses the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive and adapt to the low pH environment of the stomach. Gene sequence analysis indicates that lmo0043, an orthologue of arcA, encodes a protein containing conserved motifs and critical active amino acids characteristic of arginine deiminase that mediates an arginine deimination reaction. We attempted to characterize the role of ArcA in acid tolerance in vitro and in mice models. Transcription of arcA was significantly increased in L. monocytogenes culture subjected to acid stress at pH 4.8, as compared with that at pH 7.0. Deletion of arcA impaired growth of L. monocytogenes under mild acidic conditions at pH 5.5, and reduced its survival in synthetic human gastric fluid at pH 2.5 and in the murine stomach. Bacterial load in the spleen of mice intraperitoneally inoculated with an arcA deletion mutant was significantly lower than that of the wild-type strain. These phenotypic changes were recoverable by genetic complementation. Thus, we conclude that L. monocytogenes arcA not only mediates acid tolerance in vitro but also participates in gastric survival and virulence in mice.

Listeria monocytogenes aguA1, but not aguA2, encodes a functional agmatine deiminase: Biochemical characterization of its catalytic properties and roles in acid tolerance

Background: Listeria monocytogenes has two putative agmatine deiminase homologs, AguA1 and AguA2. Results: Only AguA1, but not AguA2, acts as functional agmatine deiminase and mediates acid tolerance in L. monocytogenes. Conclusion: Provided is the first biological insight into the roles of AgDI in acid tolerance of L. monocytogenes. Significance: We have discovered a novel residue Gly-157 other than the known catalytic triad (Cys-His-Glu/Asp) critical for L. monocytogenes AgDI activity. Listeria monocytogenes is adaptable to low pH environments and therefore crosses the intestinal barrier to establish systemic infections. L. monocytogenes aguA1 and aguA2 encode putative agmatine deiminases (AgDIs) AguA1 and AguA2. Transcription of aguA1 and aguA2 was significantly induced at pH 5.0. Deletion of aguA1 significantly impaired its survival both in gastric fluid at pH 2.5 and in mouse stomach, whereas aguA2 deletion did not show significant defect of survival in gastric fluid. With agmatine as the sole substrate, AguA1 expressed in Escherichia coli was optimal at 25 °C and over a wide range of pH from 3.5 to 10.5. Recombinant AguA2 showed no deiminase activity. Site-directed mutagenesis revealed that all nine AguA1 mutants completely lost enzymatic activity. AguA2 acquired AgDI activity only when Cys-157 was mutated to glycine. AguA1 mutation at the same site, G157C, also inactivated the enzyme. Thus, we have discovered Gly-157 as a novel residue other than the known catalytic triad (Cys-His-Glu/Asp) in L. monocytogenes that is critical for enzyme activity. Of the two putative AgDIs, we conclude that only AguA1 functionally participates in the AgDI pathway and mediates acid tolerance in L. monocytogenes.

Listeria monocytogenes 10403S Arginine Repressor ArgR Finely Tunes Arginine Metabolism Regulation under Acidic Conditions

Listeria monocytogenes is able to colonize human and animal intestinal tracts and to subsequently cross the intestinal barrier, causing systemic infection. For successful establishment of infection, L. monocytogenes must survive the low pH environment of the stomach. L. monocytogenes encodes a functional ArgR, a transcriptional regulator belonging to the ArgR/AhrC arginine repressor family. We aimed at clarifying the specific functions of ArgR in arginine metabolism regulation, and more importantly, in acid tolerance of L. monocytogenes. We showed that ArgR in the presence of 10 mM arginine represses transcription and expression of the argGH and argCJBDF operons, indicating that L. monocytogenes ArgR plays the classical role of ArgR/AhrC family proteins in feedback inhibition of the arginine biosynthetic pathway. Notably, transcription and expression of arcA (encoding arginine deiminase) and sigB (encoding an alternative sigma factor B) were also markedly repressed by ArgR when bacteria were exposed to pH 5.5 in the absence of arginine. However, addition of arginine enabled ArgR to derepress the transcription and expression of these two genes. Electrophoretic mobility shift assays showed that ArgR binds to the putative ARG boxes in the promoter regions of argC, argG, arcA, and sigB. Reporter gene analysis with gfp under control of the argG promoter demonstrated that ArgR was able to activate the argG promoter. Unexpectedly, deletion of argR significantly increased bacterial survival in BHI medium adjusted to pH 3.5 with lactic acid. We conclude that this phenomenon is due to activation of arcA and sigB. Collectively, our results show that L. monocytogenes ArgR finely tunes arginine metabolism through negative transcriptional regulation of the arginine biosynthetic operons and of the catabolic arcA gene in an arginine-independent manner during lactic acid-induced acid stress. ArgR also appears to activate catabolism as well as sigB transcription by anti-repression in an arginine-dependent way.

Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions

Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the ΔtrxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA, and plcB. Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for protein folding and activation, and therefore contributes to bacterial virulence and motility.

Aminopeptidase T of M29 Family Acts as A Novel Intracellular Virulence Factor for Listeria monocytogenes Infection.

The foodborne pathogen Listeria monocytogenes employs a number of virulence determinants including metalloproteases to infect hosts. Here for the first time, we identified an M29 family aminopeptidase T (encoded by lmo1603) from L. monocytogenes that possesses a typical feature to catalyze the cleavage of amino acids from peptide substrates, with a preference for arginine. The purified recombinant Lmo1603 was activated by Fe3+, Zn2+ and Mn2+, but strongly stimulated by Co2+, indicating that Lmo1603 is a cobalt-dependent aminopeptidase. Single mutation at any of the Glu216, Glu281, His308, Tyr315, His327, and Asp329 completely abolished the enzymatic activity of Lmo1603. More importantly, we showed that Lmo1603 was mainly involved in Listeria infection, but not required for growth in rich laboratory medium and minimal defined medium. Disruption of Lmo1603 resulted in almost complete attenuation of Listeria virulence in a mouse infection model. In addition, we demonstrated that Lmo1603 was mainly localized in the bacterial cytosol and required for invasion and survival inside human epithelial cells and murine macrophages. We conclude that Lmo1603 encodes a functional aminopeptidase T of M29 family, which acts as a novel intracellular virulence factor essential in the successful establishment of L. monocytogenes infections in a mouse model.

The viral non-structural protein 1 alpha (Nsp1α) inhibits p53 apoptosis activity by increasing murine double minute 2 (mdm2) expression in porcine reproductive and respiratory syndrome virus (PRRSV) early-infected cells.

Apoptosis is one of the most important mechanisms of pathogenesis in porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells. The tumor suppressor p53 plays a critical role in apoptotic induction in viral infections. In the present study, we found that p53 activity was inhibited at the early stage of PRRSV infection in both the highly pathogenic (HP) and lowly pathogenic (LP) PRRSV isolates. Bax expression showed a similar change pattern to that of p53. Murine double minute 2 (mdm2) expressed higher in PRRSV-infected cells than in uninfected cells at the early stage of infection and promoted p53 degradation. We show for the first time that the non-structural protein 1 alpha (Nsp1α) of PRRSV is a negative regulator of p53 activity through increasing mdm2 expression and p53 ubiquitination, while p53 is inhibitory to PRRSV replication at the early stage of infection. We conclude that PRRSV manipulates the host factors mdm2 and p53 via its Nsp1α for increased replication at the early stage of infection. These provide a novel perspective to understand the interaction between apoptosis and replication of PRRSV.

Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7.

Listeria monocytogenes encodes a transcriptional activator, PrfA, to positively regulate the expression of virulence factors. Several mutations in PrfA (PrfA*) have been found to contribute to increased regulatory activity. Here, we describe a strain, M7, containing a PrfA*(G145S) that activates expression of virulence factors but with low pathogenicity. To study this contradictory relationship, we exchanged the prfA genes between strains EGDe and M7 (designated EGDe-prfA(M7) and M7-prfA(EGDe)). The phospholipase B (PlcB) and listeriolysin O (LLO) activities were significantly upregulated in the strain EGDe-prfA(M7) (PrfA*). Constitutive activation of PrfA potentiated virulence of the pathogenic strain EGDe, shown as increased adhesion and invasion as well as enhanced cell-to-cell spread in cultured cell lines. However, the strain M7, though PrfA-activated, had significant defects in these virulence-related phenotypes and low pathogenicity in the murine infection model, as compared with EGDe or EGDe-PrfA(M7). To further uncover the possible mechanisms, we analysed abundance and distributions of InlA, InlB, LLO and ActA proteins, all regulated by PrfA, in EGDe, M7 and their prfA mutants. Western blotting showed that the PrfA-regulated genes of constitutively activated PrfA strains were overexpressed in vitro, while different distributions were observed. In contrast to the virulent strain EGDe-prfA(M7), the majority of InlB in M7 was detected in the culture supernatant and not on the bacterial surface. We suppose that the low virulence of strain M7 is due to its defects in infecting host cells, possibly as a result of failed anchorage on the bacterial cells of surface proteins like InlB, a major protein involved in adhesion and invasion of pathogenic L. monocytogenes strains. Further research is warranted to address why InlB detaches from the bacterial cells of this particular strain.