| Listeria monocytogenes is an important food-borne pathogen that can cause septicaemia, encephalitis, meningitis and gastroenteritis in humans. L. monocytogenes encompasses a diversity of strains with varying pathogenic potential. While many L. monocytogenes strains could be of high pathogenicity, others are less virulent or even avirulent and produce little harm in the host. Recombinant bacterial vaccine vectors using L. monocytogenes as the model strain is one of the heated research areas in genetically engineered vaccine development. Currently, there are few studies in China on the biodiversity of L. monocytogenes, control strategies on its contamination along the food-processing lines, its gene structure and functions as well as its utilization as vaccine vectors upon proper attenuation.Mouse or chicken embryo based 50% lethal dose assays (LD50) and plaque forming assay in cell monolayers were used to compare the virulence of twenty L. monocytogenes food and environment isolates. Most L. monocytogenes isolates were as virulent as the reference strain 10403S with the mouse LD50 varying from 103.86 to 106.74 and the chicken embryonated egg LD50 from 101.23 to 103.35. The L monocytogenes isolate H4 was of low pathogenicity as revealed by its LD50 of 108.14 in mice and 106.73 in embryoniate chicken eggs. Most L. monocytogenes isolates formed clear plaques with plaque size varying from 83.7% to 108.3% relative to the reference strain 10403S. There was no visible plaque formed in isolate H4 infected cell monolayers, which was consistent with its low virulence as described above.Twenty L. monocytogenes strains isolated from food and environment sources together with the reference strain 10403S were characterized by pulsed-field gel electrophoresis (PFGE) using Sma I for genomic DNA digestion as well as by multilocus sequence typing (MLST) based on the hypervariable region of actA, InlA and InlB. All L. monocytogenes isolates could be divided into six subgroups using actA or InlA-based sequence typing, while the InlB -based sequence typing exhibited patterns slightly different from those of actA ox InlA. All L. monocytogenes isolates could be grouped into subtypes from S1 to S8 when the above three genes were considered together, with strains 0194, 10403S and H4 belonging to an individual subgroup. With PFGE, there were six subtypes of all L. monocytogenes isolates similar to MLST, with the low pathogenicity isolate H4 forming a separated subtype. Moreover, isolates 1056, 1057 and 6 exhibited the same sequence or PFGE subtype, indicating that the isolates 1056 and 1057 from the seafood products were contaminated by the environmental isolate 6. Isolates 320 and 690 shared the same sequence or PFGE subtype, illustrating that the isolate 320 from the milk product was contaminated by the environmental strain 690 other than from the dairy farm.A low pathogenicity isolate of Listeria monocytogenes from milk, as revealed in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Its corresponding virulence genes (iap, prfA, plcA, hly, mpl, actA, plcB, InlA and InlB) were compared with the reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S with LD50 108.14 vs 105.49 in mice and 106.73 vs 101.9 in chicken embryos). The genes prfA, plcA and mpl were generally homologous among L. monocytogens strains H4, 10403S and EGD (>98%). The gene hly between strain 10403S and isolate H4 had 96.9% identity at the nucleotide level, but 98.7% at the amino acid level. The nucleotide identity of plcB between strains 10403S and H4 was 95.4%, including one important mutation from A to G at position 1 of the plcB ORF and another important mutation from C to T at position -26. The act A gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acids deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.A pair of primers was designed to amplify the plcB homologous region containing its upstream sequence according to plcB sequence from the Listeria monocytogenes milk isolate H4. The plcB fragment was then purified and cloned into pUC18 to construct the recombinant plasmid pUC18-plcB. Three pairs of primers were then designed to introduce mutations of the plcB gene at positions -26 and +1 using pUC18-plcB as the template such that G mutated to A at +1 and T to C at -26 or single mutation from T to C at -26. The mutated plasmids confirmed by sequencing were subcloned into the shuttle vector pKSV7 to construct recombinant plasmids pKSV7-ΔplcB1, pKSV7-ΔplcB2 and pKSV7-ΔplcB3. After electroporation of three recombinant shuttle vectors into competent L. monocytogenes H4 cells, homologous recombination was initiated, resulting in replacement of the target nucleotide(s). PCR amplification of the target genes and sequencing confirmed successful construction of the mutated strains H4-ΔplcBl, H4-ΔplcB2 and H4-ΔplcB3. There was no phospholipase activity of three mutants as revealed by the egg yolk agar assay, indicating that high phospholipase activity of isolate H4 might be related to the mutation at position -26 (from C to T) with its ORF starting from position -27 relative to that in strain 10403S together with 9 extra amino acids. However, mechanisms of apparent expression of plcB due to extension of amino acids at the N terminus remain further exploration. The mutant strain H4-ΔplcB1 had increased virulence to mice of about 1 log in LD50 as compared with the wild parent isolate H4, indicating that high expression of both membrane damaging genes plcB and hly might result in synergistic cytotoxic activity, exposing listerial cells to the host immune response with their resultant elimination and reduced pathogenicity to the host.L. monocytogenes is an invasive intracellular bacterium that can survive and replicate in both professional and nonprofessional phagocytic cells, and becomes an attractive vaccine vector. To construct a recombinant strain of L, monocytogenes for expression of heterologous genes, homologous recombination was utilized for insertional mutation targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then cloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenesΔhly-gfp lost its hemolytic activity as visualized on the blood agar or analyzed with the culture supernatant samples and also exhibited reduced adherence and invasiveness ability to HeLa cells. Such insertional mutation resulted in reduced virulence of about 2 logs less than its parent strain 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models.Recombinant Listeria monocyogenes mutant carrying the truncated fragment Fa of the Newcastle disease virus (NDV) fusion protein gene was constructed by homologous recombination. The fragment Fa was integrated into plcB downstream of its signal sequence. Correct orientation of the inserted fragment was verified by PCR amplification. The inserted Fa was transcribed and expressed in the recombinant Lm-ΔplcB-Fa as shown by RT-PCR, SDS-PAGE and western blot respectively. The recombinant mutant exhibited reduced virulence to embryonated eggs and mice by about 1.7-2.3 logs, as compared with the parent wild strain 10403S. It was also less adherent or invasive than strain 10403S (P<0.05). Chickens receiving the recombinant strain Lm-ΔplcB-Fa orally or intraperitoneally were partially protected from virulent NDV challenge possibly due to enhancement of non-specific immunity because the antibody titers against the homologous virus strain or the recombinant truncated fusion protein were marginal. Further research is needed in other animal models to see if the low antibody response results from insufficient expression of the heterologous genes as a result of failure of L. monocytogenes or its recombinants to persist or replicate in chickens.In summary, results from these studies have laid good foundation for further exploration of the pathogenesis of L. monocytogenes, control of its contamination along the food-processing lines as well as its potential as vaccine vectors for other passenger antigens.
|
PDF Full Text Request