Van Stelten A, Nightingale K K
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171, USA.
Appl Environ Microbiol. 2008 Dec;74(23):7365-75. doi: 10.1128/AEM.01138-08. Epub 2008 Oct 3.
The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes isolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing of inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA PMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novel inlA PMSC mutations. PMSCs in inlA were significantly (ribotypes DUP-1039C and DUP-1045B; P < 0.001) or marginally (ribotype DUP-1062D; P = 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S. L. monocytogenes isolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiate L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.
毒力因子内化素A（InlA）可促进单核细胞增生李斯特菌被表达人E-钙黏蛋白异构体的上皮细胞摄取。先前的研究在inlA中鉴定出自然发生的提前终止密码子（PMSC）突变，并证明这些突变导致毒力减弱。我们收集了来自不同来源的1700多株单核细胞增生李斯特菌分离株，代表90种EcoRI核糖型。根据核糖型频率从该分离株集合中选择了一个子集，并通过Caco-2细胞侵袭试验进行了表征。对侵袭能力减弱的分离株的inlA基因进行测序，发现了三个新的inlA PMSC，这些在美国分离株中以前未被鉴定出来。由于核糖型包括有和没有inlA PMSC的分离株，我们开发了一种多重单核苷酸多态性（SNP）基因分型检测方法，以检测inlA中具有毒力减弱PMSC突变的分离株。该SNP基因分型检测方法可检测到全世界报道的所有inlA PMSC突变，并通过用荧光标记的双脱氧核苷三磷酸延伸未标记引物在本研究中得到验证。我们实施了SNP基因分型检测方法，以表征代表与新的inlA PMSC突变相关的常见核糖型的人类临床和食品分离株。inlA中的PMSC在食品分离株中比人类临床分离株显著（核糖型DUP-1039C和DUP-1045B；P<0.001）或略微（核糖型DUP-1062D；P=0.11）更常见。SNP基因分型揭示了美国单核细胞增生李斯特菌分离株中的第四个新的PMSC突变，该突变先前在来自法国和葡萄牙的分离株中观察到。监管机构和食品行业可采用这种SNP基因分型检测方法，以区分携带inlA中毒力减弱PMSC突变的单核细胞增生李斯特菌分离株与代表最大健康风险的菌株。
