选择性捕获禽病原性大肠杆菌体内转录序列

Identification of APEC genes expressed in vivo by selective capture of transcribed sequences

摘要:

采用选择性捕获转录序列(SCOTS)方法鉴定禽病原性大肠杆菌E037株(血清型O78)在感染SPF鸡过程中的转录表达基因。通过总RNA分离、cDNAs合成、PCR扩增和SCOTS对cDNAs选择和致病性特异转录序列的富集,致病性特异的cDNAs被分离鉴定,共获得31个转录序列(命名为aec),其中分别有2、1、4、14、2和8个aec序列与黏附素、LPS的合成、铁的摄取系统、质粒编码基因、噬菌体编码基因和一些其它功能基因相关;从气囊中分离到16个aec序列,心包膜中分离到15个aec序列;有3种与质粒编码基因相关序列在气囊和心包膜中都被分离到。结果显示APEC致病性特异序列包括黏附素、LPS的合成、铁的转运、质粒编码基因、噬菌体编码基因和一些其它功能基因等。通过SCOTS方法建立了一种体内表达致病性特异基因的方法和APEC在自然宿主感染模型中致病性相关基因的表达谱的筛选方法。

Abstract:

Direct screening of bacterial genes expressed during infection in the host is limited, because isolation of bacterial transcripts from host tissues necessitates separation from the abundance of host RNA. Selective capture of transcribed sequences (SCOTS) allows the selective capture of bacterial cDNA derived from infected tissues using hybridization to biotinylated bacterial genomic DNA. Avian pathogenic E.coli strain E037 (serogroup O78) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. Three-week-old white leghorn specific-pathogen-free chickens were inoculated into the right thoracic air sac with a 0.1mL suspension containing 107CFU of APEC strain E037. Total RNA was isolated from infected tissues (pericardium and air sacs) 6 or 24h postinfection and converted to cDNAs. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-pathogenic E.coli K-12 strain ) transcripts, pathogen-specific cDNAs were identified. Randomly chosen cDNA clones derived from transcripts in the air sacs or pericardium were selected and sequenced. The clones, termed aec, contained numerous APEC-specific sequences. Among the distinct 31 aec clones, pathogen-specific clones contained sequences homologous to known and novel putative bacterial virulence gene products involved in adherence, iron transport, lipopolysaccharide (LPS) synthesis, plasmid replication and conjugation, putative phage encoded products, and gene products of unknown function. Overall, the current study provided a means to identify novel pathogen-specific genes expressed in vivo and insight regarding the global gene expression of a pathogenic E.coli strain in a natural animal host during the infectious process.