組織侵襲性克雷伯氏肺炎桿菌中莢膜血清型K1的基因決定區

The cps Region Responsible for Capsular Serotype K1 in Klebsiella pneumoniae Causing Human Primary Pyogenic Liver Abscess

莊依萍(Yi-Ping Chuang)

指導教授 ： 王錦堂

國立臺灣大學/醫學院/微生物學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00856

摘要

克雷伯氏肺炎桿菌是革蘭氏陰性的桿菌，隸屬於腸內菌科，為一重要的伺機性感染的病原菌。近20年來，一種新型的侵襲性克雷伯氏肺炎桿菌感染逐漸躍升為一個全球性的新興重要社區感染症。最常見的臨床表現為原發性肝膿瘍合併菌血症及轉移性眼內炎或腦膜炎。菌株觀察發現高黏性型態和上述克雷伯氏肺炎桿菌的侵襲性感染有關；因此利用跳躍基因所建構的突變株庫找到一個與高黏性型態有關的基因，magA（mucoviscosity-associated gene A）； magA突變菌株失去對健康人的血清及嗜中性白血球的抗性，並失去致病力。合併比較莢膜血清型的統計發現，從帶有原發性肝膿瘍病人身上分離而得的42株臨床菌株中，35株為K1；而從不帶有原發性肝膿瘍病人身上分離而得的32株臨床菌株中，只有1株為K1（35/42 vs. 1/32， p<0.001）。此36株K1的菌株經由聚合酶鏈反應分析，皆帶有magA；其餘38株非K1的菌株則不帶有magA（36/36 vs. 0/38，p<0.0001）。序列分析magA的上下游區域，顯示magA位在莢膜生合成基因組中（galF至ugd共20 ORFs：open-reading frames）；其中，包含magA的9個ORFs（共10 kb）與MGH78578（K52）、Chedid（K2）的相對區域序列歧異度很大。針對NTUH-K2044此段區域其中4個基因作插入性突變，發現突變株失去了與抗K1血清凝集的反應，但若進行異位互補，則抗K1的凝集反應可以回復；此外，包含magA的11 kb區域（wzx至rfbP）以異位互補的方式送進5株非K1的克雷伯氏肺炎桿菌菌株可將其中2株的莢膜血清型轉為K1陽性。同時利用聚合酶鏈反應分析，認為此段11 kb區域中，wzx-gmd及wcaI都是莢膜血清型K1獨有的部份。因此我們認為magA位在25 kb的莢膜生合成基因組上，其中wzx至wcaI（包括magA）為莢膜血清型K1的基因決定區域，並與克雷伯氏肺炎桿菌造成原發性肝膿瘍有高度相關。

關鍵字

克雷伯氏肺炎桿菌 ； 莢膜 ； 組織侵襲性

並列摘要

Klebsiella pneumoniae is a Gram-negative bacillus belonging to Enterobacteriace, which is a opportunistic pathogen. However, a new type of tissue-invasive K. pneumoniae infection has emerged over the past two decades. The common clinical symptoms represents primary liver abscess complicated with (or without) septic meningitis and endophthalmitis. Mucoviscosity of K. pneumoniae strains was hypothesized to be correlated with tissue-invasive infection by clinical manifestation. We constructed mutant library of NTUH-K2044 using transposon mutagenesis and screened for mucoviscosity-deficient mutants. A magA (mucoviscosity-associated gene A) gene was thus identified. magA mutant strain was susceptible to non-immune human serum as well as neutrophil phagocytosis and became avirulent. Analysis of magA and serotype revealed that thirty-five of 42 strains from patients with primary liver abscess were magA positive while only 1 of 32 non- primary liver abscess strains positive. All 36 magA-positive strains were serotype K1 and the 38 magA-negative strains were not (36/36 vs. 0/38, p<0.0001). Sequencing of magA-flanking region revealed a putative capsular polysaccharide synthesis (cps) region; this region was 25kb in length and contained 20 ORFs. Of them, there were 9 ORFs (10 kb) which were co-transcribed as part of an operon and different from both MGH78578 and Chedid strains. Mutation of 4 genes in this region turned the mutant strains into anti-K1 sero-negative. Trans-complementations restored K1 phenotype. The magA-flanking regions, wzx to rfbP, successively converted 2 of 5 non-K1 K. pneumoniae strains into K1 sero-positive by trans-complementation. Thus we concluded that the operon containing magA is responsible for capsular serotype K1 in K. pneumoniae. Several loci in the operon are unique determinants for K1 strains. These results may further help to the early detection and tracing the origin of this emerging infectious disease.

並列關鍵字

Klebsiella pneumoniae ； capsule ； K1 ； magA ； mucoid ； tissue-invasive

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