開發偵測不同致病菌上的pseudaminic acid的抗體探針

Abstract

Pse單醣屬於壬二酸家族的一種醣類，它以參與細菌的致病過程以及抑制宿主免疫反應而聞名。在最近的研究中，鮑氏不動桿菌作為人類致病菌之一，發現其胞外多醣上有Pse單醣的存在，它通常會引起院內感染並使鮑氏不動桿菌產生多重抗藥性，從而對全球健康構成威脅。在臨床研究中，Pse單醣可能扮演與生物膜形成以及抗生素治療相關的致病因子，相對於沒有Pse單醣的鮑氏不動桿菌，帶有Pse單醣的鮑氏不動桿菌會造成病患感染後較高的死亡率。因此，我們迫切需要發展出能夠檢測細菌表面上Pse單醣的抗體探針。根據先前研究得知，在鮑氏不動桿菌菌株54149的胞外多醣上發現了Pse單醣，結果證實Pse對鮑氏不動桿菌菌株54149衍生的胞外多醣結合物誘導所產生的高效血清有顯著抗原性，因此可作為探針候選物。在此研究中，我們從高效血清中純化出抗體，再藉由帶NHS基團的連接子與螢光染劑Dylight-488結合製成我們所需的Pse單醣探針。實驗後發現，鮑氏不動桿菌菌株54149的熒光強度與其細菌數呈線性關係，此結果顯示了該Pse單醣探針的可行性。此外，我們利用此Pse探針成功偵測除了鮑氏不動桿菌以外且帶有Pse單醣的致病菌陰溝腸桿菌。與傳統偵測方法相比，目前發展出的跨物種Pse抗體探針不僅具有輕微技術上限制的經濟效益，而且具有更廣泛的用途，在未來的臨床早期診斷上，有利於病患及時接受抗生素治療。

Pseudaminic acid (Pse) belongs to the family of sugars called nonulosonic acids and has known for participating in pathogenic process of bacteria, including suppression of host immune response. Currently, Pse has been found on the exopolysaccharide (EPS) of Acinetobacter baumannii that commonly caused nosocomial infection with multiple antibiotic resistance, resulting in global health threat. In the clinical study, Pse was possibly acting as a virulence factor associated with biofilm formation and antibiotic therapy. Patients infected by Ab with Pse might have higher mortality than Ab without Pse. Therefore, the requirement of a probe that can detect the Pse on the bacterial surface is urgent. In our previous study, Pse was found on the EPS of A. baumannii strain 54149 (Ab-54149) and displayed the significant antigenicity toward Ab-54149 EPS derived glycoconjugate boosted serum as a candidate of probe. Here, we purified the antibody from boosted serum and then was conjugated with fluorophore Dylight-488 through an NHS-based linker for detection. Upon Ab-54149 treatment, the linear relationship between the fluorescence intensity and the OD value indicated the feasibility of the strategy. Moreover, this probe enables to detect the surface Pse on not only other A. baumannii clinical strains but also Enterobacter cloacae. This Pse probe is cost-effective with slight technical restriction, leading to wide usage compared to the conventional method. These efforts benefit the early diagnosis in the clinic that patients might be treated by antibiotics timely.