Table_1_orf6 and orf10 in Prophage phiv142-3 Enhance the Iron-Acquisition Ability and Resistance of Avian Pathogenic Escherichia coli Strain DE142 to Serum.DOC

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), is the causative agent of avian colibacillosis, a disease that causes huge economic losses in the poultry industry and is characterized by infection through respiratory tract colonization followed by bacteraemia. A previous study in our lab demonstrated that phiv142-3 enhanced the survival ability of APEC strain DE142 in chickens serum. However, the mechanism of this affect has not been completely revealed. Here, we analyzed the transcriptional level of the prophage phiv142-3 region in DE142 when grown in chicken serum. Several upregulated genes attracted our attention, and a series of mutants were constructed. Deletion of orf6 or orf10 from phiv142-3 led to lower yields compared with WT after cultivation in serum for 10 h (P < 0.05). Furthermore, avian infection assays showed that compared with WT, the bacterial loads in blood and heart tissue of chickens challenged with DE142Δorf6 were decreased to 3.9 and 13%, while the bacterial burden in blood and heart from chickens infected with DE142Δorf10 was decreased to 7.2 and 8%, respectively (P < 0.05). DE142Δorf6 showed an obviously attenuated growth rate in the logarithmic phase when cultured in iron-deficient medium, and the transcription level of the iutA gene decreased to 43% (P < 0.05). The bactericidal assays showed that the survival of the mutant DE142Δorf10 was ~60% compared with WT in 50% chicken serum. The K1 capsule-related genes (kpsF, kpsE, kpsC, and kpsM) were down-regulated nearly 2-fold in DE142Δorf10 (P < 0.01). Together, these results suggested that orf6 affects growth by contributing to the uptake ability of iron, while orf10 increases resistance to serum by upregulating K1 capsule-related genes.

禽致病性大肠杆菌（Avian pathogenic Escherichia coli，APEC）属于肠外致病性大肠杆菌（Extraintestinal pathogenic E. coli，ExPEC），是引发禽大肠杆菌病的病原菌。该疾病给家禽养殖业造成巨额经济损失，其致病特征为经呼吸道定植后引发菌血症。本实验室此前的一项研究表明，前噬菌体（prophage）phiv142-3可增强禽致病性大肠杆菌DE142菌株在鸡血清中的存活能力，但具体作用机制尚未完全阐明。本研究中，我们对在鸡血清中培养的DE142菌株的前噬菌体phiv142-3区域的转录水平进行了分析。多个上调表达的基因引发了我们的关注，据此构建了一系列基因突变菌株。在鸡血清中培养10小时后，敲除phiv142-3的orf6或orf10基因的突变株的菌体量较野生型（Wild Type，WT）显著降低（P < 0.05）。此外，禽感染实验结果显示，相较于野生型菌株，用DE142Δorf6攻毒的雏鸡血液与心脏组织中的细菌载量分别降至原水平的3.9%与13%；而用DE142Δorf10感染的雏鸡，其血液与心脏组织的细菌负荷量分别降至7.2%与8%（P < 0.05）。在缺铁培养基中培养时，DE142Δorf6在对数生长期的生长速率明显减弱，且iutA基因的转录水平降至原水平的43%（P < 0.05）。杀菌实验结果表明，在50%鸡血清中，DE142Δorf10突变株的存活率约为野生型菌株的60%。DE142Δorf10中，K1荚膜相关基因（kpsF、kpsE、kpsC与kpsM）的表达量下调了近2倍（P < 0.01）。综合以上结果可知，orf6通过影响铁摄取能力调控菌株生长，而orf10则通过上调K1荚膜相关基因的表达，增强菌株对血清的抵抗能力。