Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization

Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization

Comprehensive Identification of Meningococcal Genes and Small Noncoding RNAs Required for Host Cell Colonization

Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N. meningitidis life cycle that is poorly understood. Here, highly saturated random transposon insertion libraries of N. meningitidis were engineered, and the fitness of mutations during routine growth and that of colonization of endothelial and epithelial cells in a flow device were assessed in a transposon insertion site sequencing (Tn-seq) analysis. This allowed the identification of genes essential for bacterial growth and genes specifically required for host cell colonization. In addition, after having identified the small noncoding RNAs (sRNAs) located in intergenic regions, the phenotypes associated with mutations in those sRNAs were defined. A total of 383 genes and 8 intergenic regions containing sRNA candidates were identified to be essential for growth, while 288 genes and 33 intergenic regions containing sRNA candidates were found to be specifically required for host cell colonization. IMPORTANCE: Meningococcal meningitis is a common cause of meningitis in infants and adults. Neisseria meningitidis (meningococcus) is also a commensal bacterium of the nasopharynx and is carried by 3 to 30% of healthy humans. Under some unknown circumstances, N. meningitidis is able to invade the bloodstream and cause either meningitis or a fatal septicemia known as purpura fulminans. The onset of symptoms is sudden, and death can follow within hours. Although many meningococcal virulence factors have been identified, the mechanisms that allow the bacterium to switch from the commensal to pathogen state remain unknown. Therefore, we used a Tn-seq strategy coupled to high-throughput DNA sequencing technologies to find genes for proteins used by N. meningitidis to specifically colonize epithelial cells and primary brain endothelial cells. We identified 383 genes and 8 intergenic regions containing sRNAs essential for growth and 288 genes and 33 intergenic regions containing sRNAs required specifically for host cell colonization

Additional file 5: of Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing

Clinical pictures of patients #8 (A) and #9 (B) (mutated in ERCC8(CSA)). (PPTX 403 kb

Surveillance on classical swine fever virus persistently infected farms and phenotypic alterations of peripheral blood mononuclear cells of swine after virus infection

中 文 摘 要豬瘟為豬之急性病毒性疾病，經常造成 嚴重的經濟損失。豬瘟病毒之持續性感染現象為豬瘟防疫及清除工作上所 需克服之難題。為配合政府豬瘟清除計畫之執行及了解豬瘟病毒在豬瘟污 染場中持續感染狀況，以病理學配合RT-PCR檢查，長期監控三場豬瘟污染 之一貫作業養豬場之病弱保育豬隻潛在感染豬瘟狀況，並配合豬瘟抗體 ELISA檢測，以了解豬場之群體免疫力。結果顯示，在持續一年的監控期 間，豬場內雖沒有典型豬瘟病例發生，但仍能持續從病弱豬隻中發現疑似 豬瘟病變及可檢測出豬瘟病毒RNA存在，證明豬瘟病毒確能在病弱豬群間 長期潛伏感染。由血清ELISA檢測結果，亦顯示在正常豬瘟活毒疫苗免疫 計畫下，仍有少數豬隻未呈現抗體反應，而易暴露於豬瘟病毒感染之威脅 下。進一步延續田間監控之試驗，在實驗室條件下，觀察免疫豬隻遭受野 外強毒攻毒後之細胞性免疫反應與淋巴次族群之變化情形，並探討豬瘟病 毒在免疫後耐過豬隻中持續性感染情形。結果指出，免疫過的豬隻在經豬 瘟病毒攻擊後，淋巴細胞對 concanvalin A 刺激引起的增殖反應，在大 部分豬隻皆不受到影響，但在少部份免疫反應不良之個體，呈現輕微抑制 的現象。在淋巴細胞次族群的變化方面，經免疫過的豬隻IgM+、CD4-CD8+ 、CD4+CD8-及CD4+CD8+淋巴細胞有暫時性抑制後上升的現象，但在不同免 疫方式下其改變有些許不同。其中以哺乳前免疫加六週齡免疫組在豬瘟病 毒攻擊後，血液中淋巴次族群的變化較小。免疫過的豬隻在攻毒後一個月 ，仍可以RT-PCR檢測出豬瘟病毒存在於淋巴臟器中，顯示免疫豬隻仍有帶 野外毒之可能。綜合各實驗結果，豬瘟病毒在豬場病弱保育豬隻及正常免 疫豬群中可長期持續感染。因此，加強豬瘟污染場之監控，完整的免疫計 畫，並配合病弱豬淘汰，有助於將豬瘟病毒由豬場中清除。AbstractClassical swine fever ( CSF ) is a highly contagious disease of swine and leads to severe economic losses. The persistence of CSF virus (CSFV) infection is the major problem in the control and eradication of the disease. In corresponding to the CSFV eradication program in Taiwan and the investigation of CSFV persistence in pig farms, a monitoring system of pathological examination and reverse transcriptase - polymerase chain reaction ( RT-PCR ) method focused on weak nursing pigs were undertaken on three CSFV contaminated farms. Over one-year monitoring, there was no typical clinical CSF case, however, presumable pathological lesions and positive reaction of RT-PCR for CSFV could be persistently detected in those random samples from all three pig farms. Moreover, a serological survey on these pig farms with routine LPC vaccination programs also indicated that there were still some portions of pigs lack of CSF ELISA antibody response, which might be susceptible for CSFV infection. Following monitoring study of CSF virus persistence, the alteration of subpopulations of peripheral blood mononuclear cells, lymphoproliferative responses, and virus persistence were carried on pigs with three different CSF vaccination programs and control pigs, which were challenged with virulent CSFV ( 2*10^7 pfu). The results indicated that the lymphoproliferative function of most vaccinated pigs was not significantly affected, however, the function in a few vaccinated pigs with poor immune response was partially impaired. The alterations of lymphocyte subpopulations, including, IgM+, CD4-CD8+, CD4+CD8-, and CD4+ CD8+ lymphocytes showed slight decrease during early phase of infection and then returned to normal level later. Pigs vaccinated before consumption of colostrum and booosted at 6-week-old showed the less effect on these alterations and less clinical signs after CSFV challenge. However, virulent virus RNA from recovered pigs could be detected by RT-PCR method over 1 month after CSFV challenged (2*107 pfu). Conclusively, CSFV could long term persistence in weak nursing pigs in CSFV contaminated pig farms. Therefore, to eradicate CSFV from contaminated pig farms, an intensive CSF vaccination program and a monitoring system on CSF persistence by RT-PCR combined with a strict culling strategy might be required