Transcriptional Profiling of Neisseria gonorrhoeae biofilm

Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms over glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of this biofilm formation, transcriptional profiles of N. gonorrhoeae biofilm were compared to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 hours in continuous flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. When biofilm was compared to planktonic growth, 3.8 % of the genome was differentially regulated. Genes highly up-regulated in biofilm included aniA, norB, and ccp, which play critical roles in anaerobic metabolism and oxidative stress tolerance. Down-regulated genes included the nuo gene cluster (NADH dehydrogenase) and the cytochrome bcI complex, which are involved in aerobic respiration and are thought to contribute to endogenous oxidative stress. Furthermore, we determined that aniA, ccp, and norB insertional mutants are attenuated for biofilm formation over glass and transformed human cervical epithelial cells (THCEC). This data suggests that biofilm formation could minimize oxidative stress during cervical infection and allow N. gonorrhoeae to maintain a nitric oxide steady state that may be anti-inflammatory. Six biofilm flow chambers were inoculated with N. gonorrhoeae strain 1291 wildype. Flow chambers were inoculated with approximately 1 ml of a cell suspension (in biofilm media) from an overnight plate that was suspended to an OD600 of 0.3. Two flow cells each were supplied by a single reservoir of media and the runoff from these two flow cells was passed through a sterile glass wool filter for the removal of detached biofilm flocs, and collected in a sterile waste flask and cultured to assess culture purity. At this time, the waste flask was replaced with a second sterile flask containing 10 ml of RNAlater® (Qiagen Corporation, Valencia, CA) and 100 µl of 10% sodium azide. For the final 24 hours of biofilm growth, the filtered planktonic effluent was collected in this solution to preserve the RNA and prevent transcriptional changes from occurring. Planktonic RNA was extracted from the collected effluent of the two flow cells, while each biofilm RNA was extracted directly from these two flow cells. RNA purity was assessed on an Agilent 2100 Bioanalyzer (Quantum Analytics, Foster City, CA), and only samples with RNA integrity numbers of 7.5 or greater were used for hybridization to microarrays. All six samples had RNA integrity numbers of 7.5 or greater. Therefore, we elected to hybridize the two samples with the highest integrity numbers.