Gene regulation and antigenic variation of pili in Neisseria gonorrhoeae and Neisseria meningitidis LinYa-Hsun 2017 Neisseria gonorrhoeae and Neisseria meningitidis are dedicated human pathogens, causing gonorrhoea and meningitis, respectively. Initial attachment to host cells during infection is absolutely dependent on the expression of type IV pili (tfp) which can either be class I (CI, expressed by N. gonorrhoeae and N. meningitidis) or class II (CII, expressed by some strains of N. meningitidis). Tfp biogenesis is complex and requires a large set of Pil proteins encoded by pil genes. The mechanisms controlling piliation are poorly understood in both species. As tfp are essential for virulence, we aimed to characterise the regulation of tfp biogenesis and functionality in N. gonorrhoeae and N. meningitidis. The gonococcal pilE gene, encoding the major pilin subunit, has been shown to be positively regulated by Integration Host Factor (IHF). Furthermore, transcription of pilE and ihf appear to decrease in the stationary phase of growth. We therefore investigated whether the expression of other pil genes in N. gonorrhoeae and the meningococcal CII pilE, are also controlled by IHF and growth phase. In contrast to what was observed with gonococcal pilE, site-directed and deletion mutagenesis studies indicated that CII pilE and other pil genes are not regulated by IHF. We have also shown that growth phase does not appear to affect pil promoter strength and transcript levels except for pilD (encoding the pre-pilin peptidase) and CII pilE. For an in-depth examination of the growth phase-dependent regulon in N. gonorrhoeae, RNA-seq was employed to compare the gonococcal transcriptomes of cultures grown to the mid-exponential and late stationary phases. Our RNA-seq data mainly identified genes encoding components involved in ribosome biogenesis as decreasing in the stationary growth phase. As ribosome biogenesis is the most energy costly process, down-regulating genes associated with this process will enable bacteria to conserve energy in order to survive under stresses associated with the stationary growth phase. As mentioned earlier, N. meningitidis expresses one of two distinct classes of tfp and this may have implications for their ability to cause disease. We performed comparative genetic studies with CI and CII tfp-expressing N. meningitidis strains and discovered that CI and CII pilin are functionally similar, and that it appears to be the extent of surface capsule on N. meningitidis that primarily determines their ability to adhere to and invade human epithelial cells. Neisserial pathogens are well known for their ability to evade the host immune system. One mechanism used is the antigenic variation of the pilin subunit. Here we wanted to gather evidence of antigenic variation in other potential antigenic variation systems, such as CII pilE, mafB and opa genes using next generation sequencing. Our data suggested that CII pilE lacks variation; mafB appears to undergo limited antigenic variation; and opaK, but not opaJ appears to be antigenically variable. This work has examined various aspects of Neisserial pathogenesis, including tfp regulation, growth phase effects on gene expression, pilus-associated phenotypes and antigenic variation of genes encoding surface structures. These findings have important impacts on the understanding of the infection process by Neisserial pathogens.