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Diversification in the face of function? Lessons from the mechanisms and results of pilus antigenic variation in Neisseria gonorrhoeae
Speaker(s): Prof. Hank Seifert, Feinberg School of Medicine, Northwestern University, USA
When: 07 January 2016 (9:30 am)
Where: SBS Classroom 3 (Level 1)
Type: Seminars

Seminar Summary (Posted on SCELSE Facebook 14 Jan 2016)

For our first SCELSE seminar of 2016, we were very privileged to have Professor Hank Seifert from Northwestern University to give his talk entitled "Diversification in the face of function? Lessons from the mechanisms and results of pilus antigenic variation in Neisseria gonorrhoeae" and present his latest insights in the transmission of the disease and its interaction with the immune system.

Prof. Seifert is a world recognised leader in the study of the genetics, physiology and pathogenesis of the obligate human pathogen N. gonorrhoeae. He was also the PhD advisor of SCELSE faculty Asst. Prof. Kimberly Kline, and she fondly recalled that: "Hank was the one who taught me how to do science."

Prof. Seifert gave a quick overview of N. gonorrhoeae – a bacterium that causes the sexually transmitted infection (STI) gonorrhoea – which he has studied for 30 years now. It is a strictly human pathogen, so it does not affect other animals and it is also not found in soil or any environmental niche. Interestingly, it is related to commensal Neisseria which is resident in the nasal pharynx, suggesting that the progenitor somehow made the jump to the urogenital tract, a notion that drew some laughter from the audience.

Prof. Seifert emphasised that N. gonorrhoeae is still mainly commensal, and in the continuum of existence it is right at the border of what it means to be a pathogen, as pathogens do not always go to the extent of killing the host. He hypothesised that the key difference between a commensal and a pathogen may be their ability to induce inflammation.

A critical feature of N. gonorrhoeae that allows it to survive the host immune response, type IV pilus, was the main focus of Prof. Seifert’s talk. In human patients, N. gonorrhoeae is always seen with type IV pilus, which is required for full virulence. It is also important for twitching motility, epithelial cell adherence, resistance to killing by polymorphonuclear neutrophils (PMNs) and for antigenic and phase variation.

Prof. Seifert presented work from his group on the detailed characterisation of pilin antigenic variation. Pathogenic N. gonorrhoeae have 4-5 silent pilus loci (pilS) in the variable region of the chromosome, and each pilS in turn carries up to six silent copies. These serve as a rich source of sequence variation for the expressed pilin locus (pilE). Mutagenesis experiments reveal that the guanine quartet (G4) is involved in this process. G4s are implicated in the modulation of transcription, replication and translation, and Prof. Seifert confessed that he is "a bit of a zealot for G4s, which is an elegant and complicated mechanism and more people should know about it."

Prof. Seifert stressed that inflammation is a key aspect of the disease. Inflammation leads to the formation of fluids called "purulent exudates" that contain viable N. gonorrhoeae cells, which PMNs are incapable of clearing. He put forward a hypothesis that N. gonorrhoeae has evolved to interact with PMNs and this actually promotes pathogenesis. A candidate gene involved in this process is the metalloprotease NGO1686, which shows a 20-fold upregulation after sublethal hydrogen peroxide (H2O2) treatment against N. gonorrhoeae. NGO1686 can cleave peptidoglycan links in vitro and so Prof. Seifert renamed it as metalloprotease active against peptidoglycan (Mpg).

Mpg deletion mutants are more sensitive to H2O2 and non-oxidative PMN killing, and these mutants are also underpiliated. PilE deletion mutants that are nonpiliated also show sensitivity to H2O2 and antimicrobial peptide LL-37. In addition, other experiments show that the differential sensitivity between piliated and nonpiliated cells is an iron-dependent process. Prof. Seifert pointed out that Mpg deletion mutants do form pili, but the pili cannot stay extended and lack of Mpg leads to the retraction of pili or pilus phase variation, resulting in H2O2 and PMN sensitivity.

Prof. Seifert raised a curious observation about gonorrhoea transmission – the infection rate from females to males is about the same as the other direction. This is unusual for STIs, which made him consider a "salmon swimming upstream" model. Prof. Seifert suspected that semen may play a role in this phenomenon. To investigate this, his group members added N. gonorrhoeae to "seminal plasma" (SP) – semen without sperm cells. SP enhances the migration of piliated bacteria, disrupts the bundling of pili and enhances their motility.

However, semen is a complex biological fluid and he wanted find out what the stimulating component is. After performing many experiments with different defined molecules, they were unable to narrow down to a specific reaction, but lactoferrin, prostate-specific antigen (PSA) and human serum albumin (HSA) stimulates motility of the bacteria. SP also stimulates the formation of biofilms under flow conditions, while inhibiting and dispersing static biofilms.

Prof. Seifert presented a "big picture" animation of the whole process. He noted that transmission is "job number one" for pathogens, otherwise nothing else is possible. He reiterated that the recruitment of PMNs is required for pathogenesis and that the bacteria are "using" the neutrophils for some as yet unknown purpose. Finally, Prof. Seifert cautioned that further research work should be done with human samples because he felt that animal models have limited relevance to a strictly human pathogen like N. gonorrhoeae.