1. Genomics and Reference Libraries

Keith A. Jolley and Martin C. J. Maiden

Whole genome sequencing is now available at a cost that means it will very soon become a routine tool in epidemiology and public health. There is a challenge, however, in making sense of the vast amount of information encoded in a bacterial genome and in being able to relate this to the large collection of legacy data that exists for many bacterial species. The Neisseria community has lead the way in embracing nucleotide sequencing for typing and epidemiology and is at the forefront of the push to apply whole genome sequencing to research and public health. In this chapter we review the platform and databases hosting Neisseria allelic diversity data and discuss how they can be used to extract relevant information and perform comparative genomics.

2. Transcriptional Regulatory Proteins in the Pathogenic Neisseria

Nadine Daou, Ryan Mcclure and Caroline A. Genco

The exclusive human pathogens Neisseria gonorrhoeae and Neisseria meningitidis are the only two pathogenic members of the Neisseria genus. N. gonorrhoeae infects the human genital tract while N. meningitidis typically infects the bloodstream or cerebrospinal fluid. Because of rapidly changing environments encountered during infection, including changing oxygen levels, reactive oxygen species, pH, and iron, both pathogens must be able to quickly and accurately control gene expression. In other microbial pathogens response to these stimuli often involves a repertoire of regulatory proteins, which collectively function to ensure tight regulation of gene expression. However, unlike other bacteria, there are relatively few classical DNA binding proteins expressed by Neisseria species. The following is a discussion of Neisseria gene regulation involving transcriptional regulatory proteins with a specific emphasis on Fur, a global regulatory protein in Neisseria species. While Fur has classically been known to respond to levels of free iron, new studies show that it engages in complex cross talk with other DNA binding proteins in response to stimuli encountered during infection.

3. The Regulatory Small RNAs of Neisseria

Yvonne Pannekoek, Dave Speijer and Arie van der Ende

The discovery and characterization of regulatory small RNAs (sRNAs) in bacteria has exploded in recent years. These sRNAs act by base-pairing with target mRNAs with which they share either limited or extended complementarity. Many of them base-pair at or near the Shine-Dalgarno (SD) sequence of their targets and block translation by preventing entry of ribosomes, while others base-pair in regions influencing the stability of their cognate mRNA. Those binding at or near SD regions are the most well studied among bacteria and carry promoters that are often responsive to environmental signals. In the last years, using a variety of approaches, among which are biocomputional prediction, high-density micro array analysis and high-throughput transcriptome analysis, novel sRNAs of Neisseria species were identified. Some of them (NrrF, AniS and a sRNA involved in pilin antigenic variation) were also functionally analysed. In this chapter we will focus on strategies used to identify sRNAs in Neisseria and will highlight studies on those sRNAs for which base-pairing mRNAs were identified and functionality has been demonstrated by experimental approaches.

4. Inter- and Intraspecies Transformation in the Neisseriae, Mechanism, Evolution and DNA Uptake Sequence Specificity

Ole Herman Ambur

The evolution of the pathogenic neisseriae: Neisseria meningitidis and Neisseria gonorrhoeae, is closely linked to their ability to be transformed with extracellular DNA. A strong tradition of studying these organisms has documented that they exchange alleles with the commensal Neisseria, and that they also repeatedly utilize DNA from their own separate clonal lineages for this purpose. This chapter present studies that describe inter- and intraspecies transformation and discuss potential effects of a flexible gene pool. The molecular mechanism for transformation is highlighted, and the most important components and recent developments in this field are presented. Neisseria is one of very few bacterial genera to exhibit sequence specific transformation, and the mechanism and evolutionary implications of this is discussed. Also, the negative influence that restriction has on transformation and sexual isolation are described. Finally, a model is proposed that emphasizes the regenerative aspects of transformation.

5. Pathogenic Neisseria: Neither Aerobes nor True Anaerobes, but Dedicated Microaerophiles

J. A. Cole

Neisseria meningitidis and N. gonorrhoeae are found in contrasting sites in the human body. Meningococci are rarely oxygen-deficient, but in women, gonococci become trapped in biofilms surrounded by anaerobic, fermentative bacteria. When starved of oxygen, both species exploit low levels of nitrite for energy generation and substrate oxidation. However, too little is known about whether they are able to exploit oxidants other than nitrite and nitric oxide to survive in anaerobic environments. Although five proteins have been implicated in resistance to nitrosative stress, only for two of them has the mechanism of protection been defined. These are the nitric oxide-binding cytochrome c′, and the di-iron protein, DnrN. Both pathogens maintain very high respiration rates: multiple mechanisms contributing to defense against oxidative stress have been identified. Only subtle differences between them have so far been identified. They include the meningococcal transcription factor, FNR, which regulates adaptation to anaerobic growth, that is more tolerant to oxygen than the gonococcal FNR. The truncated denitrification pathway is totally conserved in gonococci but not in all meningococcal strains. Only in the gonococcus is there an FNR-activated cytochrome c peroxidase, and a third heme group on the cytochrome oxidase CcoP subunit that contributes significantly to electron transfer from the cytoplasmic membrane to the nitrite reductase in the outer membrane. Finally adhC, which encodes a functional S-nitrosoglutathione reductase in the meningococcus, is a pseudogene in the gonococcus. It is proposed that both species have evolved a microaerobic rather than a fully aerobic or anaerobic metabolism: both can adapt to periods of oxygen starvation, but this ability appears to be more important for the gonococcus than for the meningococcus.

6. Peptidoglycan Metabolism and Fragment Production

Joseph P. Dillard

Peptidoglycan metabolism is involved in several processes that are crucial to the growth and lifestyle of the pathogenic Neisseria. These include: 1) cell wall growth and cell separation, 2) autolysis and release of DNA for natural transformation, 3) release of soluble peptidoglycan fragments that affect host responses and cause tissue damage. Peptidoglycan fragments shed by N. gonorrhoeae during growth cause the death of ciliated cells in human Fallopian tubes and likely contribute to the inflammatory response to gonococci generally. Gonococci and meningococci have in common almost all of the enzymes and additional factors involved in peptidoglycan breakdown, recycling, and peptidoglycan fragment transport, and meningococci also release soluble peptidoglycan fragments. This review describes the genetic, biochemical, and infection studies that have led to our understanding of the structure of gonococcal and meningococcal peptidoglycan, its breakdown, and its effects on the human host.

7. The Glycome of Neisseria spp.: How Does this Relate to Pathogenesis?

Stephanie N. Bartley and Charlene M. Kahler

The genus Neisseria consists of two important human pathogens, Neisseria gonorrhoeae and N. meningitidis in addition to nine commensal species. In both N. meningitidis and N. gonorrhoeae interactions with the human host are modulated by the glycome on the surface of the bacteria which includes the lipooligosaccharide and the glycosylation status of the pilin. Capsule, which is expressed by N. meningitidis but not N. gonorrhoeae, also modulates interaction with host epithelial cells but has a major role in enabling systemic disease by contributing to resistance to the host immune system. Because of the central importance of the glycome to the pathogenesis of gonococci and meningococci, it is important to reflect on whether these attributes are shared with commensal species or have specialised roles in pathogenesis. This review will undertake to summarize the considerable knowledge underpinning the genetics and biosynthesis pathways for the neisserial glycome in pathogenic and non-pathogenic neisseria, in addition to examining the roles of the different components of the glycome on the interaction of Neisseria sp. with the human host.

8. Neisseria Biofilms: Biology, Formation and Role in Pathogenesis

Michael A. Apicella

Both N. gonorrhoeae and N. meningitidis can form biofilms on the surfaces they inhabit. In both it is important for sustaining asymptomatic colonization and for dispersal of the organism. In the gonococcus, it is most probably a major factor resulting in the frequent emergence of antibiotic resistance as new therapies are applied. Both use eDNA as a matrix for the biofilm. The meningococcus and the gonococcus both have the ability to produce an autoinducer-2 like molecule but at present very little is known about the role of quorum sensing in their biology.

9. Molecular Mechanisms of Antibiotic Resistance Expressed by the Pathogenic Neisseria

Magnus Unemo, Robert A. Nicholas, Ann E. Jerse, Christopher Davies and William M. Shafer

Diseases caused by the pathogenic Neisseria (N. gonorrhoeae and N. meningitidis) have been successfully treated by antibiotics for the past 70-80 years. However, particularly for treatment of gonorrhea, the high prevalence of gonococcal strains with resistance to inexpensive and widely available antibiotics (e.g., penicillins, narrow-spectrum cephalosporins, tetracyclines, macrolides and fluoroquinolones) and the recent emergence of strains exhibiting resistance to the last remaining options for empiric first-line antibiotic monotherapy, i.e. the expanded-spectrum cephalosporins, is of great concern. For meningococci, decreased susceptibility/resistance to most antibiotics used for chemoprophylaxis or treatment of invasive meningococcal disease is considerably rarer worldwide. Given the global nature of gonococcal and meningococcal diseases, the high rate of usage of antibiotics, suboptimal control and monitoring of antibiotic resistance, geographical differences in treatment regimens and the extraordinary capacity of the pathogenic Neisseria (particularly gonococci) to develop and retain antibiotic resistance, it is likely that the global problem of antibiotic resistance will worsen in the foreseeable future. By understanding the molecular and phenotypic mechanisms of antibiotic resistance in gonococci and meningococci, resistance to antibiotics used clinically can be anticipated, future methods for genetic resistance testing might permit region-specific antibiotic therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. Herein, we review the genetic, phenotypic and physiologic basis by which the pathogenic Neisseria have developed resistance to historically important antibiotics and how resistance to newer, and the last remaining, antibiotics is emerging.

10. The Roadblocks in Developing a Gonococcal Vaccine and Reasons for Optimism

Lee M. Wetzler

The search for an effective vaccine to prevent gonococcal infections has been an ongoing endeavor for over three decades. Gonococcal diseases, including endometritis, pelvic inflammatory disease (PID), and their sequelae (ectopic pregnancies and infertility) in women, and urethritis and disseminated gonococcal infection in both men and women, remain one of the major reportable sexually transmitted diseases (STD) in the country (approximately 300,000 cases a year, 100.8 cases per 100,000 people). Moreover, it has been shown that concurrent inflammatory STDs, and particularly gonococcal disease, can enhance HIV replication and transmission. Of equal or greater concern is the currently rising incidence of gonococcal antibiotic resistance. Over the past decade, disease-associated gonococcal strains have developed resistance to penicillin and quinolones. Most alarming is the recent report out of Japan that pathogenic strains of gonococci have developed high-level resistance to ceftriaxone. The implication of this cannot be over-emphasized since cephalosporins are the only remaining class of antibiotics to treat this infection. The development of an effective gonococcal vaccine has many roadblocks including the dearth of immune correlates of protection; mainly due to the lack of adequate animal models. In order to aid in developing an efficacious anti-gonococcal vaccine, it is important to understand the immune response to this organism in humans, including patients with disease and their infected and uninfected sexual contacts. The purpose of this review is to describe the history of gonococcal vaccine development, previous immune studies and vaccine trials and the current development of new animal models and potential new vaccine candidates.

11. Interactions of Pathogenic Neisseria with Neutrophils in the Context of Host Immunity

Alison K. Criss

The pathogenic Neisseria species, N. gonorrhoeae and N. meningitidis, avoid clearance by cells of the human immune system in order to persist in their obligate human hosts. This chapter will explore the mechanisms used by the pathogenic Neisseria to subvert the antimicrobial actions of neutrophils. These mechanisms include manipulating phagocytosis, modulating the oxidative burst, preventing granule release, defending against toxic neutrophil products, and extending neutrophil lifespan. Interactions between Neisseria and other innate immune cells (macrophages, dendritic cells) and with components of adaptive immunity (T and B cells) will be examined in the context of the Neisseria- and neutrophil-driven inflammatory response.

12. Modelling Infection by the Pathogenic Neisseria

Epshita A. Islam and Scott D. Gray-Owen

The pathogenic Neisseria are exquisitely adapted to life within the human mucosa, their only natural niche. While biologically fascinating, this strict host specificity makes it difficult to appreciate how bacterial and host factors contribute to infection in the context of an authentic mucosa and an intact immune system. While modeling neisserial infection in a non-human host is fraught with the potential that effects seen might not be replicated in humans, basic responses are often preserved and careful interpretation of the outcomes can provide insight not achievable by other methods. This chapter summarizes past work and ongoing efforts to establish animal models that can be used to study Neisseria in their mucosal niche and/or during disseminated disease. By considering the relative benefits of each model, we aim to provide a deeper appreciation of how each may be used to address different types of research questions and, moreover, how the model employed may affect the results from each study. Then, when employed judiciously, these animal models can be used to provide direct validation of findings obtained through cellular, molecular or clinical studies and, on the other hand, can reveal previously unrecognized aspects of infection that can be studied in more detail through one or the other of these complementary approaches.