Complement Factor H Related Proteins and their Biological Role During Bacterial Infection

Neisseria meningitidis is a major cause of meningitis and sepsis worldwide despite available polysaccharide and protein vaccines. Individuals with rare defects in the terminal complement pathway are susceptible to meningococcal disease but further genetic factors that contribute to disease susceptibility are less well understood. A genome wide association study has linked polymorphisms in the Complement Factor H (CFH) locus with meningococcal disease in the general population. CFH is a negative regulator of the alternative complement pathway whereas CFH-related proteins (CFHR), encoded in the CFH locus, act as antagonists of CFH. Investigations of the biological role of the CFHRs have been hampered by lack of reagents, therefore a panel of specific monoclonal antibody reagents to CFHR2-5 were generated and characterised. Previously, N. meningitidis has been shown to bind CFH via a surface exposed lipoprotein, factor H binding protein (fHbp), at high affinity. This study demonstrates that CFHR3 binds to the bacterial surface in a fHbp dependent-manner. Furthermore, CFHR3 competes with CFH for binding to fHbp. Bound CFHR3 increases susceptibility of N. meningitidis to complement-mediated lysis which was dependent on the sequence of the fHbp variant. The ability of N. meningitidis to evade the host immune system is likely to be determined by the relative levels of CFH and CFHR3 on the bacterial surface, providing a molecular mechanism for how variation in cfhr3 may predispose individuals to meningococcal disease. Furthermore this work demonstrates that Neisseria cinerea, which colonises the respiratory mucosa, expresses fHbp and binds CFH at similar affinities as meningococcal fHbp promoting bacterial survival in serum. The recently developed meningococcal vaccine, Bexsero®, includes fHbp as an antigen and antibodies elicited by Bexsero® are bactericidal against N. cinerea suggesting that the introduction of this vaccine could affect nasopharyngeal carriage of N. cinerea

Neisseria cinerea Expresses a Functional Factor H Binding Protein Which Is Recognized by Immune Responses Elicited by Meningococcal Vaccines

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W, and Y. More recently two protein-based vaccines, Bexsero and Trumenba, against meningococcal serogroup B strains have been licensed; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface-exposed lipoprotein that binds the negative complement regulator complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the functions of these genes and how immunization with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here, we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and that it is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH with affinity similar to that of meningococcal fHbp and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea. We found that immunization with Bexsero elicits serum bactericidal activity a gainst N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis and cross-reactive responses elicited by Bexsero suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts.

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host

Distinct Binding and Immunogenic Properties of the Gonococcal Homologue of Meningococcal Factor H Binding Protein

Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups

Satellite remote sensing of surface winds, waves, and currents: Where are we now?

This review paper reports on the state-of-the-art concerning observations of surface winds, waves, and currents from space and their use for scientific research and subsequent applications. The development of observations of sea state parameters from space dates back to the 1970s, with a significant increase in the number and diversity of space missions since the 1990s. Sensors used to monitor the sea-state parameters from space are mainly based on microwave techniques. They are either specifically designed to monitor surface parameters or are used for their abilities to provide opportunistic measurements complementary to their primary purpose. The principles on which is based on the estimation of the sea surface parameters are first described, including the performance and limitations of each method. Numerous examples and references on the use of these observations for scientific and operational applications are then given. The richness and diversity of these applications are linked to the importance of knowledge of the sea state in many fields. Firstly, surface wind, waves, and currents are significant factors influencing exchanges at the air/sea interface, impacting oceanic and atmospheric boundary layers, contributing to sea level rise at the coasts, and interacting with the sea-ice formation or destruction in the polar zones. Secondly, ocean surface currents combined with wind- and wave- induced drift contribute to the transport of heat, salt, and pollutants. Waves and surface currents also impact sediment transport and erosion in coastal areas. For operational applications, observations of surface parameters are necessary on the one hand to constrain the numerical solutions of predictive models (numerical wave, oceanic, or atmospheric models), and on the other hand to validate their results. In turn, these predictive models are used to guarantee safe, efficient, and successful offshore operations, including the commercial shipping and energy sector, as well as tourism and coastal activities. Long-time series of global sea-state observations are also becoming increasingly important to analyze the impact of climate change on our environment. All these aspects are recalled in the article, relating to both historical and contemporary activities in these fields

Neisseria cinerea Expresses a Functional Factor H Binding Protein Which Is Recognized by Immune Responses Elicited by Meningococcal Vaccines

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W and Y. More recently two protein based vaccines, Bexsero® and Trumenba®, have been licenced against meningococcal serogroup B strains; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface exposed lipoprotein which binds the negative complement regulator, complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the function of these genes and how immunisation with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH at similar affinity as meningococcal fHbp, and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea We found that immunisation with Bexsero® elicits serum bactericidal activity against N. cinerea, which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis, and cross reactive responses elicited by Bexsero® suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species. </p

Thermoregulation of Meningococcal fHbp, an Important Virulence Factor and Vaccine Antigen, Is Mediated by Anti-ribosomal Binding Site Sequences in the Open Reading Frame

<div><p>During colonisation of the upper respiratory tract, bacteria are exposed to gradients of temperatures. <i>Neisseria meningitidis</i> is often present in the nasopharynx of healthy individuals, yet can occasionally cause severe disseminated disease. The meningococcus can evade the human complement system using a range of strategies that include recruitment of the negative complement regulator, factor H (CFH) <i>via</i> factor H binding protein (fHbp). We have shown previously that fHbp levels are influenced by the ambient temperature, with more fHbp produced at higher temperatures (<i>i</i>.<i>e</i>. at 37°C compared with 30°C). Here we further characterise the mechanisms underlying thermoregulation of fHbp, which occurs gradually over a physiologically relevant range of temperatures. We show that fHbp thermoregulation is not dependent on the promoters governing transcription of the bi- or mono-cistronic <i>fHbp</i> mRNA, or on meningococcal specific transcription factors. Instead, fHbp thermoregulation requires sequences located in the translated region of the mono-cistronic <i>fHbp</i> mRNA. Site-directed mutagenesis demonstrated that two anti-ribosomal binding sequences within the coding region of the <i>fHbp</i> transcript are involved in fHbp thermoregulation. Our results shed further light on mechanisms underlying the control of the production of this important virulence factor and vaccine antigen.</p></div

Table of primers

<p>Table of primers</p

αRBS sequences in the open reading frame are necessary for fHbp thermoregulation.

<p><b>(A)</b> Western blot analysis of fHbp-GFP levels <i>in E</i>. <i>coli</i> harbouring p<i>fHbp-</i>9C<i>-gfp</i> (WT) or the plasmid with modified αRBS-1, αRBS-2 or both (αRBS-1&2). Bacteria were grown at the temperatures indicated to mid-log phase, and cell lysates were subjected to Western blot analysis. The membranes were incubated with antibodies recognising fHbp or RecA. <b>(B)</b> <i>In vitro</i> translation of RNA from <i>E</i>. <i>coli</i> harbouring p<i>fHbp-</i>9C<i>-gfp</i> or plasmids containing altered RBSs (indicated). <i>in vitro</i> translation was performed at the temperatures indicated and subject to Western analysis with antibodies recognising GFP and RecA (loading control). mut-αRBS-1 and mut-αRBS-2 showed reduced thermosensing ability whereas modification of both αRBS-1 and 2 (mut-αRBS-1&2) almost completely abolishes thermoregulation both in <i>E</i>. <i>coli</i> and in <i>in vitro</i> translation assays. Relative values (rel. values) of GFP are indicated below each lane.</p

Thermoregulation of fHbp occurs post-transcriptionally.

<p><b>(A)</b><i>N</i>. <i>meningitidis</i> MC58 was grown at the temperatures indicated until mid-log, then subjected to flow cytometry analysis using anti- V1.1 fHbp pAbs to detect surface localised fHbp. Representative graph of fHbp detection at indicated temperatures. Secondary antibody only used as a control (grey). Geometric mean fluorescence of surface expressed fHbp from three independent experiments; error bars are ± SEM. Stastically significant levels of fHbp were detected between bacteria grown at 30°C and 42°C. <b>(B)</b> RNA was obtained at mid-log phase from bacteria grown at the temperatures indicated, and samples (20 μg) were separated on agarose-formaldehyde gel and subjected to Northern blot analysis with probes for <i>fHbp</i> and <i>tmRNA</i>. The position of bands corresponding to the mono- and bi-cistronic transcripts are shown. <b>(C)</b> Proteins extracted from the same cultures as in B were analysed by Western blot. Relative levels of RNA and protein are indicated. <b>(D)</b> Western blot analysis of protein stability. Bacteria were grown at 30°C or 37°C until mid-log phase, then protein synthesis blocked by the addition of spectinomycin. Samples were removed at time-points afterwards and subjected Western blot analysis. Membranes were probed with antibodies recognising fHbp or RecA (loading control).</p