HexR controls glucose-responsive genes and central carbon metabolism in Neisseria meningitidis

none6noNeisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection. N. meningitidis can utilize a restricted range of carbon sources, including lactate, glucose and pyruvate, whose concentration varies in host niches. Microarray analysis of N. meningitidis grown in chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most of these genes are implicated in energy metabolism and transport as well as some implicated in virulence. In particular, genes involved in glucose catabolism were up-regulated whereas genes involved in the TCA cycle were down-regulated. Several genes encoding surface exposed proteins were up-regulated in the presence of glucose, including the MafA adhesins and the Neisseria surface protein A. Our microarray analysis led to the identification of a glucose-responsive hexR-like transcriptional regulator that controls genes of the central carbon metabolism of N. meningitidis in response to glucose. We characterized the HexR regulon and showed the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites we propose a 17-bp pseudo-palindromic HexR consensus binding motif. Furthermore, N. meningitidis strains lacking hexR expression are deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogen in vivo.openAntunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, IsabelAntunes, Ana; Golfieri, Giacomo; Ferlicca, Francesca; Giuliani, Marzia M; Scarlato, Vincenzo; Delany, Isabe

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

K.L.S. was supported by the Australian National Health and Medical Research Council (NHMRC) C. J. Martin Fellowship and Career Development Fellowship. A.F.H. was supported by a Marie Curie Fellowship (PIEF-GA-2012-328377). F.O., L.F., and S.B. were recipients of Novartis fellowships from the Ph.D. program of the University of Siena (Siena, Italy) and University of Bologna (Bologna, Italy), respectively.GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.PostprintPeer reviewe

Molecular engineering of Ghfp, the gonococcal orthologue of neisseria meningitidis factor H binding protein

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes

Urbanizzazioni popolari e pratiche di pianificazione a Buenos Aires

Urban informality in the Global South is widely understood as an unofficial/illegal process exclusively driven by the urban poor, occurring beyond the State and solely enacted by the popular sector’s agency. On the other hand, it is considered a diametrically opposed alternative to conventional conceptions of planning, representing a form of urbanization that is independent of formal frameworks and in violation of planning rules and regulations. Yet, although a fact generally neglected by planning theory, the local dwellers of what have been called “informal settlements” comply with regulatory frameworks and the spatial outcomes of their settlement actions are frequently from the beginning in accordance with planning norms. This phenomenon is crucial for understanding how local dwellers’ rationality is often driven by their expectations in terms of accessing basic services and obtaining securing tenure. This thesis is aimed to theorize how and under what conditions various actors and their practices interact with the regulatory frameworks within the planning system to enable and sustain a mode of popular urbanisation in contemporary Buenos Aires. Therefore, this research centres the analysis on two spatial configurations of popular urbanisation in Buenos Aires – particularly Villa 20 and the toma de tierra of Guernica - and two corresponding positions assumed by policy makers and urban planners, starting from their relationships with local actors from a transversal perspective of governmentality on different focal points of encounter and disagreement. By adopting a relational and socio-material approach to the study of planning practices, the research provides a transversal reading across different actors and their rationalities at play observing their engagement with the regulatory frameworks. Through an ethnography of planning practices, the thesis provides a novel methodology for bringing into view the processes, practices, alliances, and agencies which are often invisible to policy makers. The thesis illustrates that popular urbanisation and planning need to be considered as urban assemblages that have numerous and unexpected ways of interlinking. Firstly, it recognizes the presence of conflicting, competing and conflating rationalities at play in popular urbanisation of Buenos Aires. Secondly, the thesis offers a novel insight into the strategies and tactics employed by planners in attempts to intervene in popular urbanization in Buenos Aires. Thirdly it proposes practices-centred recommendations for institutional change and social justice considering the technical as much as the political aspects of planning and interrogating the agency of materiality in urban processes. For policy makers and urban planners, a better understanding of the socio-technical configurations of popular urbanization can guide their actions to rearrange them toward coproducing urban governance.Urban informality in the Global South is widely understood as an unofficial/illegal process exclusively driven by the urban poor, occurring beyond the State and solely enacted by the popular sector’s agency. On the other hand, it is considered a diametrically opposed alternative to conventional conceptions of planning, representing a form of urbanization that is independent of formal frameworks and in violation of planning rules and regulations. Yet, although a fact generally neglected by planning theory, the local dwellers of what have been called “informal settlements” comply with regulatory frameworks and the spatial outcomes of their settlement actions are frequently from the beginning in accordance with planning norms. This phenomenon is crucial for understanding how local dwellers’ rationality is often driven by their expectations in terms of accessing basic services and obtaining securing tenure. This thesis is aimed to theorize how and under what conditions various actors and their practices interact with the regulatory frameworks within the planning system to enable and sustain a mode of popular urbanisation in contemporary Buenos Aires. Therefore, this research centres the analysis on two spatial configurations of popular urbanisation in Buenos Aires – particularly Villa 20 and the toma de tierra of Guernica - and two corresponding positions assumed by policy makers and urban planners, starting from their relationships with local actors from a transversal perspective of governmentality on different focal points of encounter and disagreement. By adopting a relational and socio-material approach to the study of planning practices, the research provides a transversal reading across different actors and their rationalities at play observing their engagement with the regulatory frameworks. Through an ethnography of planning practices, the thesis provides a novel methodology for bringing into view the processes, practices, alliances, and agencies which are often invisible to policy makers. The thesis illustrates that popular urbanisation and planning need to be considered as urban assemblages that have numerous and unexpected ways of interlinking. Firstly, it recognizes the presence of conflicting, competing and conflating rationalities at play in popular urbanisation of Buenos Aires. Secondly, the thesis offers a novel insight into the strategies and tactics employed by planners in attempts to intervene in popular urbanization in Buenos Aires. Thirdly it proposes practices-centred recommendations for institutional change and social justice considering the technical as much as the political aspects of planning and interrogating the agency of materiality in urban processes. For policy makers and urban planners, a better understanding of the socio-technical configurations of popular urbanization can guide their actions to rearrange them toward coproducing urban governance

Migrants and precarious housing : working paper 4

This working paper focuses on precarious housing experienced by migrants in Europe, a social group among those most severely affected by precariousness, particularly (but not only) in accessing adequate housing. After introducing key terms and main categories into which migrants are classified, the paper delves into the main obstacles they face in accessing the public and private housing markets, and the precarious formal or informal housing options they are pushed into as a consequence

The RNA Chaperone Hfq Is Involved in Stress Response and Virulence in Neisseria meningitidis and Is a Pleiotropic Regulator of Protein Expression▿

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the Δ2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and amodel for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence

GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the ∆2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and a model for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.—Seib, K. L., Haag, A. F., Oriente, F., Fantappiè, L., Borghi, S., Semchenko, E. A., Schulz, B. L., Ferlicca, F., Taddei, A. R., Giuliani, M. M., Pizza, M., Delany, I. The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence. Neisseria meningitidis, a gram-negative β-proteobacteria, is a leading cause of bacterial sepsis and meningitis worldwide (1). The meningococcal protein Genome-derived Neisseria Antigen 2091 (GNA2091) was first described during the N. meningitidis serogroup B reverse vaccinology project as a lipoprotein predicted to be surface exposed in some meningococcal strains, which is able to induce passive protection in the adult mouse model of meningococcal bacteraemia (2). The function of GNA2091 is still unknown, but because of its protective properties, it was selected for inclusion in the 4-component meningococcal serogroup B vaccine (4CMenB; trade name Bexsero) (3). The 4CMenB vaccine is widely licensed and used to protect against invasive meningococcal disease from MenB and has also been introduced in the United Kingdom for mass vaccination of infants (4). 4CMenB contains 3 recombinant proteins [factor H binding protein (fHbp), Neisseria heparin binding antigen (NHBA), and Neisserial adhesin A] and outer membrane vesicles (OMVs) derived from New Zealand strain NZ 98/254 (2, 5). The immunogenicity and stability of the recombinant antigens was optimized by generating protein-protein fusions of fHbp-GNA2091 and NHBA-GNA1030, which induce higher serum bactericidal activity titers than those induced by the individual antigens alone (2). fHbp, Neisserial adhesin A, and NHBA have been extensively characterized and shown to be involved in meningococcal virulence (6–11). The accessory protein GNA1030 has recently been characterized as a Neisseria ubiquinone binding protein (NUbp) (12). However, the role of GNA2091 has not yet been characterized in detail. GNA2091 has been shown to be localized at the periplasmic side of the outer membrane, where it is proposed to be required for the efficient assembly of a subset of outer membrane proteins (OMPs), including porin (Por)A, PorB, pili associated protein Q (PilQ), and the β-barrel assembly machinery (Bam) complex, with accumulation of misassembled monomeric proteins seen in a gna2091 mutant strain (13). The gna2091 mutant is also sensitive to detergent stress, indicating compromised membrane integrity (14). Here we further characterize the expression and functional role of GNA2091 in vitro and in the in vivo infant rat model of meningococcal bacteraemia

Investigating the Role of Antigen Orientation on the Immune Response Elicited by Neisseria meningitidis Factor H Binding Protein on GMMA

GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines