Escherichia coli CFT073 Fitness Factors during Urinary Tract Infection: Identification Using an Ordered Transposon Library.

Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field.IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.

Bilayer polymeric nanocapsules: A formulation approach for a thermostable and adjuvanted E. coli antigen vaccine.

One of the strategies used to improve the immunogenicity of purified protein antigens has relied on their association with synthetic nanocarriers, which, in general, have functioned as simple antigen containers. Here, we present a more advanced strategy based on the design of an antigen nanocarrier at the molecular level. The nanocarrier is composed of a vitamin E oily core, surrounded by two layers: a first layer of chitosan and a second of dextran sulphate. The selected antigen, IutA protein from Escherichia coli, was harboured between the two polymeric layers. The final bilayer nanocapsules had a nanometric size (≈ 200 nm), a negative zeta potential (< -40 mV) and a good antigen association efficiency (≈ 70%). The bilayer architecture led to an improvement on the formulation stability and the controlled release of the associated antigen. Remarkably, after being administered to mice, bilayer nanocapsules elicited higher IgG levels than those obtained with antigen precipitated with Alum. Moreover, freeze-dried nanocapsules were stable at room temperature for, at least, 3 months. These promising data, in addition to their contribution to the development of an uropathogenic E. coli vaccine, has allowed us to validate these novel bilayer nanocapsules as adequate platforms for the delivery of protein antigens.

Impact of human-associated Escherichia coli clonal groups in Antarctic pinnipeds: presence of ST73, ST95, ST141 and ST131.

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, ß1, ß2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds.

Evaluation of CpxRA as a Therapeutic Target for Uropathogenic Escherichia coli Infections.

CpxRA is an envelope stress response system found in all members of the family Enterobacteriaceae; CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR, a transcription factor. CpxR also accepts phosphate groups from acetyl phosphate, a glucose metabolite. Activation of CpxR increases the transcription of genes encoding membrane repair and downregulates virulence determinants. We hypothesized that activation of CpxR could serve as an antimicrobial/antivirulence strategy and discovered compounds that activate CpxR in Escherichia coli by inhibiting CpxA phosphatase activity. As a prelude to testing such compounds in vivo, here we constructed cpxA (in the presence of glucose, CpxR is activated because of a lack of CpxA phosphatase) and cpxR (system absent) deletion mutants of uropathogenic E. coli (UPEC) CFT073. By RNA sequencing, few transcriptional differences were noted between the cpxR mutant and its parent, but in the cpxA mutant, several UPEC virulence determinants were downregulated, including the fim and pap operons, and it exhibited reduced mannose-sensitive hemagglutination of guinea pig red blood cells in vitro In competition experiments with mice, both mutants were less fit than the parent in the urine, bladder, and kidney; these fitness defects were complemented in trans Unexpectedly, in single-strain challenges, only the cpxA mutant was attenuated for virulence in the kidney but not in the bladder or urine. For the cpxA mutant, this may be due to the preferential use of amino acids over glucose as a carbon source in the bladder and urine by UPEC. These studies suggest that CpxA phosphatase inhibitors may have some utility for treating complex urinary tract infections.

Liposomes or traditional adjuvants: induction of bactericidal activity by the macrophage infectivity potentiator protein (Mip) of Neisseria meningitidis.

Currently, one of the main approaches to achieve a vaccine for serogroup B Neisseria meningitidis is based on outer membrane proteins with low antigenic variability among strains. Since these proteins tend to be minor components of the outer membrane, recombinant production is required to obtain them in sufficient amounts for evaluation and development of vaccines. In this study, we analysed the ability of recombinant macrophage infectivity potentiator (rMip) protein to induce protective bactericidal activity in mice. The rMip protein was cloned from N. meningitidis strain H44/76 and was used to immunise mice, and the sera obtained were tested against the homologous and several heterologous N. meningitidis strains. The sera were obtained using the rMip alone, with adjuvant Al(OH)3 , or after inclusion into liposomes. Bactericidal activity was variable depending on the strain, although high titres were seen against strains H44/76 and NmP27. Liposomes enhanced fourfold the reactivity against the homologous strain. The results presented suggest that the rMip protein should be considered a promising candidate for the improvement of future protein-based vaccines.

Lambs are an important source of atypical enteropathogenic Escherichia coli in southern Brazil.

Food-producing animals can harbor Escherichia coli strains with potential to cause diseases in humans. In this study, the presence of enteropathogenic E. coli (EPEC) was investigated in fecal samples from 130 healthy sheep (92 lambs and 38 adults) raised for meat in southern Brazil. EPEC was detected in 19.2% of the sheep examined, but only lambs were found to be positive. A total of 25 isolates was characterized and designated atypical EPEC (aEPEC) as tested negative for bfpA gene and BFP production. The presence of virulence markers linked to human disease as ehxA, paa, and lpfAO113 was observed in 60%, 24%, and 88% of the isolates, respectively. Of the 11 serotypes identified, eight were described among human pathogenic strains, while three (O1:H8, O11:H21 and O125:H19) were not previously detected in aEPEC. Associations between intimin subtypes and phylogroups were observed, including eae-θ2/A, eae-ß1/B1, eae-α2/B2 and eae-γ1/D. Although PFGE typing of 16 aEPEC isolates resulted in 14 unique pulsetypes suggesting a genetic diversity, specific clones were found to be distributed in some flocks. In conclusion, potentially pathogenic aEPEC strains are present in sheep raised for meat, particularly in lambs, which can better contribute to dissemination of these bacteria than adult animals.

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil.

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep.

Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131.

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.

Virulence patterns in a murine sepsis model of ST131 Escherichia coli clinical isolates belonging to serotypes O25b:H4 and O16:H5 are associated to specific virotypes.

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.

Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans.

Escherichia coli strains O45:K1:H7 are implicated in severe human infections such as meningitis. Since an increasing prevalence of serogroup O45 among avian pathogenic (APEC) and human extraintestinal pathogenic (ExPEC) E. coli strains isolated in Spain have been noticed, the aims of the present study were to investigate similarities between poultry and human O45 isolates, and to investigate the evolutionary relationship of ST95 types. The genetic relatedness and virulence gene profiles of 55 O45 APEC obtained from an avian colibacillosis collection (1991-2011) and 19 human O45 ExPEC from a human septicemic/uropathogenic (UPEC) E. coli collection (1989-2010) were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based genotyping. Two main clonal groups were established. The most prevalent and highly pathogenic O45:K1:H7-B2-ST95 shows a successful persistence since the 90s to the present, with parallel evolution both in human and poultry, on the basis of their PFGE and virulence gene profile similarities (9 human strains and 15 avian strains showed ≥85% PFGE identity). Comparison of this group with other ST95 closely related members (O1:K1:H7 and O18:K1:H7 isolates from our collections) shows pathogenic specialization through conserved virulence genotypes. The other prevalent O45 clonal group characterized in this study, the O45:HNM/H19-D-ST371/ST2676 was only detected in APEC strains suggesting host specificity. In conclusion, poultry could be acting as a reservoir of O45:K1:H7-B2-ST95 and other pathogenic ST95 serotypes in humans. Further studies would be necessary to clarify if pathogenic mechanisms used by ST95 strains are the same in avian and human hosts.

Emergence of new variants of ST131 clonal group among extraintestinal pathogenic Escherichia coli producing extended-spectrum ß-lactamases.

Having shown that Lucus Augusti Hospital in Lugo, Spain, has been affected by Escherichia coli clone O25:H4-ST131 producing CTX-M-15, the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates and to identify novel variants of this clone. Of the 77 ESBL-producing E. coli isolated between January and April 2012, 47 (61%) were identified as belonging to the ST131 clonal group, comprising 38 O25b:H4-B2-ST131 (34 CTX-M-15, 2 CTX-M-14, 1 CTX-M-1 and 1 CTX-M-27), 7 O-non-typeable:H4-B2-ST131 (all CTX-M-15) and 2 O16:H5-B2-ST131 (both CTX-M-14). The 47 isolates of ST131 exhibited a significantly higher virulence score (mean of 9.1 virulence genes) compared with the 30 non-ST131 isolates (mean of 4.3 virulence genes). A new virulence profile (fimH, papG II, sat, cnf1, hlyA, iucD, kpsM II-K5, traT, malX, usp) was detected among O25b:H4-B2-ST131 isolates belonging to the new Pasteur sequence type PST621. To our knowledge, this is the first study to report the O-non-typeable:H4-B2-ST131 and O16:H5-B2-ST131 variants in Europe.

Four main virotypes among extended-spectrum-ß-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-ß-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.

Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes.

Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona, Spain.

OBJECTIVES: The present study was carried out to evaluate the prevalence of clonal group O25b:H4-B2-ST131 in water environments with faecal pollution (urban sewage and river water) in the north-east of Spain and to study the virulence gene content of environmental isolates and to compare them with isolates causing human extraintestinal infections in Spain. METHODS: This study was performed with 10 sewage samples (collected in Catalonia, north-eastern Spain, in autumn 2009 from the influent raw urban sewage of a wastewater treatment plant that serves a large urban area) and 6 river water samples (collected monthly from February to April 2010 in the Llobregat river catchment area, near Barcelona, a watercourse subjected to heavy anthropogenic pressure). Escherichia coli colonies were screened by PCR for the rfbO25b gene associated with the clonal group O25b:H4-B2-ST131. Sequence types (STs), serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum ß-lactamase (ESBL) enzymes were determined in 75 E. coli isolates positive for the O25b molecular subtype. RESULTS: Of the 75 O25b-positive isolates, 51 belonged to the O25b:H4-B2-ST131 clonal group and the remaining 24 belonged to clonal group O25b:H4-D-ST69. The majority of ST69 isolates (23 of 24) were isolated from urban sewage, whereas ST131 isolates were isolated from urban sewage (25 isolates) as well as from river water (26 isolates). ST131 and ST69 isolates carried 4-13 virulence genes, the majority (82%) being quinolone resistant. CONCLUSIONS: We showed the presence of E. coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona. Furthermore, we observed that the environmental O25b:H4-B2-ST131 isolates showed similar virulence and macrorestriction profiles to clinical human isolates. To our knowledge, this is the first study describing the O25b:H4-D-ST69 clonal group.

Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.

The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.

Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes.

We have analysed the structure of meningococcal outer membrane complexes and found that the main complexes are formed by different combinations of PorA and/or PorB molecules, associated to other proteins such as RmpM. In view of the growing knowledge of the importance of conformational epitopes in the immune responses to many pathogens, our aim in this study was to analyse the interactions of PorA and PorB by reconstitution of both recombinant porins into liposomes and determine the relevance of these interactions for the immune response. Recombinant PorA and PorB incorporated into liposomes associate forming complexes that are homomeric when only one of the porins is present, but heteromeric when both neisserial porins are present, mimicking those found previously in native outer membrane vesicles (OMVs). Association of PorA and PorB to form heterocomplexes modifies the immunogenicity of at least PorB, allowing the production of antibodies that recognise conformational epitopes, and produces new epitopes that react with a 50 kDa outer membrane protein not yet identified.

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild-type and specific knock-out mutant strains.

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1-D blue native (BN) electrophoresis, 2-D BN/SDS-PAGE and 2-D diagonal electrophoresis, followed by LC/MS-MS (for 1-D gels) or MALDI-TOF (for 2-D gels) revealed at least six porin complexes in the wild-type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild-type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (approximately 800 kDa). Our results suggest that PorA and PorB could form a 'basic' template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.

A cross-reactive neisserial antigen encoded by the NMB0035 locus shows high sequence conservation but variable surface accessibility.

The meningococcal NMB0035 locus encodes a 47 kDa outer-membrane protein that is highly conserved antigenically, and is able to induce antibodies during infection and bactericidal responses in vitro. This study analysed the surface exposure of this protein using specific antibodies in flow cytometry assays and determined its nucleotide sequence in 33 Neisseria strains. Genomic analyses revealed no significant differences in the nucleotide or amino acid sequences, but flow cytometry showed that surface accessibility was highly variable among the strains. These results suggest that masking by and/or association with lipo-oligosaccharides or other membrane molecules can be crucial for antigen accessibility, which must be thoroughly analysed in new vaccine candidates.