Neil J. Oldfield

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models

A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.

CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.

The moonlighting protein fructose-1, 6-bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion

Fructose‐1, 6‐bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non‐glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life‐threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane‐localized FBA was surface‐accessible to FBA‐specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA‐deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp‐2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.

Cloning, mutation and distribution of a putative lipopolysaccharide biosynthesis locus in Campylobacter jejuni.

A region encoding ORFs with homology to known lipopolysaccharide (LPS) biosynthesis genes was isolated from two strains of Campylobacter jejuni. One of the strains produces LPS, but the second strain is reported to produce only lipooligosaccharide (LOS) and therefore lacks the O-chain. The two strains shared six predicted ORFs, but an additional ORF, orfE, of unknown function was identified in the LOS-producing strain. Mutation of the shared wbeE (rfbE) homologue (orfF) or deletion of five of the seven genes reduced core reactivity with specific antiserum without affecting O-chain production. Mutation of either the capD homologue (orfG) or the unique orfE had no detectable effect on LOS or LPS production. The presence or absence of orfE in 36 isolates of C. jejuni did not correlate with LOS/LPS phenotype or serotype. However, after insertion of orfE into a LPS-producing orfE-negative strain the O-chain ladder was no longer detectable on Western blots. We were not able to disrupt the wbaP (rfbP) homologue (orfC) in C jejuni.

The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in Neisseria meningitidis adherence to human cells

BackgroundGlyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.ResultsIn this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant.ConclusionsOur data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.

Characterization of the Campylobacter jejuni Heptosyltransferase II Gene, waaF, Provides Genetic Evidence that Extracellular Polysaccharide Is Lipid A Core Independent

Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides in C. jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second L-glycero-D-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide. In this report we confirm experimentally that C. jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. The Campylobacter waaF gene complements an S. enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF in C. jejuni, waaF mutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM(1), asialo-GM(1), and GM(2) ganglioside epitopes. The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C. jejuni. The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-beta(1,3) N-acetylgalactosamine. In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species.

Persistence, Replacement, and Rapid Clonal Expansion of Meningococcal Carriage Isolates in a 2008 University Student Cohort

ABSTRACT A study of meningococcal carriage dynamics was performed with a cohort of 190 first-year students recruited from six residential halls at Nottingham University, United Kingdom. Pharyngeal swabs were obtained on four occasions between November 2008 and May 2009. Direct plating and culture on selective media were succeeded by identification and characterization of meningococci using PCR-based methodologies. Three serogroup Y clones and one serogroup 29E clone were highly prevalent in particular residential halls in November 2008, which is indicative of rapid clonal expansion since the start of the academic year. Persistent carriage of the same meningococcal strain for at least 5 to 6 months was observed in 45% of carriers, with infrequent evidence of antigenic variation in PorA. Sequential carriage of heterologous meningococcal strains occurred in 36% of carriers and involved strains with different capsules and antigenic variants of PorA and FetA in 83% of the cases. These clonal replacement strains also exhibited frequent differences in the presence and antigenic structures of two other surface proteins, NadA and HmbR. This study highlights the low level of antigenic variation associated with persistent carriage but, conversely, the importance of alterations in the repertoire of antigenic variants for sequential carriage of meningococcal strains. Rapid clonal expansion of potentially pathogenic strains in residential halls has implications for the implementation of public health interventions in university populations.

Carriage of meningococci by university students, United Kingdom.

To the Editor: Neisseria meningitidis causes septicemia and meningitis (1). Meningococci usually persist on the nasopharyngeal mucosa of asymptomatic carriers (2). Because carriers are the only reservoir of meningococci, carriage in at-risk populations should be monitored. Meningococcal carriage rates have been assessed during 1997–8 for first-year students at the University of Nottingham (3) and in autumn during 1999–2001 for >48,000 sixth-form students (pre-university, age range 15–17 years) throughout the United Kingdom (4). Serogroup B and nongroupable strains predominated; serogroup Y strains were found in only 1%–2% of participants. From November 2008 through May 2009, to investigate persistence and spread of meningococcal strains in students living in dormitories, we conducted a longitudinal study in a cohort of 190 first-year students at the University of Nottingham. We found high rates of carriage and prevalence of serogroup Y strains (5). During September 2009 (first week of term) through March 2010, we conducted a large repeated cross-sectional study analyzing pharyngeal swabs from students in all school-year groups at Nottingham University. The objective of this study was to determine the significance of changes in overall meningococcal and serogroup Y-specific carriage rates among students. In September, first-year students were recruited on the main campus during registration and subsequently in dormitories and the main library. Undergraduates not in the first year were all recruited in the main library. This September sample of 823 first-year students represents 16.5% of the 5,000 undergraduate students registered each academic year on the main campus. Although not intentional, some overlap occurred when students were resampled during subsequent visits to the same dormitories and library, e.g., among the 557 first-year students from whom swab samples were collected in December, 74 (13%) had previously provided swab samples. Our study was approved by the Nottingham University Medical School Ethics Committee, and written informed consent was obtained from all participants. Pharyngeal swab samples were spread onto GC selective agar (Oxoid, Basingstoke, UK) and incubated at 37°C in air containing 5% CO2. After 48 hours, colonies suggestive of Neisseria spp. were examined for positive oxidase reaction; single colonies were confirmed as meningococci by amplification of meningococcal genes crgA plus ctrA and/or porA (6). PCR-based serogrouping was performed as described (6,7). Chi-square tests for significance were performed by using STATCALC (Epi Info version 6.04; Centers for Disease Control and Prevention, Atlanta, GA, USA). Among first-year students, carriage rates increased from 23.2% in late September to 55.7% by mid-December and remained at a similar level in March (Table). Among second- and third-year students, carriage rates were 34.2% and 30.5% in September, respectively, and remained at similar levels throughout the academic year. The increase in carriage among first-year students from September through December was mainly the result of a significant (23%) increase in carriage of serogroup Y strains (Table). In contrast, during the same period, carriage rates of serogroup Y strains did not change significantly among second- and third-year students (Table). Table Characteristics of meningococci carriage, University of Nottingham students, United Kingdom, 2009–10* Initial carriage rates were significantly higher for incoming (first-year) students in September 2009 than in 1997 (13.9% [3]; χ2 = 14, 1 df; p<0.0001); swabbing and culture protocols and sampling sites were identical in both studies, so the increases are real. Because 83% of students at Nottingham University come from all regions of the United Kingdom and 17% from other countries, the increased rates of carriage may reflect a nationwide change (8). Furthermore, testing within the first week of term meant that recovered strains were predominately brought into the university. Serogroup Y carriage rates for incoming students (2.9%) were significantly higher than rates detected by identical genotyping methods during 1999–2001 (1.7%–1.8% [4]; χ2 = 4.6%–6.4%, 1 df; p<0.05), suggesting that meningococcal carriage by young adults, particularly of serogroup Y strains, has increased across the United Kingdom. The major increase in serogroup Y strains among first-year students during 2009–10 probably resulted from spread of clones within dormitories, as observed in the 2008–9 study (5) and may be facilitated by characteristics of the organism, lack of immunity, or a combination of these factors. The high prevalence of serogroup Y strains in carriers may help explain the recent increased incidence of serogroup Y disease in the United Kingdom: from 20 to 62 laboratory-confirmed cases in England and Wales from 2003 through 2009 (9). In the United States during the late 1990s, a similar increase in serogroup Y carriage was linked to a concomitant increase in serogroup Y disease (10). In conclusion, in a representative UK student cohort we detected high rates of carriage and elevated prevalence of serogroup Y strains of meningococci. Any further significant increase in serogroup Y disease should lead to prompt reconsideration of the current vaccine policy in the United Kingdom.

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.

T‐cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells

T‐cell stimulating protein A (TspA) is an immunogenic, T‐cell and B‐cell stimulating protein of Neisseria meningitidis. Sequence similarity between TspA and FimV, a Pseudomonas aeruginosa protein involved in twitching motility, suggested a link between TspA and type IV pili (Tfp). To determine the role of TspA an isogenic deletion mutant was created. Loss of TspA did not affect twitching motility or piliation indicating that there are functional differences between TspA and FimV. Mutation of tspA led to a significant reduction in adhesion of meningococci to meningothelial and HEp‐2 cells, which was not due to a lack of transcription of adjacent genes or pilC1. Other Tfp‐mediated phenotypes (i.e. auto‐aggregation and transformation competence) were not altered. Our results indicate that the role of TspA in adhesion is unlikely to be directly linked to the function of Tfp. TspA was expressed by all N. meningitidis and Neisseria polysaccharea strains examined but not by Neisseria gonorrhoeae or Neisseria lactamica, although sequences with homology to tspA were present in their genomes. In summary, TspA is a highly conserved antigen that is required for optimal adhesion of meningococci to human cells.