J.P.M. van Putten

Binding of syndecan-like cell surface proteoglycan receptors is required for Neisseria gonorrhoeae entry into human mucosal cells.

Bacterial invasion of human mucosal cells is considered to be a primary event in the pathogenesis of a gonococcal infection. Here we report that cell surface heparan sulfate proteoglycans may play a role in the establishment of an infection, by functioning as receptors for the invasion‐promoting gonococcal opacity protein adhesin. Chemical modification and enzymatic removal of proteoglycan receptors from cultured epithelial cells abolished opacity protein‐associated gonococcal invasion, and mutant cell lines defective in proteoglycan synthesis were poor substrates for gonococcal attachment. The addition of purified receptor and receptor analogues totally blocked gonococcal entry into the cells. Heparin‐affinity chromatography and receptor binding assays using recombinant bacteria producing defined opacity proteins and reconstituted receptor or purified receptor fragments as probes, identified one particular member of the opacity protein family (MS11‐Opa30) as the primary ligand for this novel class of receptors for bacteria. Heparan sulfate proteoglycans with gonococcal binding activity were purified from various cell types derived from target tissues of gonococcal infection, including ME‐180 endocervical cells and primary cultures of human corneal epithelium. The physico‐chemical properties of the receptor indicate that it may belong to the syndecan proteoglycan family.

Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.

Cell surface‐located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface‐located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild‐type bacteria to a capsule‐negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule‐negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element‐based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.

Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.

Neisseria gonorrhoeae is a facultative intracellular bacterium capable of penetrating into certain human epithelial cell types. In order to identify gonococcal factors essential for invading Chang human conjunctiva cells, a gentamicin selection assay for the quantification of viable intracellular bacteria was used in conjunction with microscopy. The results demonstrate a correlation between the invasive behaviour of gonococci and the expression of Opa proteins, a family of variable outer membrane proteins present in all pathogenic Neisseria species. However, only particular Opa proteins supported invasion into Chang cells as indicated by the use of two unrelated gonococcal strains. Invasion was sensitive to cytochalasin D, and strong adherence mediated by the Opa proteins appeared to be essential for the internalization of gonococci. In contrast pili, which also conferred binding to Chang conjunctiva cells, did not support cellular invasion but rather were inhibitory.

Chicken TLR21 Is an Innate CpG DNA Receptor Distinct from Mammalian TLR9

TLRs comprise a family of evolutionary conserved sensory receptors that respond to distinct classes of ligands. For one major evolutionary branch of TLRs, the ligands are still largely unknown. Here we report the cloning and function of one member of this group, chicken TLR21 (chTLR21). This TLR is absent in the human species but has homologs in fish and frog and displays similarity with mouse TLR13. Expression of chTLR21 in HEK293 cells resulted in activation of NF-κB in response to unmethylated CpG DNA, typically recognized by mammalian TLR9. Silencing of chTLR21 (but not chTLR4) in chicken macrophages inhibited the response to CpG-DNA (but not to LPS), indicating similar functionality of the endogenous receptor. ChTLR21 responded to human- and murine-specific TLR9 ligands, as well as to bacterial genomic DNA isolated from Salmonella enterica serovar Enteritidis. Confocal microscopy located chTLR21 in the same intracellular compartments as human TLR9. Inhibition of the chTLR21 response by the endosomal maturation inhibitor chloroquine suggested that the receptor is functional in endolysosomes, as known for TLR9. The analogous localization and function of the phylogenetically only distantly related chTLR21 and mammalian TLR9 suggest that during evolution different classes of TLRs have emerged that recognize the same type of ligands.

Phase variation of lipopolysaccharide directs interconversion of invasive and immuno-resistant phenotypes of Neisseria gonorrhoeae.

Phase variation of Neisseria gonorrhoeae lipopolysaccharide (LPS) controls both bacterial entry into human mucosal cells, and bacterial susceptibility to killing by antibodies and complement. The basis for this function is a differential sialylation of the variable oligosaccharide moiety of the LPS. LPS variants that incorporate low amounts of sialic acid enter human mucosal epithelial cells very efficiently, but are susceptible to complement‐mediated killing. Phase transition to a highly sialylated LPS phenotype results in equally adhesive but entry deficient bacteria which, however, resist killing by antibodies and complement because of dysfunctional complement activation. Phase variation of N. gonorrhoeae LPS thus functions as an adaptive mechanism enabling bacterial translocation across the mucosal barrier, and, at a later stage of infection, escape from the host immune defence.

Immunity to Campylobacter: its role in risk assessment and epidemiology

Abstract Acquired immunity is an important factor in the epidemiology of campylobacteriosis in the developing world, apparently limiting symptomatic infection to children of less than two years. However, also in developed countries the highest incidence is observed in children under five years and the majority of Campylobacter infections are asymptomatic, which may be related to the effects of immunity and/or the ingested doses. Not accounting for immunity in epidemiological studies may lead to biased results due to the misclassification of Campylobacter-exposed but apparently healthy persons as unexposed. In risk assessment studies, health risks may be overestimated when immunity is neglected.

Unique features of chicken Toll-like receptors.

Toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune homeostasis and the defense against infections. The research explosion that followed the discovery of TLRs more than a decade ago has boosted fundamental knowledge on the function of the immune system and the resistance against disease, providing a rational for clinical modulation of the immune response. In addition, the conserved nature of the ancient TLR system throughout the animal kingdom has enabled a comparative biology approach to understand the evolution, structural architecture, and function of TLRs. In the present review we focus on TLR biology in the avian species, and, especially, on the unique functional properties of the chicken TLR repertoire.

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

Successful Selection of Cross-Protective Vaccine Candidates for Ornithobacterium rhinotracheale Infection

ABSTRACT Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.

H-NS and Lrp serve as positive modulators of traJ expression from the Escherichia coli plasmid pRK100

Conjugative transfer of F-like plasmids is a tightly regulated process. The TraJ protein is the main positive activator of the tra operon which encodes products required for conjugative transfer of F-like plasmids. Nucleotide sequence analysis revealed potential Lrp and H-NS binding sites in the traJ regulatory region. Expression of a traJ-lacZ fusion in hns and lrp mutant strains showed that both are positive modulators of traJ expression. Competitive RT-PCR demonstrated that H-NS and Lrp exert their effect at the transcriptional level. Electrophoretic mobility-shift assays showed that H-NS and Lrp proteins bind to the traJ promoter. Conjugative transfer of pRK100 was decreased in hns but not in lrp mutant strains. Together, the results indicate H-NS and Lrp function as activators of traJ transcription.