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Colonization and invasion of human epithelia by Neisseria meningitidis. Bacterial surfacevariation and exploitation of host defense molecules
de Vries, F.P.
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Citation for published version (APA):de Vries, F. P. (2001). Colonization and invasion of human epithelia by Neisseria meningitidis. Bacterial surfacevariation and exploitation of host defense molecules.
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Download date: 23 May 2020
ChapterChapter 1
Generall introduction
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GeneralGeneral introduction
NeisseriaNeisseria meningitidis: host-parasite relationship and course of infection
NeisseriaNeisseria meningitidis
NeisseriaNeisseria meningitidis belongs to the genus Neisseria, which harbors several closely related
speciess of Gram-negative diplococci. These include the exclusively human pathogen N.
gonorrhoeaegonorrhoeae and a number of other Neisseria species that colonize the mucosae of men and
otherr mammals without causing disease (1). Pathogenic and commensal Neisseria are alike in
thatt both groups of bacteria may use similar mechanisms to facilitate successful colonization
off their particular niches. The pathogenic Neisseria and probably many commensal Neisseria
speciess are naturally transformable. DNA released from autolytic bacteria can be taken up and
integratedd into the chromosomes of recipient bacteria (34,171). Neisseria recognize a specific
DNAA sequence, 10 bp in length, commonly found in neisserial genes, that serves as a signal
forr high frequency uptake of DNA. Bacterial transformation with unrelated DNA is
exceptionall (21,61,102). Transformation can account for a continuous horizontal exchange of
geneticc material within and between different neisserial species (52,58,73,173). This
mechanismm provides a genetic plasticity that enables long-term adaptation to even gross
environmentall changes encountered during evolution and to microenvironmental changes in
thee host. In addition, it may generate mosaic genes and thus promote the development of
phenotypicc variants (52,58,73,110). Newly generated variants may rapidly spread into the
humann population in a clonal manner. Horizontal gene transfer and frequent recombination
events,, in which small segments of genomes are exchanged, result in the relatively rapid
diversificationn of clones. In case of Neisseria meningitidis, this ongoing microevolution has
ledd to a population structure where occasional clonal outgrowths occur in an otherwise
panmicticc context. In Europe and North America, for example, the genetic diversity of
(predominantlyy serogroup B and C) meningococci commonly isolated from cases and carriers
is,, with a few exceptions, extreme and the population genetic structure is non-clonal. In
contrast,, meningococci that cause epidemics and outbreaks of disease belong to fairly uniform
clonall groupings. These hypervirulent organisms include most serogroup A meningococci but
alsoo a limited number of pandemic/hyperendemic serogroup C and B lineages. Meningococci
off such subgroups often carry mosaic cell surface antigens as a result of recombination
events,, but retain distinctive epidemiologies and virulence potential (3,31). Some clonal
groupings,, like strains from the (mainly serogroup B) electrophoretic type (ET-5) and the ET-
377 complex persist in the population for decades, sometimes in the same region, without
9 9
ChapterChapter 1
evidencee for inter-genetic exchange (36). The existence of different restriction/modification
systemss within clonal lineages may represent a barrier for horizontal gene transfer between
distinctt clonal groupings and stabilize their clonal expansion(66).
Thee host-parasite relationship
Inn order to understand the host-parasite relationship, the evolutionary background of a long-
standingg adaptation between the meningococcus and its human host has to be considered.
Outsidee its host, the meningococcus is very vulnerable, it does not have the ability to become
aa spore and lacks the hardiness to survive in the environment. As the host range of the
microorganismm is limited to a single species, there is a strong negative selective pressure
againstt meningococci that are overwhelmingly virulent. Infection with such clones will result
inn decimation of the host species, a lower probability of transmission from a dead to a live
host,, and nowadays, increased chances that the clone will be eliminated by antibiotic
treatment.. The adaptive process favors a moderate meningococcal virulence, such that a
bacteriall density is reached that maximizes transmission between hosts with normal
antimicrobiall defenses. Whenever the organism encounters a host whose defenses are below
average,, serious disease ensues, but the ability of the meningococcus to find a new host is
diminishedd or abrogated (62). The dichotomy between the main reservoir where selection is
operatingg and disease is illustrated by the almost complete absence of nasopharyngeal
carriagee in children below age four, yet this population has by far the highest frequency of
systemicc meningococcal disease (33,37).
Whenn we try to understand the selected advantage afforded by certain virulence factors,
itt might be important to consider the anatomical localization at which selective forces have
operatedd in evolution (62). When meningococci enter the systemic circulation, encapsulated
organismss are protected against the bactericidal effect of normal human serum, while non-
encapsulatedd bacteria are almost invariably killed (75,82,93,206,207). However, it is very
unlikelyy that such a mechanism evolved to serve this purpose. It is more likely that the
advantagee of the capsule to the organism lies in its functions during colonization of the
mucosa,, or during transmission between hosts. Remarkably, not a single gene or bacterial
attributee has been identified and shown to contribute to or increase transmission efficiency,
althoughh this is a key event in the meningococcal life style.
10 0
GeneralGeneral introduction
Coursee of infection
Uponn acquisition, Neisseria meningitidis colonizes the mucosal surface of the human oro- and
nasopharynx,, the only known habitat and reservoir for this bacterium. Meningococci are
efficientlyy transmitted between hosts by inhalation of respiratory droplet aerosols. This is
reflectedd in a mean carriage rate of 5-15 % of the healthy human population during non-
epidemicc periods (28,60). The carrier-state normally elicits host antibodies within 7-10 days,
butt the host may remain carrier of a specific strain for months (29). Occasionally, the balance
betweenn host defenses and bacterial virulence is disturbed and the meningococcus
disseminatess to the bloodstream and may eventually reach the central nervous system. As a
resultt of released outer membrane vesicles (blebs) and fragmentation of bacterial cell
membranes,, both of which contain high amounts of lipopolysaccharide (LPS), meningococcal
septicemiaa can lead to excessively high endotoxin concentrations in plasma and cerebrospinal
fluidd (10,26,27,48). These events can induce a cascade of inflammatory and often
disproportionall anti-inflammatory responses that can lead to pathogenic changes, like
purulentt infection of the meninges, septic shock, disseminated intravascular coagulation,
multiplee organ failure and ultimately, death (9,27,48,78,138,146,208,209).
11 1
ChapterChapter 1
Virulencee repertoire of the meningococcus.
Inn order to cause systemic disease, meningococci colonizing the nasopharynx have to invade,
survivee and proliferate in the intravascular space and tissues. In case of meningitis, the blood-
brainn barrier must be crossed and the bacteria have to survive in the cerebro-spinal fluid
(CSF).. Attachment and mucosal cell invasion are probably normal events in colonization, but
alsoo logical first steps in a process that can lead to bacterial invasion of the bloodstream (175).
Inn this respect, meningococcal components mediating interaction with the mucosal epithelium
shouldd be considered as virulence factors. A major difficulty in identifying putative virulence
factorss is the strong intra- and inter-strain variation in surface constituents displayed by the
bacteria,, in vitro as well during carriage and disease. Ongoing phase switching and antigenic
variationn of factors such as capsule (33,42,76,77,88,116), pili (4,7,129,187,201), opacity
associatedd proteins (Opa and Ope) (5,6,43,135,161,212), porins (PorA and PorB)
(4,52,57,110,212)) and lipopolysaccharide (LPS) (85,88,217) have been described. The
antigenicc heterogeneity of some of these cell surface components constitute the basis of a
phenotypicc classification system for epidemiological purposes (57).
Capsulee polysaccharides
Meningococcii can be divided into serogroups based on the antigenic differences of the
polysaccharidee capsules they produce. Although for a long time considered as a stable
attribute,, an increasing body of evidence suggests that meningococci can switch their capsule
polysaccharidee type as a result of horizontal gene transfer of part(s) of the capsule gene
clusterr (35,58,109,184). Polysaccharide capsules can consist of homopolymers of sialic acid
(serogroupp B and C) (19,63), polymers of sialic acid with galactose (W135) (19) and sialic
acidd with glucose (serogroup Y) (19). Homopolymers of modified N-acetyl mannosamine-
phosphatee and of N-acetylglucosamine-phosphate can be found on serogroup A (30,63) and X
(30)) meningococci, respectively. Serogroup Z polysaccharide consists of N-
acetylgalactosaminee and glycerol-phosphate (84), while N-acetylgalactosamine and modified
KDOO form the repeating disaccharide of serogroup 29-E polysaccharide (18). In addition,
severall less frequently isolated serogroups, which are generally not associated with disease,
havee been described (D,E,H-L) (107).
Whilee the majority (>50%) of isolates obtained from healthy carriers lack a capsule
12 2
GeneralGeneral introduction
andd are thus non-groupable, meningococci isolated from blood and liquor are almost
invariablyy encapsulated (37,38,88). It has been widely accepted that the capsule is
indispensablee for survival and proliferation of the bacteria in the bloodstream of
immunologicallyy naive hosts (75,82,93,206,207) and confers resistance against the human
complementt system. Polysaccharides prevent proper insertion of the membrane attack
complexx in the bacterial outer membrane, rather than reducing the deposition of C3b (206).
Serogroupp B polysaccharide is by itself poorly immunogenic, because of structural
similaritiess with sialic acid epitopes on fetal and infant brain tissue (N-CAM) (54,55,213).
Capsulee also reduces the exposure of subcapsular antigens to the immune system. This
maskingg effect inhibits non-opsonic phagocytosis (48,119), but also the meningococcal
invasionn of epithelial and endothelial cells (44,45,75-77,203). The apparent conflict between a
bacteriall phenotype that is protected against complement mediated lysis during systemic
infectionn but hampered in its interaction with certain host cells, is resolved through phase
variationn of capsule expression. Variation in capsule synthesis appears to be controlled both
byy environmental signals (116) and reversible variation of capsule gene expression. The latter
occurss through reversible insertion of the IS 1301-like element in the first gene of the capsule
synthesiss operon (siaA) (76) and/or via a slipped strand-mispairing event in the siaD gene
encodingg the polysialyltransferase (77).
AA putative and potentially important function of the hydrophilic capsules that thus far
hass received littl e attention is the protection of meningococci against desiccation during
transmissionn between hosts.
Pili i
Pilii (fimbriae) are hair-like structures, composed of protein subunits, which emanate from the
bacteriall surface. These organelles probably play an important initial role in neisserial
colonizationn and are the prime attachment promoting factor on encapsulated meningococci
(49,129,174,176,200).. Pili also mediate bacterial auto-agglutination (45,115,159), twitching
motilityy (movement of bacterial cells across a solid surface) (25,81) and are associated with
increasedd transformation competence (20). Because of their exposed location and prime
importancee for the initiation of infection, pili are expected to be main targets for host defense.
Thiss probably explains the strong antigenic variability of pili . The molecular basis for pilin
variabilityy depends in part on a large family of (up to 20 in N. gonorrhoeae) variant pilS genes
onn the neisseria chromosome (168). These silent (pilS) genes are truncated at the amino-
terminuss and lack promoter sites. As the result of a non-reciprocal, homologous, intragenic
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ChapterChapter 1
recombinationn of silent pilin copies into the expression locus pilE, the coding sequence of the
expressedd gene copy can be changed (67,183). Alternatively, antigenic variation can arise
fromm transformation-mediated recombination or allelic replacement (59,169). N. meningitidis
cann express Class I pili which closely resemble gonococcal pili and Class II pili , with identity
too pilin genes of the human commensal species Neisseria lactamica and Neisseria cinerea
(7,144,201).. All predicted protein sequences display features typical of type IV (or N-
methylphenylalanine)) pilins. Class II pili are composed of pilin with smaller subunit size and
lackk reactivity with the broadly cross-reacting (for class I pilin) monoclonal antibody SMI
(201).. Both class I and class II pili producing meningococci can act as pathogens, and contain
silentt (pilS) genes.
Apartt from antigenic variation, the pilus biosynthesis is also subjected to phase
variation.. Reversible phase variants are frequently observed as non-piliated interfaces
betweenn the expression of two successive, antigenically distinct pilus variants. Loss of pili
cann results from an alternative processing of propilin, which results in truncated subunits that
cann not polymerize and are excreted in the growth medium as soluble (S-) pilin (67,68,101).
Followingg the identification and characterization of the minor pilin subunit PilC, a new
mechanismm for pilus phase-variation was proposed (90). Gonococcal and meningococcal
strainss usually express two pilC loci, encoding PilCl and PilC2. These variants usually are
moree than 70% identical (90-92,128,160). It has been reported that expression of PilC
correlatedd with the presence of assembled pili (P+). PilC" gonococcal variants were non-
piliatedd (P) and either secreted S-pilin or produced no pilin at all, as a result of promoter
deletionss in the pilE gene (91). PilC expression is controlled by frequent deletions and
insertionss in a tract of repetitive G residues located at the 5' end of the gene, generating
frameshiftt variants (90). Usually, the on-off-on transition of PilC and pili (PilC+P+- PilCT-
PilC+P+)) correlates with the expression of altered pilins due to rearrangements in the pilin
subunitt gene {pilE) (91). These results show that PilC may have a significant effect on pilus
variation. .
Apartt from primary sequence changes within the neisserial pilin molecule itself, post-
translationall modifications of the pilin subunit with saccharide (65,132), phosphorylcholine
(211)) and a-glycerophosphate (153,165,180) have been found. These surface-exposed
modificationss may contribute to antigenic variation (56) and immune escape (74) of the intact
piluss fibers, but as yet no noticeable effects on pili expression, maturation or functions have
beenn detected (56).Pili of pathogenic neisseria have distinct specificities for attachment to a
varietyy of different host cells, including epithelial cells (42,89,117,128,159,203), erythrocytes
14 4
GeneralGeneral introduction
(62,105,159,189)) and endothelial cells (128,200,200). Recently, the complement regulatory
proteinn CD46, a human-specific protein, has been recognized as a pilus receptor for
pathogenicc neisseria (96). The pilus molecule(s) responsible for this attachment have not been
identified.. Early studies suggested direct involvement of a conserved domain on the major
subunit,, pilin (PilE) in the adherence of Neisseria gonorhoeae to epithelial cells (165). PilE
peptidess (in the region between residues 41 and 84) were shown to bind to epithelial cells,
whilee antibodies directed against some of these peptides (residue 69-84) (50) blocked the
adherencee of an unrelated piliated gonococcal strain to endometrial ENCA-4 epithelial cells
(154).. Only a small area of this pilin sequence however, representing amino acid 73 to78 is
conservedd between all known pilin sequences (67,69,102,130,159). A peptide containing the
centrall region of the pilin molecule (CNBr-2) also competitively inhibited haemagglutination
causedd by purified pili of different gonococcal strains (62). This erythrocyte-binding ability
seemss to be conserved and independent of pilin variation (105,106,159).
Too investigate whether minor pilus subunits (mainly PilC ) are responsible for
adherencee specificity, binding studies were performed with derivatives and mutants of TV.
gonorrhoeaegonorrhoeae MS11, carrying structurally defined PilE and PilC proteins. (89,159). The
bindingg efficiencies of piliated gonococcal clones containing different pilE sequences varied
dramaticallyy for corneal and conjunctival tissues, but were unchanged when cervical and
endometriall tissues were used (89,159). Expression of either PilCl or PilC2 containing pili
didd not change binding properties. PilC expression without assembled pili did not result in
bindingg of bacteria to host cells (89). Additional information on two serogroup B
meningococcall strains indicated that modification in pilin subunit structure resulted in altered
adhesionn phenotype, although PilC was unchanged (204). Together, these data suggest that
sequencee alterations in the pilE gene can result in a quantitative change in pilus mediated
adherencee and in differences in tissue tropism. More recent data suggest a central role for the
minorr pilus subunit PilC in pilus-mediated adherence (128,156,159,160,204). Gonococci and
mostt meningococci, producing either class I or class II pili express two different pilC loci,
encodingg PilCl and PilC2 (92,128,155,160). These proteins have been located both at the tip
off pilus fibers (156) and in the outer membrane (147). They have been implicated to function
inn pilus assembly (90,128,160), adherence to epithelial cells (128,156,160) and natural
transformationn competence (155). The same PilC can function in both class I (gonococcal)
andd class II (meningococcal) pili backgrounds (160). At least in one Mc strain (8013) only
PilCll seems to be associated with adherence, regardless of PilC2 phenotype (128). Recently,
aa transient upregulation of transcription of this adhesive pilCl allele, but not pilC2, has been
15 5
ChapterChapter 1
reportedd to result from initial meningococcal-host cell interactions (46,185,186). In some
meningococcall strains, only a single PilC could be detected (145,204). The strongest support
forr a direct role of PilC in pilus mediated adherence comes from data, showing that purified
gonococcall PilC2 inhibits the adherence of either PilCl or PÏ1C2 expressing piliated
gonococcii to epithelial cells (156). In conclusion, PilC probably plays an important role in
piluss expression, antigenic variation, epithelial and endothelial cell adherence and competence
forr DNA transformation. Still, the mechanistic roles of PilCl and PilC2 in some of these
processess have not been convincingly demonstrated.
Inn analogy to the P fimbriae of uropathogenic E. coli (79,80,181), it has been suggested
thatt the type IV pili of pathogenic neisseriae can mediate pathogen-host cross-talk at the
epitheliall cell surface (2,95,215). Although direct evidence is missing, these observations
suggestt a role for both the pilus receptor CD46 and meningococcal PilCl in fimbriae
mediatedd signal transduction. The membrane co-factor protein CD46 is a human-specific
proteinn that is expressed on virtually any cell type except on erythrocytes. Pathogenic
neisseriaa however can bind erythrocytes in a pilus specific, PilC independent way
(105,106,159,166),, indicating that at least one additional binding domain exists, conserved on
thee major pilus subunit pilin, that recognizes a second (non-CD46) host cell receptor.
Outerr membrane proteins
Opa/Opc Opa/Opc
Inn vitro experiments have shown that non-encapsulated meningococci that lack pili can still
adheree to and invade host cells provided that they carry the appropriate surface
adhesins/invasins,, including the Ope protein and/or certain members of the opacity (Opa)
outerr membrane protein family (44,45,202,203). Ope and Opa are basic 24-35 kDa neisserial
surfacee proteins that share many features although their corresponding genes and predicted
two-dimensionall structures are unrelated (5,8,121,131). Both types of proteins may exist as
multimers,, confer colonial opacity and have been implicated in facilitating meningococcal
adherencee and invasion into various types of host cells (5,44,45,119,120,182,202,203). Both
typess of proteins are variable expressed. The transcription of the opcA gene (encoding Ope)
variess from zero to intermediate or high (5), dependent on the number of C residues in a
variablee homopolymer positioned between the -10 and -35 boxes of the ope A promoter
(161).. Several opc-like (pseudo-) genes have recently been described in N. gonorrhoeae and
NeisseriaNeisseria meningitidis, but expression of functional amounts of Ope protein is probably
restrictedd to a subset of meningococcal strains (216). Opa proteins are subject to both phase-
16 6
GeneralGeneral introduction
andd antigenic variation (40,97). Multiple, unlinked opa genes each carrying their own
promoterr can be found in N. gonorrhoeae (strain MS11, 11 genes), in N. meningitidis (3-4
genes)) and in N. lactamica (2 genes) and are constitutively expressed (8,17,97,104,178,179).
Phasee variation results from frequent phase transitions that occur via a RecA-independent
DNA-slippagee mechanism involving a variable number of CTCTT repeats in the signal-
peptidee encoding region of the genes (126,177,178). In this way, a heterogeneous population
off bacteria, expressing none, one or multiple different opa proteins can be generated.
Recombinationn events between opa genes do occur and are probably responsible for the often
mosaicc structure of the genes, in which (parts of) semi variable (SV), but especially of
hypervariablee regions (HV1 and HV2) are exchanged between individual Opa's
(8,17,40,177).. The variable expression of Ope and Opa's at the meningococcal surface occurs
inn the human host during infection (5,6,43,135,212). It may serve as an adaptive mechanism
thatt enables the bacteria to spread to different anatomical niches by creating phenotypes with
differentt receptor specificities.
MajorMajor porins, Por A and PorB
Twoo major classes of neisserial outer membrane proteins are the pore forming proteins PorA
(onlyy in Neisseria meningitidis) and PorB, which enable the flux of ions and small
macromoleculess across the outer membrane (24,214). These proteins form a trimeric
configurationn of porin monomers, which span the membrane in amphipatic P-sheets (41).
Theirr trimeric structure and sequence similarities place them in a group with other gram-
negativee bacterial porins like OmpC, OmF (cation selective) (16,124) and PhoE (anion
selective)) (15). Topological models for neisserial porins predict 16 membrane spanning
regionss with 8 surface exposed loops (47,195). Several of these loops carry variable regions
(VRs).. Within each serogroup (capsule type), these VRs form the basis for a serological
typingg (PorB) or subtyping (PorA) scheme for the Mc (57). Genetically, two different porB
alleless have been described for N. meningitidis, porB2 (class 2 OMP: 40-42 kDa) and porB3
(classs 3 OMP: 37-39 kDa), which correspond with the porB la (PI A) and porB lb (P1B)
alleless of the single gonococcal PI porin, respectively (14,53,210). Expression of the different
allelicc forms of porB is mutually exclusive so that a strain produces either one of these porins
(57). .
Besidess its function as a transporter molecule, the PorB protein has been implicated in
thee interaction of bacteria with mammalian cells. Purified PorB can insert into model lipid
bilayerss and into plasma membranes of eukaryotic cells, where they can cause a transient
17 7
ChapterChapter 1
changee in transmembrane potential and modulate cell signaling events (22,70,71,157,193).
Thee observation that these proteins can transfer from viable bacteria directly into target cell
membraness further contributed to the idea that PorB can trigger uptake of neisseria into host
cellss (108). Direct evidence for a role of PorB in neisserial invasion of human cells was
obtainedd with the identification of a new invasion mechanism, driven by a subtype of the
PorBB proteins, the gonococcal PI A (PorB la). This invasion event mediated by Gc PI A but
nott P1B (PorBlb) was only apparent under conditions of low phosphate availability (197).
Currentt hypotheses assume that non-covalent binding of phosphate by PI A porin prevents its
interactionn with or proper insertion into mammalian cell membranes. Alternatively, phosphate
mayy interfere with the gating function of inserted porins, reminiscent of the reported closing
effectt of nucleoside triphosphates on neisserial porins (158). As yet, no porin driven invasion
mechanismm has been described for the meningococcus or commensal neisseria.
Expressionn of the PI A porin by gonococci is correlated with serum resistance (32) and
thee ability to cause disseminated gonococcal infection (DGI) (125). Preferential binding of the
complementt regulatory factor H to loop 5 of the gonococcal PI A porin correlates with an
increasedd conversion of C3b to iC3b and decreased complement mediated killing of the
bacteriaa (149). It is currently unclear whether meningococcal PorB (class 2 or class 3) porins
cann bind factor H. A binding site identical to the one present in the loop 5 of the gonococcal
porinn is absent from meningococcal porins (206).
Althoughh a porA pseudogene has been identified in N. gonorrhoeae, PorA is only
expressedd by N. meningitidis (53). In contrast to PorB, the expression level of the
meningococcall PorA protein is subject to phase variation, caused by a change in the number
off nucleotides positioned between the -10 and -35 region of the porA promoter (194). In
addition,, even within a group of genetically related meningococci as the ET5 complex, gene
replacement,, horizontal exchange of gene fragments, and accumulation of new mutations
havee been observed. These events have resulted in the evolvement of a wide variety of
antigenicallyy different PorA proteins among clinical isolates. Despite its antigenic diversity
PorAA is a prime vaccine candidate antigen. This choice is largely based on the observation
thatt porA elicits bactericidal antibodies. These antibodies are usually directed against the
mostt exposed, variable regions (VRs) of the protein (23,122). Sero-subtyping monoclonal
antibodiess which are used for phenotyping of disease strains, are directed against these same
epitopess (118,195), are bactericidal and confer protection in infant rat infection experiments
(162,163).. To overcome the induction of strain-specific protection by a PorA-based vaccine,
thee most prevalent disease associated PorA variants (representing 70-80% of Dutch serogroup
18 8
GeneralGeneral introduction
BB and C case isolates) have been included in a vesicle vaccine (39,196). Field trials with this
hexavalentt PorA based vaccine have been performed (137).
Phasee switching of PorA does occur in vivo, especially during carriage but also in
clinicall isolates (4,212). It is currently unclear whether PorA contributes to meningococcal
virulence.. Meningococci grow much faster on solid culture media than the closely related
gonococci,, which do not carry a PorA protein. Although the growth of cultured
meningococcall PorA mutants is not reduced compared to the wildtype (188), the presence of
thiss additional meningococcal porin may allow faster growth under certain environmental
conditionss in vivo. It has been argued that meningococci can express PorA/PorB porin
heterotrimerss (123). In this respect, PorA may contribute to the functionality of the major
meningococcall porin. It can however not be excluded that PorA has developed into an
immunologicall decoy, directing host immune defenses to a phase variable antigen without
essentiall function. Unlike PorB epitopes, which are immunologically shielded by full chain
lengthh LPS (1,141), the highly immunogenic PorA epitopes are not shielded or protected.
Lipopolysaccharide e
Thee outermost layer of the asymmetric outer membrane of the neisserial cell envelope
consistss largely of lipopolysaccharide (LPS). Neisseria express LPS lacking O-specific side-
chains.. The glycolipid consists of a hydrophilic oligo-saccharide core, linked to lipid A,
whichh anchors the molecule in the outer membrane. Lipid A of the pathogenic neisseria
consistss of a l,4'-biphosphorylated, p(l-6)-linked glucosamine disaccharide backbone,
substitutedd with six (predominantly) or five fatty acid residues (103,136). Neisserial lipid A
differss from the well-studied enterobacterial lipid A in the nature and the position of fatty
acids.. N.meningitidis lipid A differs from its gonococcal counterpart in that the phosphate
groupss of the lipid A backbone are largely substituted with 0-phosphorylethanolamine
(103,136).. In some (serogroup B) isolates the 4' phosphate may be lacking (148).
Thee oligosaccharide core region of meningococci contains a conserved part consisting
off two heptoses (the first being the one fixed to KDO) and two KDO molecules, which link
thee variable terminal saccharide chain(s) to the lipid A. Structural studies (83,139,140,199),
electromorphicc profiles and the use of monoclonal antibodies have demonstrated considerable
heterogeneityy of the oligosaccharide moiety of neisserial LPS (11,12,114). This variation
formss the basis for the identification of twelve different LPS immunotypes (LI-LI2 )
(99,192,217,218).. Observed variations include the presence, length and composition of the
oligosaccharidee antennas (64). Additional structural differences may arise from the presence
19 9
ChapterChapter 1
orr absence of phosphorylethanolamine substituents and their location in the inner core
(136,199).. A single bacterium may simultaneously carry a heterogeneous population of low
molecularr mass LPS molecules (164,190). LPS variation is caused by a high frequency
switchingg in the availability and/or activity of glycosyltransferases (85,86,94). Some of the
enzymess involved in oligosaccharide synthesis are variably expressed as a result of
transcriptional/translationall frame shifting in genes encoding those transferases (94). The
availabilityy of LPS precursor molecules (like modified hexoses), depends on many genes of
centrall metabolism and can be influenced by growth conditions and growth phase of the
bacteriaa (141,190).
AA determinant present at the non-reducing end of the L2, L3, 7,9, L4 and L5
immunotypee LPS and also highly conserved among strains of N. gonorrhoeae, is the terminal
tetrasaccharidee lacto-iV-neotetraose (LNnT) (87,98,112,191). This structure,
(Gaipi^4GlcNAcpl-»3Galfil-»-4Glc)) is identical to the carbohydrate portion of
paraglobosidee (nLc4Cer), a glycophingolipid precursor of the major human (ABH)
bloodgroupp antigens (72,172). Monoclonal antibodies (3F11, 06B4) demonstrated the
immunochemicall similarity between epitopes on this neisserial LPS and antigens present on
humann erythrocytes (111). This molecular mimicry may prevent an effective immune
responsee to this bacterial antigen.
Ann additional in vivo modification of neisserial LPS was first discovered for N.
gonorrhoeae.gonorrhoeae. It was observed that gonococci in urethral exudates are serum resistant, a
virulencee factor that is lost upon subculture onto an artificial medium (13,113,127,133,134).
Thee component responsible for this sero-conversion was identified as N-acetylneuraminic- or
sialicc acid, a-(2-3)- linked to the terminal galactopyranosyl residue of the 4.5 kDa
paragloboside-likee LPS component. A bacterial membrane associated oc-2,3-sialyltransferase
mediatedd this sialylation, using the host derived nucleotide sugar, cytidine 5'-monophospho-
yV-acetylneuraminicc acid (CMP-NANA) as a substrate (113). Most strains of meningococci
thatt synthesize a sialic acid capsule (serogroups B, C, W135 and Y) have the intrinsic ability
too synthesize CMP-NANA, and thus sialylate their LPS when the 4.5 kDa LPS acceptor is
presentt (112,191). Mutants unable to produce CMP-NANA can not endogenously sialylate
theirr LPS, but sialylation is restored when exogenous CMP-NANA is available (112).
Inn gonococci, sialylation of LPS results in an increased binding of complement factor H
whichh causes a reduced deposition of C3b and contributes to the inducible serum resistance
phenotypee (150). Thus, the increased binding of factor H by two closely associated surface
20 0
GeneralGeneral introduction
moleculess in the gonococcal outer membrane, the sialylated LPS and the PI A porin (loop 5,
seee before), protects the bacterium from alternative pathway-mediated killing (206). In
addition,, LPS sialylation reduces complement dependent opsonophagocytosis of pathogenic
neisseriaa by human neutrophils (100,151). In meningococci, the biological effects of
sialylationn are less well defined. LPS sialylation appears to be of minor importance for serum
resistancee compared with the polysaccharide capsules. Isogenic oc-2,3 sialyltransferase knock-
outt mutants of several capsulated serogroup B and C strains, only marginally differed in
serumm resistance from their wild type counterparts (93,205). However, in several serum-
sensitivee serogroup C isolates, obtained from healthy carriers, exogenous sialylation of the
LPSS enhanced serum resistance (51). So, it is hypothesized that the more susceptible the
neisseriall strain, the higher the benefit of LPS sialylation for serum resistance (205).
Interestingly,, truncation of the LNnT structure by a galE mutation, rendered several
previouslyy serum resistant encapsulated serogroup B and C strains (B1940, MC58, 2120,
C:NT:P1.2,5)) highly susceptible to normal human serum (NHS). The massive C3b deposition
onn these encapsulated strains with truncated LPS resulted from the concerted action of the
alternativee and classical pathway and correlated with increased binding of natural IgM and
mannosee binding lectin (MBL) (207). GalE mutation however, had no effect on the serum
resistancee of another serogroup B strain (NMB) or on the gonococcal strain MSI 1 (93,152).
Thesee results indicate that strain-specific differences have to be taken into account, when
investigatingg meningococcal serum resistance.
Theree are data implying that the LPS phenotype of the pathogenic neisseria may be a
criticall factor for cellular invasion. Studies in experimental models indicated that mutations
resultingg in LPS truncations that extend into the core region result in reduced or abrogated
invasionn of Chang epithelial cells, despite expression of an invasion-promoting Opa protein
(167,198).. Other experiments indicate that the paraglobosyl-like LPS (LNnT) is necessary for
invasionn of genitourinary (170) and HEP2 liver cells (142,143) by gonococci.
21 1
ChapterChapter 1
Aimss and outline of this thesis
Meningococcall disease probably represents an evolutionary dead end and the bacteria are
optimallyy adapted to the human nasopharynx, their sole habitat and natural reservoir.
Colonizationn of the human host generally elicits a specific immune response towards the
bacteria.. For variable periods of time however, such antibodies seem unable to eliminate the
homologouss strain from the nasopharynx, as the bacteria may persist in the human upper
airwayss for periods of over 20 months. An important factor that may contribute to the
prolongedd carrier state is the extensive variation in surface constituents expressed by
meningococcii colonizing the human host. It can be envisioned that this variability serves as
ann immune escape mechanism. At the time of design of this study, knowledge of putative
otherr biological functions of the phenotypic variation was rudimentary. However, in several
instancess reversible phase transitions of surface antigens had been observed during the course
off the carriage state. This may reflect a functional adaptation of the bacteria to frequent, often-
recurrentt microenvironmental changes encountered during colonization and transmission.
Inn the underlying study we investigated whether the phenotypic variation of
meningococcii surface constituents contributed to the ability of meningococci to adhere to and
invadee nasopharyngeal epithelial cells, and whether local host cell factors are major
determinantss of the bacteria-host cell interaction. Identification and characterization of
invasivee meningococci should answer the question whether all meningococci or only distinct
phenotypess are able to invade cells of the mucosal epithelium, a logical first step in the onset
too meningococcal disease.
Inn chapter 2, we describe the development of a new experimental infection model,
monolayerss of primary nasopharyngeal epithelial cells. Infection experiments indicated that
meningococcall phenotypes like those found in the bloodstream or liquor do not interact with
thee nasopharyngeal epithelium. Invasion of these epithelial cells however did occur following
concurrentt phase variation of multiple surface antigens. Comparison of invasive
meningococcii selected on epithelial cells originating from different anatomical sites indicated
thatt meningococcal class 5 proteins (Opa and Ope) may promote tissue tropism.
Inn chapter 3, we investigated the interaction of Ope and a distinct Opa protein with
certainn epithelial cells in more detail. We could show that both bacterial proteins bound to
epitheliall cell surface proteoglycan receptors, but only Opc-expressing bacteria could exploit
thiss newly identified meningococcal host cell receptor to gain access to the cell interior.
22 2
GeneralGeneral introduction
Inn chapter 4 we describe the discovery that human neutrophil defensins, present in neutrophil
granulee extract stimulate meningococcal invasion of epithelial cells, irrespective of the Mc
phenotype. .
Inn chapter 5 we could show that the invasion stimulating effect of neutrophil defensins
iss associated with the formation of defensin rich intermembrane contact sites between
bacterial-- and host cell membranes and involves a novel mechanism of bacterial invasion of
mammaliann cells.
Finally,, in chapter 6, the implications of our findings are discussed, in the context of
theirr relevance for colonization and development of meningococcal disease.
23 3
ChapterChapter 1
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38 8