Transcript medmicro-neisseria

MIC 328 Lectures 38
Aims:
To provide overview of
- Antigenic variation in Neisseria
Neisseria
• Gram-negative, nonmotile diplococcus
• Oxidase producer – a key test to identify them
• Two very closely related, important species:
– Neisseria meningitidis
– Neisseria gonorrhoeae
N. meningitidis
N. gonorrhoea
Have polysaccharide capsules
Have plasmids
Found typically in upper resp T
No capsules
No plasmid
Cause genital infections
The Neisseriae
• Gram – cocci; usually in pairs, sometimes piliated. 2
major species: gonococci and meningococci –
differentiated by the usual clinical presentations of the
diseases they cause.
• Cultures: after 48h on enriched media (Mueller-Hinton)
form convex, glistering, elevated, mucoid colonies, 15mm; non-haemolytic, transparent colonies.
• Produce oxidase. Test: paper soaked with tetramethylparaphenylenediamine hydrochloride → dark purple
rapidly.
• Rapidly killed by drying, sunlight, moist, heat and
many disinfectants.
Neisseria gonorrhoeae
• Gonorrhoea "flow of seed"; in ancient times it
was thought that the pus discharge associated
with the disease contained semen.
• Antigenic switching mechanism:
pilin ↔Opa ↔ lipooligosaccharide (LOS)
Rapid and occurs 1 in every 103 organisms
Different mechanisms applied - Multiple genes
encode for pilin,removal part of DNA for Opa.
Neisseria gonorrhoeae
• Clinical specimens are looked for Opa protein;
colonies form transparent or opaque.
• Pathogenesis: gonococci attack mucous
membranes of GUT, eyes, rectum:
• Acute suppuration→tissue invasion→ chronic
inflammation→fibrosis; spread to other
tissues.
• Gonococcal bacteriaemia leads to skin lesions,
haemorrhagic papules and pustules.
• Newborn – gonococcal ophthalmia neonatum.
Antiphagocytic Mechanism
• To escape phagocytes (macrophages and
neutrophils), and prevent from opsonisation,
Neisseria gonorrhoeae develops by antigenic
variation of its surface structure.
• Two major variable surface proteins: pilin and
outer membrane protein, Opa
• N. gonorrhoeae can create up to 1 million pilin
antigenic variants,
Neisseria gonorrhoeae
• Rx: localised infection – serum-sensitive
(killed by Abs and c’) bacteria; whereas
bacteriaemia – serum-resistant.
• Diagnostic lab tests: taken from pus &
secretions from urethra, cervix, throat,
synovial fluid and smears from endocervix
and urethral are stained.
• Disease: worldwide, sexually, multiple
sexual partners, asymptomatic infection.
Meningitis
• A life-threatening infection of the CNS,
characterised by the inflammation of the
meninges and invasion of the subarachnoid
space
• N. meningitidis is the commonest cause of
meningitis in the UK
• Access to the meninges requires that bacteria
invade the endothelium and exit the blood
vessels. Role of Opc?
Meningococcus
• Piliated cocci; meningococci pathogenic to
human only.
• Symptoms with upper Resp T, high fever, sudden
intense headache, vomiting, stiff neck, coma
within hours. Meningococcemia – thrombosis of
many small blood vessels in multiple organs.
• Specimens: blood, spinal fluid, nasopharyngeal
swab; all are tested with Gram staining and
oxidase test. Further test: Abs to meningococcal
polysaccharides by latex agglutination or
haemagglutination.
• Pustules Pustules are well circumscribed elevations of the superficial
layers of the epidermis. Like papules, they may be follicular or
interfollicular in distribution. The most common cause is bacterial
infection, where the pustules are filled with neutrophils, bacteria,
debris, and possibly a few loose keratinocytes (acantholytic cells). It
must be kept in mind that pustules, due to the thinness of the epidermis
are usually transient. What is often observed is a transition from
erythematous macules to papules to relatively few pustules to small
crusts and scale.
• Papules P Papules are circumscribed, solid elevations of the skin, up to
1 cm in diameter. The elevation may be due to accumulation of cells,
fluid, debris or metabolic deposits and may be follicular or
interfollicular in orientation. Papules often begin as erythematous
macules.
Bacterial Pili
• Type IV pili are essential virulence factors for many
gram-negative bacteria, playing key roles in surface
motility, adhesion, formation of microcolonies and
biofilms, natural transformation, and signaling. We
have determined structures for the type IV pilin
subunits and for the assembled pilus fiber.
Currently, we are investigating the type IV pilus
assembly system, including the assembly ATPase,
the membrane anchor protein interactions, and the
assembled pilus fiber (Fig. 2).
Fig. 2. A schematic
view of the assembly
machinery of type IV
pili: the electron
cryomicroscopy
structure of the pilus of
Neisseria gonorrhoeae
(GC); crystal structures
of full-length
Pseudomonas
aeruginosa (P.a) pilin;
BfpC, the binding
partner protein to
ATPase from
enteropathogenic
Escherichia coli; and
GspE2, the hexameric
assembly ATPase from
Archaeoglobus
Opa (PII)
 Required for full virulence in human volunteers
 Adhesins + invasins, but receptor(s) not on apical surfaces
- roles in vivo ?
 Multiple opa genes in c/s – depends on strain (some > 10)
 Sequencing showed:
HV1
SV
HV2
ATG
= constant
----(CTCTT)n----
SV = semi-variable ‘loops’ exposed
HV = hyper-variable on OM surface
 All copies intact and transcribed – but translation of Opa
proteins depends on number (n) of ‘CTCTT’ repeats
Opa (PII) - Mechanism of variation
HV1
SV
HV2
ATG
----(CTCTT)n----
n varies up to 30 copies
 Slipped-strand mispairing
change n
frame-shifts
• premature termination of translation
• restoration of correct reading frame
 Occurs at random, in any of the 3 - 11 (or so) opa genes
• phase-variants (no Opa expressed)
• antigenic variants (different Opa’s expressed)
 Overall frequency: 10 - 3 to 10 - 4
GONOCOCCI PILI
 Comparison of pil gene sequences
• Conserved sequences (
) + semi- & hyper-variable
‘minicassettes’ ( ) in all pilin genes
mc6
mc5
mc4
mc3
mc2
c
mc1
c
• Short (7-residue) ‘leader’ peptide in propilin
• During export, Phe @ position 8 methylated, producing
N-Me-Phe at N-terminus of ‘mature’ pilin
GONOCOCCI PILI
 Multiple copies (17– 20) of pil sequences scattered thro’
genome, but only 1 expressed – called pilE ( )
 Other copies (
) lack 5’ ends of pil gene, and are silent
• called pilS1, pilS2, pilS3,…..etc
• Each has different ‘minicassette’
sequences
Repertoire’ of variable sequences,
which can be introduced into pliE
by homologous recombination
 ca. 20 pilS, with 6 ‘mc’ each
107 variants
GONOCOCCI PILI
 Homologous recombination between pilE and pilS
- generates new variants @ frequencies of 1 x 102 to 104
pilE
mc6
mc5
mc4
mc3
mc2
mc1
pilS
New pilE
+
 Original (not new) pilS sequence often retained in c/s
- recombination usually involves a copy of pilS
 Transformation by DNA released from other cells in population
plays significant role
PILI PHASE-VARIATION
 S-Phase variants
• ‘Short’ or ‘soluble’ phase variants
• Results from incorrect processing during pili biogenesis
 Propilin ‘leader’ normally removed by cleavage @ a.a. 7 - 8
+1
+40
mc6
mc5
mc4
mc3
mc2
c
mc1
c
 Some (random) combinations of ‘mc’s result in cleavage
between residues 39 & 40 - possibly due to different folding
‘Truncated’ (short) pilin not assembled – ‘soluble’ pilin released
PILI PHASE-VARIATION
 L-Phase variants
• Express longer (L) than normal pilin - cannot be assembled
 pil genes posses repeated sequences between minicassettes
pilE
pilS
L-pilin
 Arise randomly + revert randomly to P+
PILI PHASE-VARIATION
 Pil C variants
• PilC required for initiation of pili assembly
• pilC gene contains run of ‘G’s at 5’ end
• Slipped-strand mispairing insert/delete ‘G’
• Resulting frame-shifts cause premature termination
of PilC translation
THE END