موجز عن البحث:
|
Asymptomatic and persistent colonization of
the upper respiratory tract by Neisseria meningitidis occurs despite
elicitation of adaptive immune responses against surface antigens. A putative
mechanism for facilitating host persistence of this bacterial commensal and
pathogen is alterations in expression of surface antigens by simple sequence
repeat (SSR)-mediated phase variation. We investigated how often phase
variation occurs during persistent carriage by analyzing the SSRs of eight
loci in multiple isolates from 21 carriers representative of 1 to 6 months
carriage. Alterations in repeat number were detected by a GeneScan analysis
and occurred at 0.06 mutations/gene/month of carriage. The expression states
were determined by Western blotting and two genes, fetA and nadA, exhibited
trends toward low expression states. A critical finding from our unique
examination of combinatorial expression states, “phasotypes,” was for
significant reductions in expression of multiple phase-variable surface
proteins during persistent carriage of some strains. The immune responses in
these carriers were examined by measuring variant-specific PorA IgG
antibodies, capsular group Y IgG antibodies and serum bactericidal activity
in concomitant serum samples. Persistent carriage was associated with high
levels of specific IgG antibodies and serum bactericidal activity while
recent strain acquisition correlated with a significant induction of
antibodies. We conclude that phase-variable genes are driven into lower
expression states during long-term persistent meningococcal carriage, in part
due to continuous exposure to antibody-mediated selection, suggesting
localized hypermutation has evolved to facilitate host persistence.
|
موجز عن البحث:
|
Neisseria
meningitidis is a human nasopharyngeal commensal capable of causing
life-threatening septicemia and meningitis. Many meningococcal surface
structures, including the autotransporter proteins NalP and MspA, are subject
to phase variation (PV) due to the presence of homopolymeric tracts within
their coding sequences. The functions of MspA are unknown. NalP
proteolytically cleaves several surface-located virulence factors including
the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the
meningococcal outer membrane and secretome whose expression may reduce
isolate susceptibility to 4CMenB-induced immune responses. To improve our
understanding of the contributions of MspA and NalP to meningococcal-host
interactions, their distribution and phase-variable expression status was
studied in epidemiologically relevant samples, including 127 carriage and 514
invasive isolates representative of multiple clonal complexes and serogroups.
Prevalence estimates of .98% and .88% were obtained for mspA and nalP,
respectively, with no significant differences in their frequencies in disease
versus carriage isolates. 16% of serogroup B (MenB) invasive isolates,
predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion
of nalP often resulted from recombination events between flanking repetitive
elements. PolyC tract lengths ranged from 6–15 bp in nalP and 6–14 bp in
mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of
invasive nalP-positive MenB isolates were nalP phase ON. The frequency of
this phenotype was not significantly different in serogroup Y (MenY) carriage
strains, but was significantly higher in invasive MenY strains (86.3%;
p,0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA
phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%)
isolates. This differential expression resulted from different mode mspA
tract lengths between the serogroups. Our data indicates a differential
requirement for NalP and MspA expression in MenB and MenY strains and is a
step towards understanding the contributions of phasevariable loci to
meningococcal biology.
|
ملخص المشاركة:
|
Many bacterial pathogens and commensals
exhibit phase variation or antigenic variation of surface antigens due to
mutations in hypermutable sequences. Alterations in these surface structures
mediate adaptation to fluctuations in the selective pressures encountered
during colonisation and persistence in their hosts. Neisseria meningitidis is
a major agent of meningitis and septicaemia but is usually found as an
asymptomatic coloniser ofthe human nasopharynx. Several genes in this species
are subject to phase variation due to alterations in simple sequence repeat
(SSR)tracts. Multiple isolates of Neisseria meningitidis were collected from
21 individuals subject to persistence colonisation for up to six months. SSRs
of nine outer membrane proteins were analysed for alterations in repeat
number by fragment analysis. Two genes exhibited trends towards lower
expression levels. Reductions in expression were also detected for
combinations of these phase variable genes. The levels of phase variation
were compared to recombination-mediated antigenic variation of pilE, encoding
the major pilin sub-unit, and to allelic variation as detected from whole
genome sequences of these isolates. We conclude that localised hypermutation
in contingency loci is the major mechanism mediating adaptation of
meningococci during persistent carriage in the upper respiratory tract of
humans.
|
ملخص المشاركة:
|
Introduction:
Meningococcal carriage rates are in the range of 10-15% but can increase in
closed and semi-closed populations. Meningococci can persist in a carrier for
6-9 months and this persistence may be enabled by antigenic or phase
variation of outer membrane antigens. Phase variation of the five surface
proteins, FetA, Opc, NadA , HpuA and PorA, are associated with changes in
repeat tract lengths. The genesfetA,opcandporAhave polyG tracts located in
the core promoter whilst thenadArepeats (TAAAs) are located upstream of the
core promoter.ThehpuApolyG repeats are in the open reading frame. The aim of
this study is to determine whether these five genes exhibit frequent
alterations in repeat length during persistent carriage. Methods: Multiple
colonies were isolated of persistent nasopharyngeal meningococcal carriage
strains obtained from university students over a 6 month period at three/four
time points. PCR and sequencing methodologies were using to characterise the
capsular group, fetA, porAand MLST types. DNA was extracted from 6 colonies
for each time point. Length variation of the repeat tract was measured by
GeneScan analysis and direct DNA sequencing. Results: Five different
meningococcal strains (CC174, CC167, CC23, CC60, CC32) from 21 volunteers were
examined for changes in repeat tract length of 5 genes in 368 isolates. Tract
lengths ranged between 8-13, 8->15,9-14, 9-14 and 10-20 forfetA, opc,
nadA, hpuA, and porA, respectively. The CC60 strain did not exhibit changes
in thefetArepeat length (6G) during carriage. The nadAgene was absent in
CC60, CC167 and CC23 strains but exhibited an ON-to-OFF switch in 4 of 8
CC174 carriers. A 9G tract was observed for fetAat the first time point in
all carriers of strains CC174 and CC32 and exhibited switches to other
lengths in 6 of 9 of these carriers. Overall phase variation occurred in
11/21, 13/21, 4/9, 11/21 and 11/21 of volunteers for fetA, opc, nadA, hpuA
and porA, respectively. Conclusion: This study indicates that high
frequencies of phase variation occur during persistent meningococcal
carriage. We are currently investigating the expression status of these phase
variants but speculate that the phase variable changes in the proteins
encoded by these genes are affected by different conditions in each individual
and may be mediating avoidance of host immune responses.
|