Context Nontypeable Haemophilus influenzae strains
form part of the normal flora of the human upper respiratory tract but are
also implicated in a wide range of diseases. Infections caused by nontypeable H influenzae are major health and socioeconomic burdens.
No single bacterial trait has been associated with disease as opposed to colonization.
Objectives To compare IgA1 protease activity in nontypeable H influenzae strains isolated from patients with symptomatic Haemophilus infection (sputum, cerebrospinal fluid, blood,
or normally sterile tissue) vs strains from throat swabs of asymptomatic carriers
and to compare iga gene carriage and variability
in nontypeable H influenzae strains.
Design and Setting Retrospective study of 63 strains (44 clinical and 19 carriage) collected
between 1991 and 2000 and maintained at the Public Health Laboratory, Gwynedd
General Hospital, Bangor, Wales.
Main Outcome Measures Levels of IgA1 protease activity produced by carriage strains and clinical
isolates from symptomatic patients; the determination of the size and sequence
of a variable region of the iga gene.
Results Bacterial IgA1 protease activity was significantly higher (P<.001) in strains isolated from sputum, blood, cerebrospinal fluid,
or normally sterile tissue of symptomatic individuals (median, 155 mU; interquartile
range [IQR], 80-220 mU; mean, 169 mU; 95% confidence interval [CI], 126-211
mU) than in those isolated from throat swabs of asymptomatic carriers (median,
30 mU; IQR, 15-90 mU; mean, 56 mU; 95% CI, 26-86 mU; assayed on secretory
IgA). The iga gene was detected in 97% of all strains
examined. Variations in the sizes and sequences of part of the iga genes were also apparent. This variable region encodes a polypeptide
linker connecting the protease domain to the β-core autotranslocator,
a porelike structure required for secretion of the protease.
Conclusions These findings reveal the importance of iga
gene variability and expression levels in the establishment of disease phenotype.
They identify nontypeable H influenzae IgA1 protease
as a virulence factor and as a potential target for the development of new
strategies to fight these important pathogens.
Haemophilus influenzae causes a number of diseases
in humans, including both localized and systemic infections.1
Early work established that not all isolates of this gram-negative bacterium
possess the same pathogenic potential. Lower respiratory tract infections
caused by nontypeable H influenzae (NTHi) strains
are responsible for significant mortality in both infants and children in
developing countries.2 They also represent
a major cause of morbidity in both developed and developing countries. In
general, carriers of NTHi are healthy but occasionally develop localized acute
respiratory tract infections (eg, otitis media, sinusitis, pneumonia, and
conjunctivitis). In addition, NTHi is associated with exacerbations of underlying
lung disease (eg, chronic bronchitis, bronchiectasis, and cystic fibrosis).
Less frequently, it has been reported to cause septicemia, endocarditis, epiglottitis,
septic arthritis, and meningitis, illnesses more usually associated with H influenzae type b (Hib).3
Also, NTHi is reported as causal in female genital tract infection and postpartum
and neonatal infection (including septicemia).4
Brazilian purpuric fever is the most serious disease exclusively caused by
strains of NTHi.5
Attempts to identify a particular virulence factor responsible for NTHi
pathogenesis have so far been unsuccessful. Evidence suggests that NTHi relies
on more than 1 specific factor or mechanism to colonize the human nasopharyngeal
tract and then, on occasion, to invade other sites and cause localized infections.6
Immune exclusion is the most important defense mechanism involved in
protection of mucosal membranes.7 This is an
essentially mechanical process in which secretory IgA (sIgA) agglutinates
the colonizing bacteria, resulting in steric hindrance of the adhesin-epithelial
receptor interaction. Once agglutinated, bacterial complexes, entrapped in
mucus, are expelled by mucociliary clearance. The key element in this defense
mechanism, IgA1 is the most abundant antibody associated with human upper
respiratory tract mucosal surfaces.8
Isolates of NTHi produce specific enzymes able to cleave human IgA1.9 Bacterial IgA1 proteases (IgA1-specific postproline
endopeptidases) are extracellular enzymes able to cleave human IgA1 exclusively
within the hinge region of the α-heavy chain, thus separating the antigen
recognition fragments (Fab) and constant region (Fc).10
This cleavage results in decoupling of the recognition function (mediated
by Fab fragments) from effector functions (mediated by the Fc fragment) and
ultimately in impedance of agglutination and mechanical clearance. Released
Fab fragments still have the capacity to bind cognate antigen,11
resulting in the masking of specific epitopes on the bacterial surface and
prevention of subsequent recognition by intact antibodies.
The link between IgA1 protease production by bacterial mucosal pathogens
and the key immunologic role of IgA1 suggests these enzymes may be virulence
determinants in bacterial pathogenesis.12 The
literature contains conflicting reports on the importance of IgA1 proteases.13,14 To date, no quantitative studies
have been reported comparing levels of IgA1 protease in carriage and disease
isolates of NTHi. Studies involving quantitative variations in any virulence
factors are rare. The aim of this retrospective study was to compare the levels
of IgA1 protease activity in individual strains of NTHi isolated from cases
of clinical infection with those obtained from throat swabs of healthy individuals.
Strains of NTHi (44 clinical and 19 carriage, collected between 1991
and 2000) were randomly chosen from the collection maintained at the Public
Health Laboratory, Gwynedd General Hospital, Bangor, Wales. Clinical strains
(n = 44), isolated throughout Wales, were defined as those isolated from symptomatic
patients diagnosed as having current infections, such as pneumonia (from blood
samples), exacerbation of chronic obstructive pulmonary disease, bacteremia,
meningitis, chest infection, subdural abscess, and ear infection. They included
samples from purulent sputum (n = 20), blood (n = 18), cerebrospinal fluid
(n = 3), and other normally sterile sites (n = 3). These strains were collected
as a part of an ongoing epidemiologic survey begun in 1988 to evaluate the
patterns of invasive disease before and after introduction of Hib vaccination.
Carriage strains were defined as those isolated from throat swabs of healthy
volunteer laboratory staff (n = 11) or preschool-aged children attending 4
local crèches (n = 8) with no recent history of respiratory disease.
The strains used in this study are presented in Table 1 and Table 2.
Strain 2509 is a nontypeable strain of unknown origin also isolated in this
laboratory. Strains HK368, HK61, HK393, HK715, and Rd (all capsulated, except
nontypeable HK61), used for comparative purposes, were described previously.15-17
Isolation and Taxonomic Identification
Clinical samples were isolated according to standard laboratory procedures.
The sputum samples were microscopically examined for the presence of polymorphonuclear
leukocytes and epithelial cells to establish their suitability before further
analysis. A sample was considered bronchopulmonary in origin and suitable
for culture if it contained less then 10 oropharyngeal squamous epithelial
cells and 25 or more polymorphonuclear leukocytes per low-power magnification
(×10) field. Samples of purulent or mucopurulent sputum (mucoid and
salivary samples were excluded), submitted from patients with acute chest
infection or exacerbation of chronic obstructive pulmonary airways disease,
were vortexed with an equal volume of sterile phosphate-buffered saline, and
the resultant emulsion was further diluted 1:100 with peptone water. Ten-microliter
aliquots of each dilution (ie, 1:2 and 1:200) were cultured on blood agar
and heated blood (chocolate) agar, containing 10 mg/L of bacitracin to inhibit
normal gram-positive flora. Body fluids (subdural, cerebrospinal fluid, and
vitreous humor) were centrifuged for 5 minutes at 3000 rpm and the deposit
subcultured onto blood and chocolate agars. Blood culture isolates were derived
from 5 to 10 mL of venous blood, inoculated into 50 to 100 mL of tryptone
soya broth, and incubated for up to 5 days before subculture. All plates were
incubated at 37°C in an atmosphere enhanced with 5% carbon dioxide for
up to 48 hours. Throat swabs from healthy volunteers were inoculated onto
chocolate agar containing 10 mg/L of bacitracin and incubated for 48 hours
under the same conditions.
Nutritional requirements, hemolytic activity, porphyrin production,
and serotyping were performed according to standard laboratory methods. A
commercial identification system (API NH; BioMerieux Ltd, Marcy l'Etoile,
France) was used to confirm the identity of the strains. This system includes
detection of indole, urease, and ornithine decarboxylase production from which
the biotype may be derived.18
IgA1 protease activities were measured using an enzyme-linked immunosorbent
assay (ELISA) originally described by Reinholdt and Kilian19
and modified as previously described.20 Briefly,
a 10-mL culture was started by inoculating a loopful of bacteria from chocolate
agar plates. Bacterial cultures were grown until late log phase, and absorbance
at 550 nm (A550) of each culture was monitored. A secondary 10-mL
culture was started using 0.1 mL of the primary culture as an inoculum. This
culture was grown to mid log phase (0.4-0.5 A550). Four milliliters
of each bacterial culture was subsequently filtered through a 0.22-mm disposable
filter unit (Pall Gelman Laboratory Inc, Ann Arbor, Mich) and the filtrate
used directly in the ELISA.
IgA1 protease substrates (purified IgA1, 3.2 µg/mL; Calbiochem,
San Diego, Calif) or purified human colostrum IgA (3.2 µg/mL; Sigma,
St Louis, Mo) were bound to the surface of a polystyrene microtitration plate
(Immulon 2; Dynex Technologies, Chantilly, Va) through their Fab domain using
rabbit anti–human λ light chain antibody (Dako, Glostrup, Denmark).
Immobilized IgA substrates were exposed to filtered culture supernatants (150
µL) for 3 hours. Incubation of bound substrates with bacterial culture
supernatants results in the release and loss of the Fcα region on washing
and the retention of the Fab fragment. Loss of Fcα was detected indirectly
through a reduced binding of peroxidase-conjugated rabbit anti–human
Fcα antibody (Dako) as assayed with the chromogenic substrate o-phenylenediamine dihydrochloride (Sigma). The difference
in absorbance at 490 nm (ΔA490nm) between the undigested
substrate molecules in control wells and the well containing bacterial supernatants
was recorded for each bacterial isolate. Relative activity was calculated
by dividing the ΔA490nm value by the absorbance of the same
culture (measured at 550 nm). One unit of enzyme activity was defined as the
amount of enzyme able to effect a change in optical density of 1 absorbance
unit (at 490 nm) in 1 hour at 37°C. Each bacterial strain was assayed
in triplicate for both IgA1 and sIgA.
The nonparametric Mann-Whitney U test was used
to analyze data, and the null hypothesis assumes that both groups have the
same capacity to produce IgA1 protease.
DNA Isolation and Polymerase Chain Reaction Amplification
We used the polymerase chain reaction (PCR) to examine strains for presence
of the iga protease gene and to assess the genetic
variability of this locus in individual isolates. Four sets of primers were
used to identify 3 separate domains of the iga gene
in each strain examined. All primers were derived from the published sequence
of the H influenzae HK368 IgA1 protease gene (GenBank
sequence database accession No. M87492)15:
linker domain (LF, GTTCCACCACCTGCGCCTGCTAC and LR1, GTTTTCTCTGTTTCTACTTTAGC
or LR2, GTTATATTGCCCCTCGTTATTCAT), mature protease (PF1, ACGCCGTGAAGACTACTATATG
and PR1, CTCGTTGTTGATATGGTTCAT), and β-core domain (CF1, GCAGAATTCAAAGCACAATTTGTTGCA
and CR1, TTATAACGTTAATTCAAACAGGCTT). The primers were designed to bind to
positions 3334-3356, 3595-3617, 4123-4146, 741-762, 2182-2202, 4051-4077,
and 4889-4913, respectively, of the HK368 sequence. CR1 was designed to overlap
with the sequence immediately downstream from the iga
gene. Genomic DNA was prepared by suspending a loopful of bacteria from a
single plate derived from a single colony in 0.5 mL of 10-mM Tris hydrochloride
(pH 8) buffer and boiling for 10 minutes. Amplification was performed using
1 U of AmpliTaqDNA polymerase (Perkin Elmer, Norwalk, Conn) and buffer supplied
by the manufacturer supplemented with 250 µM of each deoxynucleoside
triphosphate, 100 ng of each primer, and 5 µL of crude genomic DNA in
a total volume of 50 µL. Negative controls were included using PCR mixtures
lacking either bacterial DNA or primers. The PCR products were analyzed by
electrophoresis of 10 mL of the amplification mixture on 1% agarose gel and
detected by staining with ethidium bromide. Amplification of Haemophilus 16S ribosomal RNA genes was performed as a control for
DNA concentration and quality for each strain before IgA1 protease gene domain
detection using previously published primer sequences.21
Fragment sizing was performed by constructing calibration curves by
plotting the distance traveled on the gel in millimeters against log10 number of base pairs (bp) for each DNA size standard fragment. The
size of the variable region fragment, in bp, was then calculated from the
relative distances of migration through the gel. The PCR fragment sequences
were determined using the dye termination method with an ABI Taq FS sequencing
kit (Applied Biosystems, Cheshire, England) analyzed on an ABI 373 A Stretch
automated sequencing machine (Applied Biosystems). DNA sequence analysis was
performed using MacVector and AssemblyLIGN (International Biotechnologies,
Cambridge, England) software packages.
Strains were accepted as H influenzae if they
were gram-negative bacilli, hemin, and nicotine adenine dinucleotide dependent,
nonhemolytic on horse blood agar, non–carbon dioxide requiring, and
unable to produce porphyrin from δ-aminolevulinic acid. Serotype was
determined by agglutination with anticapsular-type antiserum. The characteristics
of the strains used in this study are presented in Table 1 and Table 2.
Strains were further characterized using the API NH kit (BioMerieux) (data
not shown). These results suggested that the isolated strains represent a
diverse group, as expected for NTHi, and were not clonally related as are
Hib strains.22
Measurement of IgA1 Protease Activity
The levels of IgA1 protease activity detected in individual strains
are presented in Table 1 and Table 2 and a summary appears in Figure 1. Analysis of these data revealed
a wide variation in detectable levels of enzyme activity. For example, a 60-fold
higher level of enzyme activity was detected in clinical strain 77412 compared
with carriage strain C12. IgA1 protease activity was detected in 39 of the
44 clinical strains. Using a myeloma-derived IgA1 substrate, high levels of
protease activity were observed for the clinical isolates (median, 155 mU;
mean, 169 mU [95% confidence interval (CI), 126-211 mU]). Similar results
were obtained using sIgA substrate (median, 150 mU; mean, 166 mU [95% CI,
127-206 mU]). Measurable activity was detected in 15 of 19 carriage strains
using either substrate (median, 30 mU; mean, 56 mU [95% CI, 26-86 mU]; median,
30 mU; mean, 49 mU [95% CI, 23-76 mU]). The differences between the levels
of IgA1 protease activity between the clinical and carriage strains is statistically
significant at the P< .001 level for both substrates
using the Mann-Whitney U test. All protease-negative
supernatants were incubated with substrate for extended periods (up to 12
hours) to detect low levels of activity but still failed to show detectable
activity.
The presence of an iga gene was detected in
61 (97%) of the 63 strains examined (all clinical and 17 of 19 carriage),
based on the positive amplification of at least 1 iga
gene fragment (protease, linker, or β-core) using 4 different primer
sets. Results of individual amplifications for each strain are expressed as
an overall positive or negative score in Table 1 and Table 2.
A positive signal for β-core domain was obtained in 86% (54 of 63) of
strains and for protease domain in 67% (42 of 63) of strains. The PCR products
corresponding to the linker region were detected in 43 of 44 clinical isolates
but only 4 of 19 carriage isolates.
The β-core domain and protease domain fragments from the 63 strains
had minimal variation in size, as judged by agarose gel electrophoresis (865
and 1464 bp ± 5%). Eleven clinical strains were used to more accurately
demonstrate the size variability of the amplified linker fragment. The sizes
of the PCR products of the amplified variable fragments using the same set
of primers (LF1 and LR1) were between 200 and 1000 bp (210, 200, 440, 460,
235, 1005, 460, 480, 420, 613, and 231 bp in length for isolate numbers 1428,
1958, 2005, 5220, 6338, 6350, 7244, 7693, 8304, 8625, and 77688, respectively).
The sequenced linker region iga PCR products of H influenzae strains 6338, 77688, 8625, and 2509 were composed
of 235, 231, 613, and 749 nucleotides, respectively (GenBank database accession
Nos. AF274859, AF274860, AF274861, and AF274862). Linker region sequences
of iga genes from HK368, HK393, HK715, Rd, and HK61
were available from GenBank (accession Nos. X64357, M87490, M87489, U32779,
and M87491, respectively). Percentage similarities between nucleotide sequences
were generated using CLUSTAL W and results are presented in Table 3.23 Comparison with available H influenzae linker domain sequences revealed varying levels
of homology between equivalent domains of different strains (59%-100% identity
on nucleotide level). Sequence similarities were much higher among encapsulated
strains (96%-100%) compared with NTHi strains (59%-92%).
In this article, we provide quantitative evidence that suggests that
IgA1 protease activity is an important virulence factor in NTHi-mediated infections.
This finding parallels our previous report20
showing increased levels of IgA1 protease activity in invasive strains of Neisseria meningitidis. Taken together, these 2 results
indicate that IgA1 protease contributes significantly to the pathogenic potential
of these bacteria.
The first step toward investigation of the potential role of IgA1 protease
in infections caused by NTHi was to verify the presence of iga genes in all isolates by PCR amplification. Failure to detect a
PCR product in some strains could be explained by the absence of a suitable
complementary nucleotide sequence caused by a mutation abolishing the priming
reaction. The high percentages of strains with positive signals for β-core
and protease domains, together with the uniform size of the amplified fragments,
illustrates the conserved nature of these domains. The PCR fragments corresponding
to the linker region in contrast were detected in almost all of the clinical
isolates but in only 4 of the 19 carriage strains, and exhibited the widest
variation in size. We further characterized 4 iga
protease genes by sequencing their linker domain PCR products and found much
lower sequence similarities than among encapsulated strains. This result supports
previous observations regarding the clonal population structure of encapsulated
strains in contrast to the existence of extensive genetic diversity among
NTHi strains.22 In particular, our results
underline previous findings regarding the limited antigenic and genetic diversity
among Hib IgA1 proteases and more apparent antigenic heterogeneity of NTHi
IgA1 proteases.24,25 Routine biotyping
results (data not shown), together with the PCR and sequencing data, confirm
the relatively wide genetic variability of the samples used in this study.
Quantitative ELISA was used to assay IgA1 protease activity. All strains
belonging to each of the 2 groups were examined in triplicate for IgA1 protease
activity using 2 different substrates (monoclonal IgA1 and polyclonal sIgA).
This guarded against the potential problem of the presence of enzyme-neutralizing
antibodies, although this complication did not materialize. Two main observations
were made: (1) the level of detectable IgA1 protease activity varies between
individual strains (10-600 mU) and (2) strains isolated in association with
clinical NTHi infections demonstrated significantly higher levels of IgA1
protease activity than colonizing strains (median values of 155 mU vs 30 mU
for IgA1 and 150 mU vs 30 mU for sIgA). These findings, including the significant
difference in protease activity for the 2 groups (P<
.001), suggest that IgA1 protease is an important factor in NTHi pathogenesis.
In this article, we have shown that NTHi strains differ remarkably in
their IgA1 protease activities and in the size and nucleotide sequence of
the linker domain of their iga genes. However, it
will be of interest to see whether these observations hold true for other
populations of NTHi from different geographic regions. Indeed, the difference
in IgA1 protease activity was previously reported for 3 NTHi strains, all
clinical isolates.26 What molecular mechanisms
underlie this variability and what are the consequences of this variability
for bacterial pathogenesis? Haemophilus is a naturally
competent genus able to incorporate exogenous DNA via transformation. The
observed variations in IgA1 protease activity levels could be explained by
polymorphisms in the iga gene or promoter.27,28 These polymorphisms arise during
natural transformation and subsequent homologous recombination of DNA fragments
containing the IgA1 protease gene. New variants of bacterial IgA1 protease
with increased activity could result from this process, providing the mutated
strain with survival advantages (eg, avoidance of immune surveillance in the
hostile mucosal membrane environment).
Earlier indirect evidence implicating IgA1 protease in pathogenesis
has been reported; nonpathogenic species of Neisseria
and Haemophilus lack IgA1 protease activity.9,29 The products of IgA1 cleavage have
been found in the cerebrospinal fluid of patients with bacterial meningitis,
in vaginal washings from gonorrhea cases, and in other secretions from individuals
infected with known IgA1 protease–producing bacteria.12
Animal model and tissue culture experiments have been cited for dismissal
of IgA1 proteases as virulence factors. However, animal models of infection
caused by IgA1 protease–producing bacteria are unsatisfactory because
of the exclusive specificity of the enzyme for human IgA1.12
Animal IgA1 does not possess the same hinge region and cannot be cleaved by
NTHi IgA1 protease. Experiments based on infection of organ cultures with
wild-type and isogenic iga mutants again do not mimic
the situation in vivo on human mucosal membranes because they do not produce
or contain human IgA1 or sIgA strain-specific antibodies for bacteria used
in the experiment.30,31
Recently, new roles for bacterial IgA1 protease have been identified.
Human lysosome-associated membrane protein, a heavily glycosylated protein
forming the protective lining of terminal phagolysosomes, is cleaved by Neisseria gonorrhoeae IgA1 protease.32,33
In addition, the participation of N gonorrhoeae IgA1
protease in transepithelial trafficking34 and
the ability of a fragment of the iga gene product
(the α-peptide) to migrate into the nucleus of epithelial cells were
also reported.35 It was suggested that this
protein might be directly involved in regulation of host cell functions via
interaction with nuclear DNA. Thus, bacterial IgA1 proteases could contribute
to bacterial adaptive fitness not only via avoidance of immune exclusion,
but also via intracellular survival and possible regulation of host cell functions.
The recent results showed that IgA1 protease is a potent inducer of proinflammatory
cytokines, further underlining the importance of this enzyme in pathogenesis.36 Thus, high-level IgA1 protease production could assist
NTHi in avoiding the immune response by aiding longer or more extensive colonization
of the host. The presence of larger numbers of organisms, possibly for extended
periods, would presumably increase the likelihood of pathogenic consequences
being displayed, such as localized inflammation or penetration of sterile
sites. Possession of an enzyme with enhanced activity could clearly contribute
to the pathogenic potential of a particular bacterial strain.
Our increased appreciation of the roles of IgA1 protease in pathogenicity
suggests that the enzyme may be a target for development of vaccines against
NTHi infection. Alternatively, enzyme inhibitors could be developed to interfere
with the protease. Elucidation of the exact molecular mechanisms governing
the interaction of IgA1 protease–producing bacteria with the host will
enhance our capacity to develop new strategies for fighting these important
pathogens.
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