BACKGROUND
strains of non-typeable  haemophilus influenzae asymptomatically colonize the human pharynx, but are also opportunistic pathogens that cause localized respiratory tract infections such as otitis media, pneumonia, bronchitis, sinusitis, and copd exacerbation  <cit> .
bacterial factors that differentiate disease from commensal strains are largely unknown since the population structure of nt h. influenzae is genetically heterologous  <cit> .
the association of bacterial factors with disease-causing strains can be inferred, however, by comparing the prevalence of genetic traits between epidemiologically defined collections of disease and commensal strains  <cit>  or, alternatively, between the pathogenic species and a phylogenetically close but non-pathogenic relative  <cit> .
haemophilus haemolyticus is a phylogenetically close relative of nt h. influenzae, but has not been associated with disease  <cit> .
the two species reside in the same host niche, overlap extensively by both taxonomic and phylogenetic analyses  <cit> , and exchange dna through natural transformation  <cit> .
given their close relationship, but difference in disease potential, nt h. influenzae and h. haemolyticus likely possess common genes or genetic traits for commensal growth but differ in genes or traits that facilitate disease  <cit> .
historically, h. haemolyticus has been considered a rarely encountered commensal that was easily differentiated from nt h. influenzae by its hemolytic phenotype  <cit> .
recent studies, however, have shown that 20-40% of isolates in various nt h. influenzae collections were miss-classified, and found to be non-hemolytic h. haemolyticus  <cit> .
these observations suggest that h. haemolyticus is significantly more prevalent in the pharynges than previously thought, and that clinical differentiation of the species from throat and sputum samples is inadequate  <cit> .
therefore, we recently sought to differentiate the species by their relative proportions of selected nt h. influenzae virulence genes and observed that a probe made to lica, a nt h. influenzae gene necessary for phosphorylcholine  modification of los, hybridized to 96% of nt h. influenzae isolates and to 42% of h. haemolyticus isolates  <cit> .
the relationship of chop expression between nt h. influenzae and h. haemolyticus is unknown but differences between the species may highlight important roles in nt h. influenzae virulence.
in studies addressing nt h. influenzae virulence, chop-modified los has been shown to promote bacterial adherence and invasion of host cells through interaction with the platelet activating factor receptor, to increase bacterial resistance to host antimicrobial peptides such as cathelicidin , and to modulate the host inflammatory response directed toward bacteria present in biofilms  <cit> .
paradoxical to its role in enhancing colonization and virulence, chop can bind c-reactive protein  which initiates c1q binding that leads to activation of the classical complement pathway and bactericidal killing  <cit> .
the concentration of crp  dramatically increases during inflammation, and has been proposed to facilitate clearance of chop-expressing bacteria in the respiratory tract  <cit> .
human chop-specific antibodies capable of eliciting in vitro bactericidal activity against some h. influenzae strains have also been identified, suggesting a further liability of h. influenzae chop expression  <cit> .
h. influenzae may avoid crp and anti-chop antibody binding, however, by phase varying chop expression and by strain-dependent localization of chop substitutions within los  <cit> .
in h. influenzae, chop expression is controlled by a contingency locus, lic <dig>  that contains the lica, licb, licc, and licd genes   <cit> .
contingency loci, such as lic <dig>  contain simple sequence repeats  that provide an organism with the ability to phase vary specific phenotypes in response to host challenges  <cit> .
in lic <dig>  the ssr are tetranucleotide  and are present at the 5' end of lica, the first gene in the locus  <cit> .
during replication, intragenic ssr repeats undergo slipped-strand mispairing which results in translational phase variation, and the rate of these mutations is proportional to the length of the repeat region  <cit> .
de bolle et al  <cit>  found that mutation rates of a h. influenzae type iii restriction modification gene  engineered to contain 17- <dig> tetranucleotide  intragenic repeats increased linearly with the number of repeats.
in contrast, the same gene containing 5- <dig> repeats demonstrated rare, if any, phase-variation.
thus, higher numbers of repeats in a contingency locus may protect the bacteria by decreasing the response time to host challenges  <cit> .
among h. influenzae strains, however, the number of lica gene 5'-caat-3' repeats range from 3- <dig>  and patterns pertaining to virulence have not been identified  <cit> .
depending on the h. influenzae strain, chop may be substituted at different positions within los.
substitutions may occur on oligosaccharides that extend from any one of the three conserved inner-core heptose residues  or, alternatively, directly to heptose iv, an outer core heptose that extends from heptose i  <cit> .
these substitutions are dictated largely by the diphosphonucleoside choline transferase encoded by the licd gene.
three licd gene alleles mediate chop substitutions at different positions within los and, for simplification, we have named the alleles to reflect their association with a given heptose-residue: licdi, licdiii, and licdiv.
although chop has been associated with heptose ii residues in selected strains, a specific licd allele mediating these substitutions has not been experimentally documented  <cit> .
the deduced licd proteins are 265- <dig> amino acids in length and range in sequence identity from 74-88% with much of the variation occurring in the central part of the primary structure  <cit> .
although most nt h. influenzae strains possess a single licd allelic gene that facilitates one chop substitution, fox et al  <cit>  recently reported that 4/ <dig>  of nt h. influenzae middle ear strains possessed two different licd alleles, each present in a separate, phase-variable lic <dig> locus, that together could produce up to two chop substitutions in the strain's los.
both the number and position of chop substitutions within los may affect binding of host clearance molecules such as crp or natural chop antibodies  <cit> .
for instance, h. influenzae strains with dual chop substitutions bind more crp, and h. influenzae strains with chop substitutions positioned from the distal heptose iii residue are 10-fold more sensitive to crp-initiated bactericidal killing than chop associated with the proximal heptose i in the same strains  <cit> .
consequently, strains with proximal chop substitutions  may be more protected from crp-mediated clearance, and los structural studies on selected nt h. influenzae strains have found that chop predominate at this position  <cit> .
the overall prevalence of these substitutions in the nt h. influenzae population, however, is not known.
differences in the prevalence of single or combined licd gene alleles between nt h. influenzae and h. haemolyticus may reflect the importance of chop structures in nt h. influenzae virulence.
the presence of a lica gene in h. haemolyticus suggests that it may contain a lic <dig> locus and express chop in a manner similar to h. influenzae  <cit> .
since chop expression among nt h. influenzae strains can vary greatly due to genetic factors listed above, we speculated that differences in the prevalence of these factors between strain populations of h. influenzae and h. haemolyticus may highlight, in part, which ones provide an advantage to h. influenzae in transcending from commensal to disease-related growth.
RESULTS
chop expression in h. haemolyticus
although h. influenzae is known to modify its los with chop, expression and surface localization of chop in h. haemolyticus has not been demonstrated.
to investigate chop expression in h. haemolyticus, we obtained los profiles on silver-stained tricine sds-page from whole-cell lysates on three h. influenzae control strains, six h. haemolyticus strains containing a lica gene, and five h. haemolyticus strains lacking a lica gene  <cit> .
as seen in figure  <dig> , both nt h. influenzae and h. haemolyticus demonstrated intra-and inter-strain variability in los migration.
a duplicate gel was transferred to a western immunoblot and chop was detected with tepc- <dig>  a mab that recognizes chop on a number of pathogenic bacteria  <cit> .
tepc- <dig> reacted with los-associated bands in all h. influenzae control strains and in the six h. haemolyticus strains that contained a lica gene .
the antibody, however, did not react to five h. haemolyticus strains lacking a lica gene .
the association of chop epitopes with h. haemolyticus los was further supported by proteinase k digestion experiments.
tepc- <dig> reactivity was still present on western immunoblots containing h. influenzae strain rd and h. haemolyticus strain m07- <dig> that were pre-treated with proteinase k, although no proteins were visible in these preparations when they were run on glycine sds-page and stained with coomassie .
together these results suggest that, similar to h. influenzae, some strains of h. haemolyticus can express a chop epitope that is localized within its los.
h. haemolyticus contains a lic <dig> locus similar to h. influenzae
the ability of h. haemolyticus to hybridize with a h. influenzae lica gene probe suggests that h. haemolyticus contains a lic <dig> locus  <cit> .
in h. haemolyticus strains m07- <dig> and 60p3h <dig>  lica-licd gene probes were each found to hybridize with one restriction fragment on southern blots, suggesting that all genes were confined to a single locus in each strain .
pcr designed to amplify overlapping regions of h. influenzae lic <dig> locus genes also amplified similar products in h. haemolyticus strains m07- <dig> and 60p3h <dig>  and dna sequencing of these products revealed that the strains contained tandem lica-licd genes spanning  <dig> and  <dig> bp , respectively, similar in size to the lic <dig> loci present in the genome-sequenced h. influenzae strains rd  and 86-028np   <cit> .
further comparisons of the lic <dig> loci between h. haemolyticus and h. influenzae  <cit>  revealed that, in both species, the loci were flanked by the same chromosomal genes, contained lica α, β, and γ start codons positioned immediately upstream of tandemly arranged tetranucleotide  repeats, and contained licb and licc start codons that overlapped each preceding gene .
the lica, licb, and licc amino-acid sequences for the two h. haemolyticus strains m07- <dig> and 60p3h <dig> were deduced and found to be  <dig>   <dig>  and 95% identical, respectively, between the strains .
amino-acid sequences comparisons of the putative lica, licb, and licc proteins between h. haemolyticus and h. influenzae  revealed identities that were somewhat lower, ranging from 87-94% for all comparisons .
as mentioned above, three licd protein alleles  have been described for h. influenzae.
the licd protein of h. haemolyticus strain m07- <dig> was  <dig> and 87% identical to the licdi allele of h. influenzae strains rd and 86-028np, respectively, but was 95% identical with and contained a  <dig> amino-acid insertion similar to the licdiii allele of h. influenzae strain e1a, suggesting that this h. haemolyticus strain possessed a licdiii allele .
in contrast, the putative licd protein of h. haemolyticus strain 60p3h <dig> averaged only 69% identity with the licd alleles of h. haemolyticus strain m07- <dig> and the three h. influenzae strains .
blast analysis, however, revealed that it was 99% identical to the deduced licdiv protein of nt h. influenzae strain r <dig>  suggesting that h. haemolyticus strain 60p3h <dig> contained a licdiv allele.
together, these data suggest that h. haemolyticus possess lic <dig> loci that are very similar to the lic <dig> loci described for h. influenzae.
prevalence of lic <dig> loci in h. influenzae and h. haemolyticus
as mentioned, the prevalence of the lica gene has been reported for a phylogenetically defined nt h. influenzae and h. haemolyticus strain collection  <cit> .
we therefore determined the distribution of the remaining lic <dig> locus genes  among the same strains by dot-blot hybridization.
the licb-licd gene probes each hybridized to three h. influenzae positive control strains , to 81/ <dig>  nt h. influenzae strains and to 46/ <dig>  h. haemolyticus strains.
four nt h. influenzae strains  that previously hybridized with the lica gene probe did not hybridize with the licb-licd probes.
in addition, one nt h. influenzae strain  that did not previously hybridize with the lica gene probe did hybridize with the licb-licd probes in this study.
repeat hybridization of these discrepant strains with the lica gene probe revealed that lica hybridization was concordant with licb-licd hybridization, and that all strains either lacked or possessed all four lic <dig> locus genes.
the probes did not hybridize to a negative control species  or to any of the remaining nt h. influenzae or h. haemolyticus strains that previously failed to hybridize with the lica gene probe .
the absence of the lica-licd genes in these strains suggests that 8% of nt h. influenzae and  <dig> % of h. haemolyticus strains lack a lic <dig> locus for chop expression, and that absence of a lic <dig> locus is  <dig>  times more prevalent in h. haemolyticus than in nt h. influenzae .
a prevalence ratios  were calculated for h. influenzae using h. haemolyticus as the referent group.
b logit,  <dig>  used in place of  <dig> for pr and statistical calculations.
c p <  <dig>  is considered statistically significant using χ <dig> analysis.
the prevalence of nt h. influenzae and h. haemolyticus strains possessing single or duplicate lic <dig> loci is not known.
similar to the method reported by fox et al  <cit> , we screened our  <dig> nt h. influenzae and  <dig> h. haemolyticus lic1-containing strains for duplicate lic <dig> loci using southern hybridization of mfe <dig> digested genomic dna to identify two restriction fragments that hybridized with a licd gene probe.
strains with two licd-hybridizing bands were present in seven nt h. influenzae strains and in none of the h. haemolyticus strains.
further hybridization using a lica gene probe on the seven nt h. influenzae strains also revealed two lica hybridizing bands in these strains, suggesting that they possessed two complete lic <dig> loci.
assessing the population prevalence of lic <dig> locus copy number among the species, the data suggest that 74/ <dig>  nt h. influenzae and 46/ <dig>  h. haemolyticus possess one copy of lic <dig>  and that strains with one lic <dig> locus are  <dig>  times more prevalent in nt h. influenzae than in h. haemolyticus  .
duplicate lic <dig> loci were present in 7/ <dig>  nt h. influenzae and 0/ <dig>  h. haemolyticus strains, suggesting that duplicate lic <dig> loci in h. haemolyticus are rare or altogether absent .
prevalence of the three licd alleles in nt h. influenzae and h. haemolyticus
determining the prevalence of the three previously described licd alleles among the two species was initiated by pcr amplification and dna sequence analysis of the licd genes from the  <dig> nt h. influenzae and  <dig> h. haemolyticus strains in our collection that contained a single lic <dig> locus.
the deduced licd amino-acid sequences of these strains were determined  and the licd gene from one nt h. influenzae strain  was repeatedly found to possess a nonsense mutation that would result in gene termination.
a minimum-evolution dendrogram  was created from the remaining licd amino-acid sequences of the nt h. influenzae and h. haemolyticus strains.
the dendrogram revealed three distinct clusters, each containing a different h. influenzae prototype licd allele  .
these results suggest that the three previously defined licd alleles represent the major allelic variants found among the h. influenzae and h. haemolyticus species.
next, we determined the population prevalence of specific licd alleles in our nt h. influenzae and h. haemolyticus strains.
among the  <dig> total nt h. influenzae strains in the collection,  <dig>  possessed a single licdi allele,  <dig>  possessed a single licdiii allele, and  <dig>  possessed a single licdiv allele .
in contrast, only  <dig> of the  <dig>  h. haemolyticus strains possessed a licdi allele while  <dig>  possessed a single licdiii allele and  <dig>  possessed a single licdiv allele.
although the prevalence of single licdi alleles was statistically different between nt h. influenzae and h. haemolyticus , the prevalence of the licdiii and licdiv alleles was not statistically different between the species .
assessment of licd gene alleles among the seven dual lic <dig> locus-containing nt h. influenzae strains was determined by pcr amplifying and sequencing licd from agarose gel slices of strain dna digested with mfe <dig>  the results revealed that 4/ <dig>  strains had licdiii-licdiv alleles, while only 1/ <dig>  strains each were found to possess combinations of licdi-licdiii, licdi-licdiv, and licdi-licdi alleles .
together, these results suggest that the licdi allele is rarely present in h. haemolyticus, and that the proportions of licdiii and licdiv alleles are similar between the species.
chop phase variation and the number of lica tetranucleotide  repeats among nt h. influenzae and h. haemolyticus
phase variation of chop expression is similar between nt h. influenzae and h. haemolyticus.
the lica genes of h. haemolyticus strains m07- <dig> and 60p3h <dig> contained a number of 5'-caat-3' repeats that would place the lica gene in a correct translational open reading frame .
chop expression in these two strains was corroborated by western immunoblot where tepc- <dig> reactive epitopes were present in each strain .
in addition, phase-negative variants could be isolated from each h. haemolyticus strain, and dna sequence analysis revealed that each lica repeat region gained one 5'-caat-3' repeat, placing the lica gene out of frame .
mutation rates in contingency loci are proportional to the length of the repeat region in the loci and the repeat region length may therefore affect the ability of bacteria to respond to a host immunologic challenge  <cit> .
to determine if a general population difference of lica repeat length exists between the species in this study, we compared the number of lica 5'-caat-3' repeats between the  <dig> nt h. influenzae and  <dig> h. haemolyticus strains that contained a single lic <dig> locus.
dna sequence analysis of pcr amplified repeat regions from these strains revealed a wide range in repeat numbers for both species  .
the average number of lica repeats between the species, however, was statistically different with nt h. influenzae having a mean of  <dig> repeats and h. haemolyticus having a mean of  <dig> repeats  .
these results suggest that, at the population level, the contingency response for chop expression may be slower for h. haemolyticus than for nt h. influenzae.
* p < .
<dig> using the student's t-test
** p < .
<dig> for each comparison using the student's t-test
h. influenzae strains that express chop at more distal positions in los  have been shown to be more vulnerable to crp binding, leading to activation of complement mediated bactericidal killing  <cit> .
therefore, strains may differ in their lica mutation rates depending on which los structure is modified with chop.
to test this, we further stratified the number of lica gene repeats between strains with different licd alleles for each species.
among nt h. influenzae, the range of repeats was similar among strains that possessed a licdi, licdiii, or licdiv allele  .
the average number of repeats was significantly different, however, for strains that possessed a licdiii allele  than for strains that possessed a licdi or licdiv allele   .
among h. haemolyticus, the range of lica repeats was more variable between strains with licdiii and licdiv alleles , due mainly to three licdiii-containing strains with lica genes that contained  <dig>   <dig>  and  <dig> repeats .
in contrast to nt h. influenzae, however, the average number of repeats was not significantly different between h. haemolyticus strains possessing licdiii or licdiv alleles  .
these results suggest that nt h. influenzae strains that substitute chop on more proximal, exposed oligosaccharides chains may tend to have increased mutation rates within the repeat region of the lica gene.
discussion
the strain population structure of nt h. influenzae is genetically very diverse and clones or clusters of nt h. influenzae strains that differentiate virulent from commensal strains have not been identified  <cit> .
given this diversity, together with the high prevalence of nt h. influenzae colonization in the healthy human population, it is reasonable to hypothesize that not all nt h. influenzae strains possess the same ability to cause disease, but rather, that a proportion of strains possess a range of variable genetic traits that allow for infection and disease under the right host conditions  <cit> .
thus, comparison of genetic trait prevalence between populations of nt h. influenzae and the closely related but strictly commensal species, h. haemolyticus, will highlight traits within the species' gene pools that may offer clues to the virulence pathways of nt h. influenzae.
for instance, chop expression in nt h. influenzae is strongly implicated as a virulence factor  <cit>  and is thought to enhance virulence though increased epithelial cell adherence, inhibition of bactericidal peptides, and modulation of the immune system during biofilm growth  <cit> .
in this study, 58% of h. haemolyticus strains lacked a lic <dig> locus  while only 8% of nt h. influenzae strains lacked a lic <dig> locus, suggesting that, at a population level, chop expression may provide an advantage for more nt h. influenzae strains to cause disease.
furthermore, the trend of shorter lica gene repeat regions in h. haemolyticus strains that possess a lic <dig> locus , may suggest that those strains have a slower phase-variable response to host defences targeting chop , potentially limiting their survival in inflammatory environments.
obviously, prevalence differences in chop expression alone do not account for all differences in disease potential between the species since many other virulence factors have been described for nt h. influenzae.
rather, the differential prevalence of genetic traits between the species highlight factors that may be further studied for their roles in virulence using in vitro and in vivo models of nt h. influenzae infection.
although the structure of h. haemolyticus los is unknown, the assumption has been made that basic los structures and biosynthesis of chop modifications, mediated by the phosphocholine transferase, licd, are comparable between nt h. influenzae and h. haemolyticus.
some evidence suggests that these assumptions are reasonable.
in the tricine sds-page experiments of this study, h. haemolyticus los migrated at a rate similar to the los of nt h. influenzae, and h. haemolyticus los also presented intra and inter-strain structural heterogeneity similar to the los of nt h. influenzae .
recent structural analysis on the los of haemophilus parainfluenzae, a member of the pasteurellaceae family that is phylogenetically more distant to nt h. influenzae than h. haemolyticus, revealed that the inner core structure was nearly identical to that of nt h. influenzae  <cit> .
furthermore, the licdiii and licdiv alleles of the two h. haemolyticus strains in this study demonstrated higher sequence identity  to their cognate proteins in nt h. influenzae than similar comparisons of lica, licb, and licc proteins , suggesting a functional equivalence of the licd protein alleles.
although these observations are circumstantial, they argue for more detailed comparisons of los structures between nt h. influenzae and h. haemolyticus to identify dissimilarities between the structures that may be associated with the ability of nt h. influenzae to cause disease.
the results of this study suggest that genotypes facilitating los-chop structures that are not conducive to crp binding predominate among the strain populations of both species; the majority of h. haemolyticus strains  lacked a lic <dig> locus  and the majority of nt h. influenzae strains either lacked a lic <dig> locus or possessed a single licdi allele  .
in comparison, strains possessing single licdiii and licdiv alleles were in smaller, but similar fractions in the strain populations of both species , indicating that these allelic distributions are still maintained in the species despite possible increased vulnerability to crp binding.
further studies that assess the prevalence of licd alleles between epidemiologically comparable collections of virulent and commensal nt h. influenzae strains may highlight which alleles are important in nt h. influenzae disease.
one chop genotype that may be associated with nt h. influenzae disease isolates is the possession of two lic <dig> loci in the same strain where each locus contains a different licd allele, providing the bacteria with two independently phase-variable chop substitutions.
fox et al  <cit>  demonstrated that 4/ <dig>  nt h. influenzae middle ear strains had dual lic <dig> loci.
in the current study, only nt h. influenzae and not h. haemolyticus possessed dual lic <dig> loci.
although only  <dig> of  <dig>  total nt h. influenzae strains had dual loci, six were present among  <dig>  middle ear strains present in this collection .
fox et al.
<cit>  also noted that the genome sequenced nt h. influenzae strain, r <dig>  possessed a complete and partial lic <dig> loci, each containing a different licd allele, raising the possibility that other strains may have a similar genotype.
an extensive search on the lic1-containing strains in this collection using licd-specific pcr and hybridization, however, did not identify any strains  that contained more than one licd allele, suggesting that the nt h. influenzae population contains mainly complete copies of lic <dig> .
although nt h. influenzae los structural studies have identified chop modifications on oligosaccharides extending from the heptose ii position  <cit> , specific licd alleles mediating this arrangement have not been characterized.
it is possible that one or more of the current licd alleles may overlap in this process or that stochastic factors in los biosynthesis may play a role.
in addition, the clustering analysis of licd protein alleles present in figure  <dig> suggests that sub-variants may exist within the major allelic groups, and it is possible that one of these variants may facilitate heptose ii-associated chop substitutions.
as reviewed by moxon et al  <cit> , strains that are genetically and epidemiologically unrelated vary widely in the lengths of ssr , while individual strains that transmit within an outbreak or are extensively subcultured over time maintain a central modality in repeat numbers  <cit> .
using a larger number of samples from a phylogenetically defined collection of nt h. influenzae strains has allowed us to partially resolve distribution trends for the lica repeat region in the nt h. influenzae and h. haemolyticus populations  and make statistical comparisons between and within species .
we found statistically significant trends toward the increased length of lica tetranucleotide repeats in nt h. influenzae compared to h. haemolyticus, and in nt h. influenzae strains with licdiii alleles compared to nt h. influenzae strains with licdi or licdiv alleles.
longer repeat regions are predicted to increase lic <dig> loci mutation rates and chop phase variation, providing increased resistance to host clearance mechanisms such as crp or antibodies that bind chop and initiate complement mediated bactericidal killing.
the presence of the longest repeat  in a h. haemolyticus strain and only five repeats in a licdiii-containing nt h. influenzae strain, however, are reminders that these trends must be considered in the light of numerous other factors that contribute to the commensal life style of both species and disease potential of nt h. influenzae.
CONCLUSIONS
in summary, the lic <dig> locus is not part of the conserved "core" genome of the h. influenzae population but is part of the flexible gene pool that exists among different strains  <cit> .
nonetheless, the conserved chemical nature of chop and the discovery of anti-chop antibodies in human serum provides reasonable credence to chop as a vaccine candidate that may inhibit h. influenzae at some point in the infectious process.
knowledge of how chop expression varies both genetically and structurally within the nt h. influenzae strain population is critical for designing intervention strategies that will effectively target disease-related strains.
furthermore, contrasting the genetic properties of nt h. influenzae chop expression with those of h. haemolyticus, a closely related but non-pathogenic species, has highlighted a number of chop expression differences  that may provide an advantage to disease-related growth in nt h. influenzae.

 strains of non-typeable haemophilus influenzae asymptomatically colonize the human pharynx, but are also opportunistic pathogens that cause localized respiratory tract infections such as otitis media, pneumonia, bronchitis, sinusitis, and copd exacerbation. bacterial factors that differentiate disease from commensal strains are largely unknown since the population structure of nt h. influenzae is genetically heterologous. the association of bacterial factors with disease-causing strains can be inferred, however, by comparing the prevalence of genetic traits between epidemiologically defined collections of disease and commensal strains or, alternatively, between the pathogenic species and a phylogenetically close but non-pathogenic relative. haemophilus haemolyticus is a phylogenetically close relative of nt h. influenzae, but has not been associated with disease. the two species reside in the same host niche, overlap extensively by both taxonomic and phylogenetic analyses, and exchange dna through natural transformation. given their close relationship, but difference in disease potential, nt h. influenzae and h. haemolyticus likely possess common genes or genetic traits for commensal growth but differ in genes or traits that facilitate disease. historically, h. haemolyticus has been considered a rarely encountered commensal that was easily differentiated from nt h. influenzae by its hemolytic phenotype. recent studies, however, have shown that 20-40% of isolates in various nt h. influenzae collections were miss-classified, and found to be non-hemolytic h. haemolyticus. these observations suggest that h. haemolyticus is significantly more prevalent in the pharynges than previously thought, and that clinical differentiation of the species from throat and sputum samples is inadequate. therefore, we recently sought to differentiate the species by their relative proportions of selected nt h. influenzae virulence genes and observed that a probe made to lica, a nt h. influenzae gene necessary for phosphorylcholine modification of los, hybridized to 96% of nt h. influenzae isolates and to 42% of h. haemolyticus isolates. the relationship of chop expression between nt h. influenzae and h. haemolyticus is unknown but differences between the species may highlight important roles in nt h. influenzae virulence. in studies addressing nt h. influenzae virulence, chop-modified los has been shown to promote bacterial adherence and invasion of host cells through interaction with the platelet activating factor receptor, to increase bacterial resistance to host antimicrobial peptides such as cathelicidin, and to modulate the host inflammatory response directed toward bacteria present in biofilms. paradoxical to its role in enhancing colonization and virulence, chop can bind c-reactive protein which initiates c1q binding that leads to activation of the classical complement pathway and bactericidal killing. the concentration of crp dramatically increases during inflammation, and has been proposed to facilitate clearance of chop-expressing bacteria in the respiratory tract. human chop-specific antibodies capable of eliciting in vitro bactericidal activity against some h. influenzae strains have also been identified, suggesting a further liability of h. influenzae chop expression. h. influenzae may avoid crp and anti-chop antibody binding, however, by phase varying chop expression and by strain-dependent localization of chop substitutions within los. in h. influenzae, chop expression is controlled by a contingency locus, lic that contains the lica, licb, licc, and licd genes. contingency loci, such as lic contain simple sequence repeats that provide an organism with the ability to phase vary specific phenotypes in response to host challenges. in lic the ssr are tetranucleotide and are present at the 5' end of lica, the first gene in the locus. during replication, intragenic ssr repeats undergo slipped-strand mispairing which results in translational phase variation, and the rate of these mutations is proportional to the length of the repeat region. de bolle et al found that mutation rates of a h. influenzae type iii restriction modification gene engineered to contain 17- tetranucleotide intragenic repeats increased linearly with the number of repeats. in contrast, the same gene containing 5- repeats demonstrated rare, if any, phase-variation. thus, higher numbers of repeats in a contingency locus may protect the bacteria by decreasing the response time to host challenges. among h. influenzae strains, however, the number of lica gene 5'-caat-3' repeats range from 3- and patterns pertaining to virulence have not been identified. depending on the h. influenzae strain, chop may be substituted at different positions within los. substitutions may occur on oligosaccharides that extend from any one of the three conserved inner-core heptose residues or, alternatively, directly to heptose iv, an outer core heptose that extends from heptose i. these substitutions are dictated largely by the diphosphonucleoside choline transferase encoded by the licd gene. three licd gene alleles mediate chop substitutions at different positions within los and, for simplification, we have named the alleles to reflect their association with a given heptose-residue: licdi, licdiii, and licdiv. although chop has been associated with heptose ii residues in selected strains, a specific licd allele mediating these substitutions has not been experimentally documented. the deduced licd proteins are 265- amino acids in length and range in sequence identity from 74-88% with much of the variation occurring in the central part of the primary structure. although most nt h. influenzae strains possess a single licd allelic gene that facilitates one chop substitution, fox et al recently reported that 4/ of nt h. influenzae middle ear strains possessed two different licd alleles, each present in a separate, phase-variable lic locus, that together could produce up to two chop substitutions in the strain's los. both the number and position of chop substitutions within los may affect binding of host clearance molecules such as crp or natural chop antibodies. for instance, h. influenzae strains with dual chop substitutions bind more crp, and h. influenzae strains with chop substitutions positioned from the distal heptose iii residue are 10-fold more sensitive to crp-initiated bactericidal killing than chop associated with the proximal heptose i in the same strains. consequently, strains with proximal chop substitutions may be more protected from crp-mediated clearance, and los structural studies on selected nt h. influenzae strains have found that chop predominate at this position. the overall prevalence of these substitutions in the nt h. influenzae population, however, is not known. differences in the prevalence of single or combined licd gene alleles between nt h. influenzae and h. haemolyticus may reflect the importance of chop structures in nt h. influenzae virulence. the presence of a lica gene in h. haemolyticus suggests that it may contain a lic locus and express chop in a manner similar to h. influenzae. since chop expression among nt h. influenzae strains can vary greatly due to genetic factors listed above, we speculated that differences in the prevalence of these factors between strain populations of h. influenzae and h. haemolyticus may highlight, in part, which ones provide an advantage to h. influenzae in transcending from commensal to disease-related growth.  chop expression in h. haemolyticus although h. influenzae is known to modify its los with chop, expression and surface localization of chop in h. haemolyticus has not been demonstrated. to investigate chop expression in h. haemolyticus, we obtained los profiles on silver-stained tricine sds-page from whole-cell lysates on three h. influenzae control strains, six h. haemolyticus strains containing a lica gene, and five h. haemolyticus strains lacking a lica gene. as seen in figure, both nt h. influenzae and h. haemolyticus demonstrated intra-and inter-strain variability in los migration. a duplicate gel was transferred to a western immunoblot and chop was detected with tepc- a mab that recognizes chop on a number of pathogenic bacteria. tepc- reacted with los-associated bands in all h. influenzae control strains and in the six h. haemolyticus strains that contained a lica gene. the antibody, however, did not react to five h. haemolyticus strains lacking a lica gene. the association of chop epitopes with h. haemolyticus los was further supported by proteinase k digestion experiments. tepc- reactivity was still present on western immunoblots containing h. influenzae strain rd and h. haemolyticus strain m07- that were pre-treated with proteinase k, although no proteins were visible in these preparations when they were run on glycine sds-page and stained with coomassie. together these results suggest that, similar to h. influenzae, some strains of h. haemolyticus can express a chop epitope that is localized within its los. h. haemolyticus contains a lic locus similar to h. influenzae the ability of h. haemolyticus to hybridize with a h. influenzae lica gene probe suggests that h. haemolyticus contains a lic locus. in h. haemolyticus strains m07- and 60p3h lica-licd gene probes were each found to hybridize with one restriction fragment on southern blots, suggesting that all genes were confined to a single locus in each strain. pcr designed to amplify overlapping regions of h. influenzae lic locus genes also amplified similar products in h. haemolyticus strains m07- and 60p3h and dna sequencing of these products revealed that the strains contained tandem lica-licd genes spanning and bp, respectively, similar in size to the lic loci present in the genome-sequenced h. influenzae strains rd and 86-028np. further comparisons of the lic loci between h. haemolyticus and h. influenzae revealed that, in both species, the loci were flanked by the same chromosomal genes, contained lica α, β, and γ start codons positioned immediately upstream of tandemly arranged tetranucleotide repeats, and contained licb and licc start codons that overlapped each preceding gene. the lica, licb, and licc amino-acid sequences for the two h. haemolyticus strains m07- and 60p3h were deduced and found to be and 95% identical, respectively, between the strains. amino-acid sequences comparisons of the putative lica, licb, and licc proteins between h. haemolyticus and h. influenzae revealed identities that were somewhat lower, ranging from 87-94% for all comparisons. as mentioned above, three licd protein alleles have been described for h. influenzae. the licd protein of h. haemolyticus strain m07- was and 87% identical to the licdi allele of h. influenzae strains rd and 86-028np, respectively, but was 95% identical with and contained a amino-acid insertion similar to the licdiii allele of h. influenzae strain e1a, suggesting that this h. haemolyticus strain possessed a licdiii allele. in contrast, the putative licd protein of h. haemolyticus strain 60p3h averaged only 69% identity with the licd alleles of h. haemolyticus strain m07- and the three h. influenzae strains. blast analysis, however, revealed that it was 99% identical to the deduced licdiv protein of nt h. influenzae strain r suggesting that h. haemolyticus strain 60p3h contained a licdiv allele. together, these data suggest that h. haemolyticus possess lic loci that are very similar to the lic loci described for h. influenzae. prevalence of lic loci in h. influenzae and h. haemolyticus as mentioned, the prevalence of the lica gene has been reported for a phylogenetically defined nt h. influenzae and h. haemolyticus strain collection. we therefore determined the distribution of the remaining lic locus genes among the same strains by dot-blot hybridization. the licb-licd gene probes each hybridized to three h. influenzae positive control strains, to 81/ nt h. influenzae strains and to 46/ h. haemolyticus strains. four nt h. influenzae strains that previously hybridized with the lica gene probe did not hybridize with the licb-licd probes. in addition, one nt h. influenzae strain that did not previously hybridize with the lica gene probe did hybridize with the licb-licd probes in this study. repeat hybridization of these discrepant strains with the lica gene probe revealed that lica hybridization was concordant with licb-licd hybridization, and that all strains either lacked or possessed all four lic locus genes. the probes did not hybridize to a negative control species or to any of the remaining nt h. influenzae or h. haemolyticus strains that previously failed to hybridize with the lica gene probe. the absence of the lica-licd genes in these strains suggests that 8% of nt h. influenzae and % of h. haemolyticus strains lack a lic locus for chop expression, and that absence of a lic locus is times more prevalent in h. haemolyticus than in nt h. influenzae. a prevalence ratios were calculated for h. influenzae using h. haemolyticus as the referent group. b logit, used in place of for pr and statistical calculations. c p < is considered statistically significant using χ analysis. the prevalence of nt h. influenzae and h. haemolyticus strains possessing single or duplicate lic loci is not known. similar to the method reported by fox et al, we screened our nt h. influenzae and h. haemolyticus lic1-containing strains for duplicate lic loci using southern hybridization of mfe digested genomic dna to identify two restriction fragments that hybridized with a licd gene probe. strains with two licd-hybridizing bands were present in seven nt h. influenzae strains and in none of the h. haemolyticus strains. further hybridization using a lica gene probe on the seven nt h. influenzae strains also revealed two lica hybridizing bands in these strains, suggesting that they possessed two complete lic loci. assessing the population prevalence of lic locus copy number among the species, the data suggest that 74/ nt h. influenzae and 46/ h. haemolyticus possess one copy of lic and that strains with one lic locus are times more prevalent in nt h. influenzae than in h. haemolyticus. duplicate lic loci were present in 7/ nt h. influenzae and 0/ h. haemolyticus strains, suggesting that duplicate lic loci in h. haemolyticus are rare or altogether absent. prevalence of the three licd alleles in nt h. influenzae and h. haemolyticus determining the prevalence of the three previously described licd alleles among the two species was initiated by pcr amplification and dna sequence analysis of the licd genes from the nt h. influenzae and h. haemolyticus strains in our collection that contained a single lic locus. the deduced licd amino-acid sequences of these strains were determined and the licd gene from one nt h. influenzae strain was repeatedly found to possess a nonsense mutation that would result in gene termination. a minimum-evolution dendrogram was created from the remaining licd amino-acid sequences of the nt h. influenzae and h. haemolyticus strains. the dendrogram revealed three distinct clusters, each containing a different h. influenzae prototype licd allele. these results suggest that the three previously defined licd alleles represent the major allelic variants found among the h. influenzae and h. haemolyticus species. next, we determined the population prevalence of specific licd alleles in our nt h. influenzae and h. haemolyticus strains. among the total nt h. influenzae strains in the collection, possessed a single licdi allele, possessed a single licdiii allele, and possessed a single licdiv allele. in contrast, only of the h. haemolyticus strains possessed a licdi allele while possessed a single licdiii allele and possessed a single licdiv allele. although the prevalence of single licdi alleles was statistically different between nt h. influenzae and h. haemolyticus, the prevalence of the licdiii and licdiv alleles was not statistically different between the species. assessment of licd gene alleles among the seven dual lic locus-containing nt h. influenzae strains was determined by pcr amplifying and sequencing licd from agarose gel slices of strain dna digested with mfe the results revealed that 4/ strains had licdiii-licdiv alleles, while only 1/ strains each were found to possess combinations of licdi-licdiii, licdi-licdiv, and licdi-licdi alleles. together, these results suggest that the licdi allele is rarely present in h. haemolyticus, and that the proportions of licdiii and licdiv alleles are similar between the species. chop phase variation and the number of lica tetranucleotide repeats among nt h. influenzae and h. haemolyticus phase variation of chop expression is similar between nt h. influenzae and h. haemolyticus. the lica genes of h. haemolyticus strains m07- and 60p3h contained a number of 5'-caat-3' repeats that would place the lica gene in a correct translational open reading frame. chop expression in these two strains was corroborated by western immunoblot where tepc- reactive epitopes were present in each strain. in addition, phase-negative variants could be isolated from each h. haemolyticus strain, and dna sequence analysis revealed that each lica repeat region gained one 5'-caat-3' repeat, placing the lica gene out of frame. mutation rates in contingency loci are proportional to the length of the repeat region in the loci and the repeat region length may therefore affect the ability of bacteria to respond to a host immunologic challenge. to determine if a general population difference of lica repeat length exists between the species in this study, we compared the number of lica 5'-caat-3' repeats between the nt h. influenzae and h. haemolyticus strains that contained a single lic locus. dna sequence analysis of pcr amplified repeat regions from these strains revealed a wide range in repeat numbers for both species. the average number of lica repeats between the species, however, was statistically different with nt h. influenzae having a mean of repeats and h. haemolyticus having a mean of repeats. these results suggest that, at the population level, the contingency response for chop expression may be slower for h. haemolyticus than for nt h. influenzae. * p <. using the student's t-test ** p <. for each comparison using the student's t-test h. influenzae strains that express chop at more distal positions in los have been shown to be more vulnerable to crp binding, leading to activation of complement mediated bactericidal killing. therefore, strains may differ in their lica mutation rates depending on which los structure is modified with chop. to test this, we further stratified the number of lica gene repeats between strains with different licd alleles for each species. among nt h. influenzae, the range of repeats was similar among strains that possessed a licdi, licdiii, or licdiv allele. the average number of repeats was significantly different, however, for strains that possessed a licdiii allele than for strains that possessed a licdi or licdiv allele. among h. haemolyticus, the range of lica repeats was more variable between strains with licdiii and licdiv alleles, due mainly to three licdiii-containing strains with lica genes that contained and repeats. in contrast to nt h. influenzae, however, the average number of repeats was not significantly different between h. haemolyticus strains possessing licdiii or licdiv alleles. these results suggest that nt h. influenzae strains that substitute chop on more proximal, exposed oligosaccharides chains may tend to have increased mutation rates within the repeat region of the lica gene. discussion the strain population structure of nt h. influenzae is genetically very diverse and clones or clusters of nt h. influenzae strains that differentiate virulent from commensal strains have not been identified. given this diversity, together with the high prevalence of nt h. influenzae colonization in the healthy human population, it is reasonable to hypothesize that not all nt h. influenzae strains possess the same ability to cause disease, but rather, that a proportion of strains possess a range of variable genetic traits that allow for infection and disease under the right host conditions. thus, comparison of genetic trait prevalence between populations of nt h. influenzae and the closely related but strictly commensal species, h. haemolyticus, will highlight traits within the species' gene pools that may offer clues to the virulence pathways of nt h. influenzae. for instance, chop expression in nt h. influenzae is strongly implicated as a virulence factor and is thought to enhance virulence though increased epithelial cell adherence, inhibition of bactericidal peptides, and modulation of the immune system during biofilm growth. in this study, 58% of h. haemolyticus strains lacked a lic locus while only 8% of nt h. influenzae strains lacked a lic locus, suggesting that, at a population level, chop expression may provide an advantage for more nt h. influenzae strains to cause disease. furthermore, the trend of shorter lica gene repeat regions in h. haemolyticus strains that possess a lic locus, may suggest that those strains have a slower phase-variable response to host defences targeting chop, potentially limiting their survival in inflammatory environments. obviously, prevalence differences in chop expression alone do not account for all differences in disease potential between the species since many other virulence factors have been described for nt h. influenzae. rather, the differential prevalence of genetic traits between the species highlight factors that may be further studied for their roles in virulence using in vitro and in vivo models of nt h. influenzae infection. although the structure of h. haemolyticus los is unknown, the assumption has been made that basic los structures and biosynthesis of chop modifications, mediated by the phosphocholine transferase, licd, are comparable between nt h. influenzae and h. haemolyticus. some evidence suggests that these assumptions are reasonable. in the tricine sds-page experiments of this study, h. haemolyticus los migrated at a rate similar to the los of nt h. influenzae, and h. haemolyticus los also presented intra and inter-strain structural heterogeneity similar to the los of nt h. influenzae. recent structural analysis on the los of haemophilus parainfluenzae, a member of the pasteurellaceae family that is phylogenetically more distant to nt h. influenzae than h. haemolyticus, revealed that the inner core structure was nearly identical to that of nt h. influenzae. furthermore, the licdiii and licdiv alleles of the two h. haemolyticus strains in this study demonstrated higher sequence identity to their cognate proteins in nt h. influenzae than similar comparisons of lica, licb, and licc proteins, suggesting a functional equivalence of the licd protein alleles. although these observations are circumstantial, they argue for more detailed comparisons of los structures between nt h. influenzae and h. haemolyticus to identify dissimilarities between the structures that may be associated with the ability of nt h. influenzae to cause disease. the results of this study suggest that genotypes facilitating los-chop structures that are not conducive to crp binding predominate among the strain populations of both species; the majority of h. haemolyticus strains lacked a lic locus and the majority of nt h. influenzae strains either lacked a lic locus or possessed a single licdi allele. in comparison, strains possessing single licdiii and licdiv alleles were in smaller, but similar fractions in the strain populations of both species, indicating that these allelic distributions are still maintained in the species despite possible increased vulnerability to crp binding. further studies that assess the prevalence of licd alleles between epidemiologically comparable collections of virulent and commensal nt h. influenzae strains may highlight which alleles are important in nt h. influenzae disease. one chop genotype that may be associated with nt h. influenzae disease isolates is the possession of two lic loci in the same strain where each locus contains a different licd allele, providing the bacteria with two independently phase-variable chop substitutions. fox et al demonstrated that 4/ nt h. influenzae middle ear strains had dual lic loci. in the current study, only nt h. influenzae and not h. haemolyticus possessed dual lic loci. although only of total nt h. influenzae strains had dual loci, six were present among middle ear strains present in this collection. fox et al. also noted that the genome sequenced nt h. influenzae strain, r possessed a complete and partial lic loci, each containing a different licd allele, raising the possibility that other strains may have a similar genotype. an extensive search on the lic1-containing strains in this collection using licd-specific pcr and hybridization, however, did not identify any strains that contained more than one licd allele, suggesting that the nt h. influenzae population contains mainly complete copies of lic. although nt h. influenzae los structural studies have identified chop modifications on oligosaccharides extending from the heptose ii position, specific licd alleles mediating this arrangement have not been characterized. it is possible that one or more of the current licd alleles may overlap in this process or that stochastic factors in los biosynthesis may play a role. in addition, the clustering analysis of licd protein alleles present in figure suggests that sub-variants may exist within the major allelic groups, and it is possible that one of these variants may facilitate heptose ii-associated chop substitutions. as reviewed by moxon et al, strains that are genetically and epidemiologically unrelated vary widely in the lengths of ssr, while individual strains that transmit within an outbreak or are extensively subcultured over time maintain a central modality in repeat numbers. using a larger number of samples from a phylogenetically defined collection of nt h. influenzae strains has allowed us to partially resolve distribution trends for the lica repeat region in the nt h. influenzae and h. haemolyticus populations and make statistical comparisons between and within species. we found statistically significant trends toward the increased length of lica tetranucleotide repeats in nt h. influenzae compared to h. haemolyticus, and in nt h. influenzae strains with licdiii alleles compared to nt h. influenzae strains with licdi or licdiv alleles. longer repeat regions are predicted to increase lic loci mutation rates and chop phase variation, providing increased resistance to host clearance mechanisms such as crp or antibodies that bind chop and initiate complement mediated bactericidal killing. the presence of the longest repeat in a h. haemolyticus strain and only five repeats in a licdiii-containing nt h. influenzae strain, however, are reminders that these trends must be considered in the light of numerous other factors that contribute to the commensal life style of both species and disease potential of nt h. influenzae.  in summary, the lic locus is not part of the conserved "core" genome of the h. influenzae population but is part of the flexible gene pool that exists among different strains. nonetheless, the conserved chemical nature of chop and the discovery of anti-chop antibodies in human serum provides reasonable credence to chop as a vaccine candidate that may inhibit h. influenzae at some point in the infectious process. knowledge of how chop expression varies both genetically and structurally within the nt h. influenzae strain population is critical for designing intervention strategies that will effectively target disease-related strains. furthermore, contrasting the genetic properties of nt h. influenzae chop expression with those of h. haemolyticus, a closely related but non-pathogenic species, has highlighted a number of chop expression differences that may provide an advantage to disease-related growth in nt h. influenzae. 
in addition, nt h. influenzae strains that possessed a licdiii allele had increased numbers of repeats compared to nt h. influenzae with other licd alleles. crp is known to bind more to chop positioned distally than proximally in los, and the position of chop within los is dictated by specific licd alleles that are present in a lic locus. using dot-blot hybridization, 92% of nt h. influenzae and % of h. haemolyticus strains possessed a lic locus. eight percent of nt h. influenzae and none of the h. haemolyticus strains possessed dual copies of lic the licdiii and licdiv gene alleles were distributed similarly among the nt h. influenzae and h. haemolyticus strains while licdi alleles were present in % of nt h. influenzae but in less than 1% of h. haemolyticus strains. these data demonstrate that genetic similarities and differences of chop expression exist between nt h. influenzae and h. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of nt h. influenzae. although non-typeable haemophilus influenzae and haemophilus haemolyticus are closely related human commensals, h. haemolyticus is non-pathogenic while nt h. influenzae is an important cause of respiratory tract infections. phase-variable phosphorylcholine modification of lipooligosaccharide is a nt h. influenzae virulence factor that, paradoxically, may also promote complement activation by binding c-reactive protein. nt h. influenzae had an average of tetranucleotide repeats in lica compared to repeats in h. haemolyticus. the lic locus contains the lica-licd genes, and chop-host interactions may also be influenced by a second lic locus that allows for dual chop substitutions in the same strain, or by the number of lica gene tetranucleotide repeats that reflect phase-variation mutation rates.
 although non-typeable haemophilus influenzae and haemophilus haemolyticus are closely related human commensals, h. haemolyticus is non-pathogenic while nt h. influenzae is an important cause of respiratory tract infections. phase-variable phosphorylcholine modification of lipooligosaccharide is a nt h. influenzae virulence factor that, paradoxically, may also promote complement activation by binding c-reactive protein. crp is known to bind more to chop positioned distally than proximally in los, and the position of chop within los is dictated by specific licd alleles that are present in a lic locus. the lic locus contains the lica-licd genes, and chop-host interactions may also be influenced by a second lic locus that allows for dual chop substitutions in the same strain, or by the number of lica gene tetranucleotide repeats that reflect phase-variation mutation rates.  using dot-blot hybridization, 92% of nt h. influenzae and % of h. haemolyticus strains possessed a lic locus. eight percent of nt h. influenzae and none of the h. haemolyticus strains possessed dual copies of lic the licdiii and licdiv gene alleles were distributed similarly among the nt h. influenzae and h. haemolyticus strains while licdi alleles were present in % of nt h. influenzae but in less than 1% of h. haemolyticus strains. nt h. influenzae had an average of tetranucleotide repeats in lica compared to repeats in h. haemolyticus. in addition, nt h. influenzae strains that possessed a licdiii allele had increased numbers of repeats compared to nt h. influenzae with other licd alleles.  these data demonstrate that genetic similarities and differences of chop expression exist between nt h. influenzae and h. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of nt h. influenzae. 