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Infection and Immunity, May 2004, p. 2457-2461, Vol. 72, No. 5
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.5.2457-2461.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Haemophilus influenzae: Genetic Variability and Natural Selection To Identify Virulence Factors

Janet R. Gilsdorf,1,2,3* Carl F. Marrs,2,3 and Betsy Foxman2,3

Department of Pediatrics and Communicable Diseases, University of Michigan Medical School,1 Department of Epidemiology,2 Center for Molecular and Clinical Epidemiology of Infectious Diseases, University of Michigan School of Public Health, Ann Arbor, Michigan3


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INTRODUCTION
 
The evolutionary processes of natural selection govern the nucleotide sequences of bacterial genes such that during replication over many generations, bacterial virulence factor genes change and their products become diverse. This diversity, which both facilitates and reflects an organism's ability to survive in a variety of ecologic niches and under different environmental conditions, occurs through two general strategies—variation in gene expression, which is driven by mechanisms governing gene transcription or translation, and variation in gene content, which is driven by either vertical or horizontal evolution. In this regard, vertical evolution refers to the passage of genetic material from parent to offspring through cell division, with its attendant mutations resulting from mistakes in replication such as point mutations, gene inversions, or spontaneous deletions. Horizontal evolution in bacterial cells occurs by the acquisition of new genetic material from transformation of native DNA, transduction by phages, or conjugation by plasmids; this new genetic material is then passed on to subsequent generations through vertical evolution.

Until recently, bacterial pathogens were characterized solely by their phenotypic characteristics, which describe only gross strain-to-strain differences. While extremely useful, these techniques are limited in their ability to identify unique members of widely variable bacterial populations. Recent advances in bacterial genomics, furthered by the availability of complete genomic sequences from a growing number of organisms, suggest that some "clonal" designations may be misguided, given the high level of genetic diversity of bacterial strains from the same species. For example, sequence comparisons have shown up to 25% differences in gene content among strains of Neisseria meningitidis, Helicobacter pylori, and Escherichia coli (5).

While individual fitness characteristics of bacteria foster the survival of individual organisms, the population dynamics of bacteria encompass fitness characteristics that foster the survival of the group. As in all populations, not every member of a bacterial population needs to succeed in all possible environments; rather, the sum of the specialized fitnesses of individual bacteria ensures the survival of the population in variable environments. Thus, gene products required for bacterial survival in one environmental niche may not be required in another niche. Over time and under the influence of natural selection, the gene contents of organisms from the same species living in different niches will be altered to reflect the necessity for certain genes and the dispensability of others. Bacterial factors that are highly diverse are most susceptible to this process of selection. In this paper we describe how the results of evolutionary processes, as reflected by bacterial population characteristics, may be used to identify potential bacterial virulence factors. We use Haemophilus influenzae, whose known virulence factors are highly variable, as an example.


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H. INFLUENZAE AND DIVERSITY
 
H. influenzae, a gram negative coccobacillus whose environmental niche is primarily restricted to the human respiratory tract, is classified on the basis of production of a polysaccharide capsule: strain types a through f produce antigenically distinct capsules, and nontypeable strains produce no capsule. In addition to colonizing the nasopharynges of healthy humans, H. influenzae causes respiratory infections such as acute otitis media, sinusitis, bronchitis, and pneumonia, as well as invasive blood-borne infections such as meningitis, septic arthritis, and cellulitis. Measurements of the gene contents of two H. influenzae strains has revealed that the clinical type b strain Eagan possesses 270 kb of additional genomic material relative to strain Rd, a type d strain that has lost its capsule and is highly laboratory adapted (9). More-detailed analysis has shown that strain Eagan possesses gene regions not found in Rd, including the cap region genes, encoding the type b capsular polysaccharide; the hif gene cluster, encoding the pilus adhesin; the hmw1 and hmw2 gene clusters, encoding the high-molecular-weight adhesions (18); and the tryptophanase gene cluster (25). Although, to date, the entire genome sequence of only one H. influenzae strain (Rd) is available (16), genetic sequence analysis of a nontypeable H. influenzae (NTHi) strain is in progress (http://www.microbial-pathogenesis.org) and, when complete, will add important information on the extent of H. influenzae genomic diversity.

Because phenotypic typing is too limited to optimally describe bacterial diversity, several genotyping systems have been developed for H. influenzae, including enterobacterial repetitive intergenic consensus typing (3, 19, 31, 41), pulsed-field gel electrophoresis (19, 31, 38, 43), and ribotyping (31, 46). Recently, Meats et al. (28) used multilocus sequence typing to genotype H. influenzae; they identified 19 sequence types among 26 nontypeable strains and only 12 sequence types among 50 type b strains. The average number of alleles for the seven enzymes tested was 27, with a range of 22 to 36. In addition, repetitive-elements PCR-based genotyping systems using the H. influenzae uptake signal sequences and intergenic dyad sequences have been developed (7; M. Patel, I. Z. Ecevit, J. R. Gilsdorf, and C. F. Marrs, submitted for publication). These studies all demonstrate significant genetic diversity among strains, with nontypeable strains showing more diversity than typeable strains.

In adapting to its limited ecologic niches in humans, H. influenzae most likely relies on environmental sensing strategies, such as the two-component regulatory systems, less commonly than enteric organisms do (47); the genomic sequence of H. influenzae strain Rd contains 4 sets of sensor-regulator gene pairs, whereas E. coli has 40 (16). Rather, H. influenzae relies on fitness selection and clonal expansion of those population members that express the phenotypic characteristics important for survival in a particular human niche.

The sources of selective pressure that drive phenotypic, and thus genotypic, diversity among various populations of H. influenzae include host immune responses (8), nutritional resources in the various microenvironments of the human respiratory mucosa (45) or bloodstream (20), and host factors necessary for bacterial adherence (34), cellular penetration (34), and host-to-host transmission (4). The following genetic mechanisms that alter either H. influenzae gene expression or gene content generate H. influenzae strains with fitness characteristics optimal for survival under various selective pressures, thus allowing the bacteria to adapt to rapidly changing microenvironments.


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ALTERED GENE EXPRESSION: PHASE VARIATION
 
Phase variation is a strategy frequently used by H. influenzae to alter the gene expression of virulence factors through slipped-strand mispairing, which is mediated by short DNA repeats (typically 2 to 7 nucleotides each) in either the coding regions or the upstream promoter regions of virulence genes. Spontaneous gain or loss of repeat units in these unstable regions either results in a translational frameshift or alters the distance spanned by the promoter, thus turning gene expression off or on. The H. influenzae pilus gene hifA (44) and adhesin genes hmw1A and -2A (13) utilize repeat units of 2 and 7 bp, respectively, in their promoter regions to vary the efficiency of transcription. In addition, five lipooligosaccharide (LOS)-modifying genes (lic1A, lic2A, lic3A, lgtC, and lex2A), four iron acquisition genes with homology to hemoglobin binding proteins, and the yopA homologue demonstrate tetrad repeats of various lengths in their coding regions; the number of repeats correlates with off or on expression of the genes (47). Weiser and Pan (48) have shown that Gal{alpha}1-4Gal, which is modified at the terminal galactose by a putative galactosyltransferase encoded by lic2, may be expressed more commonly by strains infecting patients with pneumonia than by strains from colonized individuals without respiratory tract infections, suggesting that LOS-modifying genes may be host site specific in their expression.


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ALTERED GENE EXPRESSION: UNSTABLE MRNA
 
A recent study of H. influenzae biotype IV strains (a subset of NTHi associated with genital and neonatal infections) demonstrates their failure to express pili in spite of the presence of complete hif operons and promoter regions (6). The authors conclude that a deletion of 10 of the 44- or 45-nucleotide repetitive palindromic extragenic sequences in the genital strains relative to a piliated H. influenzae type b (Hib) strain generated unstable mRNA, resulting in transcription but not translation of hifB through hifE. Thus, H. influenzae appears to have at least two mechanisms by which pilus expression may be modulated, resulting in populations of organisms with variable piliation.


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ALTERED GENE CONTENT: POINT MUTATIONS
 
Sequence analysis of H. influenzae genes that encode several surface proteins reveals multiple point mutations, especially throughout their variable regions. These single-nucleotide polymorphisms in genes encoding pathogenic factors reflect spontaneous point mutations that occur because of replication mistakes during cell division (vertical evolution) or because of acquisition by transformation and homologous recombination of gene regions containing very limited gene differences (horizontal evolution). In the highly variable H. influenzae proteins P5 (15), P1 (30), and HifA (10), nonsynonymous (amino acid-changing) mutations are seen more commonly than synonymous (silent) mutations, suggesting that mutations resulting in altered protein structure offer a selective advantage to the bacteria.


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ALTERED GENE CONTENT: INSERTIONS AND DELETIONS
 
Comparisons of gene sequences from multiple H. influenzae strains reveal many insertions and deletions in genes or gene regions encoding virulence factors (18). The hif gene region encoding H. influenzae pili, for example, shows remarkable strain-to-strain heterogeneity (17, 29, 36), with insertions, deletions, and duplications of genes, open reading frames, and intergenic regions. Among 20 H. influenzae isolates, nine different gene arrangements were found in this region (29).

While gene insertions may result from rearrangements within the bacterial chromosome, in H. influenzae most insertions appear to occur through introduction of new DNA by lateral gene transfer through horizontal evolution. As a result, several H. influenzae genes encoding virulence factors, such as hifA and hifE (which encode the major structural subunits and adhesive molecules, respectively, of pilus adhesins) (10, 27), iga (which encodes immunoglobulin A1 protease) (33), hap (which encodes an H. influenzae adhesive/penetration protein) (21), rfaF (encoding an LOS heptosyltransferase), and lic1D (encoding LOS phosphocholine transferase) (11) exhibit intragenic mosaicism, an apparent mixing and matching of gene regions acquired from other H. influenzae strains that differ in the genetic configurations of these regions. Meats et al. (28) observed that allelic differences among seven multilocus sequence typing loci occurred more commonly from recombination than from point mutation and that mosaic gene structure was found in four of the seven loci.

H. influenzae, like other human mucosal pathogens such as Neisseria gonorrhoeae, N. meningitidis, Streptococcus pneumoniae, Moraxella catarrhalis, and H. pylori, are naturally competent and possess specific genetic mechanisms for taking up nonepisomal DNA from their environment (14). In H. influenzae, horizontal gene transfer through transformation and homologous recombination is facilitated by the presence of the so-called uptake sequences, 5'-AAGTGCGGT-3', of which 1,465 copies are randomly distributed throughout the H. influenzae strain Rd genome; if extended uptake sequences and singly mutated sites are considered, 2,229 uptake sequence sites occupying approximately 3.5% of the genome are seen (40). As evidence of their potential role in facilitating the horizontal transfer of H. influenzae virulence factors, direct repeats of uptake sequences flank the H. influenzae tryptophanase gene cluster (25).

The role of insertion elements (IS) and phage elements in the horizontal transfer of virulence genes is shown by the 711-bp IS1016 direct repeats that flank the cap (polysaccharide) region (22), the 32-bp direct repeats flanking the hif-1 gene cluster present in certain H. influenzae biogroup aegyptius strains (36), the 59-bp direct repeats flanking the hif region (29), and the duplicated inverted repeats flanking the hif region (29, 36). Furthermore, as described for Staphylococcus aureus (39), phage- and transposon-like elements may catalyze deletional events in H. influenzae. Comparison of H. influenzae strains by subtractive hybridization has revealed a relatively high number of phage-like genes that are present in one strain and not in the subtracted strain (32, 42), supporting the notion that many differences in gene content between H. influenzae strains may be associated with phage-related horizontal transfer into, or deletion out of, a strain.


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ALTERED GENE CONTENT: DUPLICATIONS
 
Gene duplications have been shown to result in alterations in H. influenzae pathogenicity. The cap locus, which encodes H. influenzae capsule production by strain types a through f, is present as a tandem duplication. A deletion of the carbohydrate transport gene bexA in one copy of the cap locus in type b strains appears to stabilize the duplication (12, 24). The resulting increased production of the type b capsule contributes to the increased virulence of type b strains. Recombination events resulting in additional copies of the cap locus generate Hib variants with increased pathogenicity proportional to the number of copies present and the amount of capsule produced (12, 23). Because of the presence of a second copy of the hif locus, some H. influenzae biogroup aegyptius strains (including strains isolated from patients with Brazilian purpuric fever) display abundant piliation, reflecting the decreased probability that, at any point in time, both copies of the phase-variable pilus promoters are present in the off orientation (35, 37).


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ALTERED GENE CONTENT: EVIDENCE FOR EVOLUTION-DRIVEN DIFFERENCES IN VIRULENCE FACTORS
 
Through the mechanisms described above, diverse populations of H. influenzae isolated from different human body sites show variable expression of virulence factors due to altered gene content or gene expression. For example, although pathogenic H. influenzae strains colonize the nasopharynx prior to establishing infection at another body site, the population of H. influenzae causing acute otitis media (derived from a subset of the throat population) may exhibit characteristics not required for survival in the throat. Examination of H. influenzae isolates from the middle-ear cavities of children with acute otitis media and from the throats of healthy children demonstrates variable prevalences of genes encoding several virulence factors (15a, 32), suggesting that certain factors may improve H. influenzae fitness in specific ecologic niches, whereas in other niches these factors may be lost through evolution with no deleterious effect on the ability of H. influenzae to survive (Table 1). These results suggest that H. influenzae strains possessing the lic2A, lic2B, and hmw genes have a fitness advantage in the middle ear, whereas H. influenzae strains possessing the hif cluster have a fitness advantage in the throat or nasopharynx. These observations suggest that the lic2A, lic2B, and hmw gene products are virulence factors for H. influenzae acute otitis media and that the hif gene products are virulence factors for respiratory tract colonization. The precise role these genes play in infection or colonization remains unclear.


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  		TABLE 1. Prevalences of virulence genes in H. influenzae isolates from the middle-ear cavities of children with acute otitis media and from the throats or nasopharynges of healthy childrena


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USE OF NATURAL SELECTION TO IDENTIFY H. INFLUENZAE VIRULENCE FACTORS
 
In contrast to genetic diversity resulting from variations in gene expression (in which genes are present but not transcribed or translated under certain environmental conditions), diversity resulting from variations in gene content occurs either because genetic material no longer essential to the survival or pathogenicity of an organism in a specific environment is lost (with no impact on survival) or because genetic material that offers a fitness advantage in a specific environment has been acquired. Various comparative genomics techniques have been used to identify genomic differences among bacteria occupying unique environmental niches. In the case of H. influenzae, several strategies based on gene content, including genomic analysis and mapping by in vitro mutagenesis (GAMBIT) (1, 2), have identified genes essential for survival under various environmental conditions. In addition, differential fluorescence induction has been used to identify H. influenzae genes expressed in vivo in a chinchilla model of acute otitis media (26). Signature-tagged mutagenesis has also been used to identify genes required for H. influenzae survival in an infant rat model of H. influenzae bacteremia (20). Among the 25 H. influenzae genes essential for invasive disease in the rat were 4 previously described H. influenzae virulence factors: hia (adhesin), licA (biosynthesis of LOS), IS1016 flanking the capsule locus, and a gene homologous to PBP-7. The genes identified by these techniques require further study of their specific roles in the pathogenesis of H. influenzae infection and disease.

Two studies have used subtractive hybridization to detect potential virulence factors. This technique identifies genes of one H. influenzae strain (the tester strain, representing a population of bacteria associated with illness or a specific epidemiologic niche) that are not present in another strain (the driver strain, which is not associated with that illness or epidemiologic niche). Smoot et al. (42) subtracted the genome of a noninvasive H. influenzae biogroup aegyptius isolate from that of Brazilian purpuric fever strain F3031. Twenty-one tester-specific clones (present in the BPF strain and absent in the noninvasive H. influenzae strain) were identified, including those possessing iga (encoding immunoglobulin protease), hmcD (encoding hemocin), various phage-related genes, and hypothetical genes of unknown function. From this study no conclusions could be drawn about the relative prevalence of these genes in the two bacterial populations represented by the tester and driver strains, thus limiting the ability to understand their real roles in H. influenzae virulence.

Pettigrew et al. (32) subtracted the genome of the fully sequenced H. influenzae laboratory strain Rd (driver strain) from that of an NTHi strain isolated from a child with acute otitis media (tester strain). Of the 36 tester (otitis media strain)-specific DNA fragments, 17 were phage-related proteins, underscoring the importance of horizontal transfer of genetic material in genetic variability between strains. In addition, clones containing 5 known H. influenzae genes were identified, including a methyltransferase-encoding gene of H. influenzae strain Rd, a gene encoding a LOS biosynthesis enzyme, the LOS biosynthesis gene lic2B, and the H. influenzae adhesin genes hmw1 and hmw2, as well as 14 clones containing genetic material with homology to 13 genes of other microbes. To assess the potential relevance of these genes to the pathogenesis of acute otitis media, the prevalence of these tester-specific genetic regions among H. influenzae otitis media isolates was compared to their prevalence in H. influenzae isolates from throat specimens. lic2B was found in 14% of NTHi throat isolates from healthy children, 39% of NTHi throat isolates from ill children with respiratory tract infections, 52% of NTHi middle-ear isolates from children, and 88% of invasive Hib isolates. Additional studies utilizing traditional methods to define virulence factors will be required in order to understand the precise role of these factors in the pathogenesis of H. influenzae infection and disease.

In summary, the convergence of the investigative techniques used in epidemiological and microbiological studies offers a largely untapped, fruitful field for the identification of bacterial virulence factors by utilizing the power of natural selection that has resulted in diverse bacterial populations.


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ACKNOWLEDGMENTS
 
This paper was supported by National Institutes of Health grants DC 005840 and AI 25630 and by a grant from the Deafness Research Foundation (all to J.R.G.).


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FOOTNOTES
 
* Corresponding author. Mailing address: L2225 Women's Hospital, Box 0244, University of Michigan, Ann Arbor, MI 48109-0244. Phone: (734) 763-2440. Fax: (734) 936-7635. E-mail: gilsdorf{at}umich.edu. Back

Editor: J. B. Kaper


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Infection and Immunity, May 2004, p. 2457-2461, Vol. 72, No. 5
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.5.2457-2461.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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