Lex2B, a phase-variable glycosyltransferase, adds either a glucose or a galactose to Haemophilus influenzae lipopolysaccharide.

Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second beta-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections.

Pathogenic microbes have evolved highly sophisticated mechanisms for colonizing host tissues and evading or deflecting assault by the immune response. The ability of these microbes to avoid clearance prolongs infection, thereby promoting their long-term survival within individual hosts and, through transmission, between hosts. Many pathogens are capable of extensive antigenic changes in the face of the multiple constitutive and dynamic components of host immune defenses. As a result, highly diverse populations that have widely different virulence properties can arise from a single infecting organism (clone). In this review, we consider the molecular and genetic features of antigenic variation and corresponding host-parasite interactions of different pathogenic bacterial, fungal, and protozoan microorganisms. The host and microbial molecules involved in these interactions often determine the adhesive, invasive, and antigenic properties of the infecting organisms and can dramatically affect the virulence and pathobiology of individual infections. Pathogens capable of such antigenic variation exhibit mechanisms of rapid mutability in confined chromosomal regions containing specialized genes designated contingency genes. The mechanisms of hypermutability of contingency genes are common to a variety of bacterial and eukaryotic pathogens and include promoter alterations, reading-frame shifts, gene conversion events, genomic rearrangements, and point mutations.

Bacterial evolution: bacteria play pass the gene.

DNA transfer between related bacterial species is enhanced by species-specific uptake sequences. These sequences have been used to identify genes that have been transferred from Haemophilus to Neisseria, providing a clear example of interspecific transfer of DNA in the evolution of the pathogenic Neisseria.

Adaptive evolution of highly mutable loci in pathogenic bacteria.

Bacteria have specific loci that are highly mutable. We argue that the coexistence within bacterial genomes of such 'contingency' genes with high mutation rates, and 'housekeeping' genes with low mutation rates, is the result of adaptive evolution, and facilitates the efficient exploration of phenotypic solutions to unpredictable aspects of the host environment while minimizing deleterious effects on fitness.

Haemophilus influence: the impact of whole genome sequencing on microbiology.

The publication of the Haemophilus influenzae genome sequence in 1995 was a landmark in microbiological research. It has changed our understanding of the prokaryotic world, and will influence the approach and focus of research on microorganisms over the next few years. In this article we outline what has been learned from this and other genome sequencing projects, and discuss some of the potential avenues of investigation that will follow in the 'post-genome era'.

Elaboration of type b capsule by Haemophilus influenzae as a determinant of pathogenicity and impaired killing by trimethoprim-sulfamethoxazole.

In vitro, Haemophilus influenzae strains have two distinct patterns of susceptibility to trimethoprim-sulfamethoxazole (TMP/SMZ); strains with low minimum inhibitory concentration and high minimum bactericidal concentration (tolerant) and those with both low minimum inhibitory concentration and minimum bactericidal concentration (kill-sensitive). Tolerant H. influenzae strains were found to elaborate significantly more type b capsular polysaccharide, a linear polymer of ribosyl ribose phosphate (PRP), than kill-sensitive strains. Tolerant strains became susceptible to killing by TMP/SMZ when type b capsule was physically removed, but reacquired tolerance following growth and reversion to original (mucoid) phenotype. Susceptibility of wild (type a, b, c), isogenic (type b and untypable), and transformed (type b and d) strains indicated that elaboration of type b capsule was associated with TMP/SMZ tolerance. In a second series of studies, virulence of H. influenzae in the infant rat model was correlated with in vitro tolerance. Tolerant strains (13/13) caused systemic disease while none (0/7) of kill-sensitive strains were pathogenic. The efficacy of TMP/SMZ in the treatment of invasive infection was evaluated in rats with established bacteremia and meningitis. TMP/SMZ failed to eradicate H. influenzae b from the blood in 85% (17/20) or from the cerebrospinal fluid in 95% (19/20) of infected animals. Thus, in vitro tolerance correlated with therapeutic failure in vivo.

Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence.

Haemophilus influenzae may make any one of six chemically distinct capsular polysaccharides, but only strains of capsular serotype b commonly cause systemic infection (e.g., meningitis) in humans. Molecular cloning of DNA was used to investigate the expression of type b capsule and its association with H. influenzae virulence. A virulent H. influenzae type b strain was used to construct a lambda library of chromosomal DNA in Charon 4. Two independently isolated recombinant phage were isolated from the library and were found to possess DNA necessary for expression of type b capsule. Using a well-characterized rat model of H. influenzae systemic infection, we showed that type b transformants elicited by the cloned DNA were pathogenic, causing bacteremia and meningitis, whereas the untransformed capsule-deficient H. influenzae organisms were not. A 4.4-kb EcoRI fragment, common to both DNA clones, was used to characterize clinical isolates representing all six encapsulated serotypes as well as several capsule-deficient H. influenzae by Southern hybridization analysis. The probe hybridized to an identical sized (4.4 kb) fragment of EcoRI-digested chromosomal DNA from eight independently isolated type b strains. Single bands of homology to the probe were also found in EcoRI fragments of chromosomal DNA obtained from 33 encapsulated, nontype b H. influenzae. However, the size of these EcoRI fragments proved to be characteristic for each of the different capsular serotypes. These studies provide a basis for pursuing the molecular analysis of the epidemiology and virulence of pathogenic H. influenzae.

Role of cerebrospinal fluid pleocytosis and Haemophilus influenzae type b capsule on blood brain barrier permeability during experimental meningitis in the rat.

The influence of leukocytes and Haemophilus influenzae type b (Hib) capsule on blood brain barrier permeability (BBBP) to circulating 125I-albumin in normal and leukopenic rats was assessed after intracisternal inoculation of encapsulated (Rd-/b+/02) or unencapsulated (Rd-/b-/02) isogenic strains of Hib. Both normal and leukopenic animals had increased BBBP 18 h after inoculation, with normal rats demonstrating significantly increased BBBP after challenge with the encapsulated strain. Despite cerebrospinal fluid (CSF) pleocytosis in normal rats, CSF bacterial concentrations were not lower. Normal rats cleared unencapsulated Rd-/b-/02 more effectively than leukopenic rats, with BBBP correlating with CSF bacterial density and not leukocyte concentrations. Challenge with heat-killed Rd-/b+/02 resulted in increased BBBP in both normal and leukopenic rats, with greater BBBP at higher bacterial concentrations. The data suggest: (a) significant increases in BBBP occur in the near absence of CSF leukocytes; (b) CSF leukocytes can augment changes in BBBP; (c) type b capsule inhibits host clearance mechanisms within the CSF; and (d) BBBP appears to correlate with bacterial concentrations within the CSF.

Microbial genome sequencing and pathogenesis.

The year 1997 saw the publication of the complete nucleotide sequence of Helicobacter pylori and Escherichia coli. It is conceivable that the complete nucleotide sequence for all the major human bacterial pathogens will be available by the end of the century. Database alignments have been used to ascribe the putative functions of open reading frames in the sequenced isolates and to define the differences between bacterial species at the nucleotide level. The most striking finding from all genome projects has been the high proportion of open reading frames that have no known function. Experimental data demonstrating the utility of the genome sequencing projects are only just beginning to emerge.

Phenotypic variation of carbohydrate surface antigens and the pathogenesis of Haemophilus influenzae infections.

Phenotypic variation of two major carbohydrate surface antigens of Haemophilus influenzae, the capsule and lipopolysaccharide, exemplifies some of the genetic mechanisms used by pathogenic bacteria in interacting with host microenvironments. The ability to generate phenotypic variety at high frequency within clonal populations of microorganisms provides an adaptive mechanism to combat the polymorphisms and immune repertoires of the host.

Virulence and transmissibility of pathogens: what is the relationship?

The fitness of most pathogenic microorganisms depends on transmission from host to host. This requires adaptation for dissemination, translocation and survival between hosts, as well as for colonization. A complex relationship exists between these components of microbial fitness and virulence. Understanding this relationship has important implications for research and public health.

Mapping bacterial glycolipid complexity using capillary electrophoresis and electrospray mass spectrometry.

This chapter presents the application of capillary electrophoresis coupled to electrospray mass spectrometry (CE-ES-MS) for the analysis of complex bacterial lipopolysaccharides (LPS) from pathogenic strains of Haemophilus influenzae and Neisseria meningitidis. A discussion is included of the development of electrophoretic conditions conducive to trace-level enrichment and separation of closely related glycoforms and isoforms, which provided sensitive detection of glycolipids from as little as five bacterial colonies. The chapter also describes the use of mixed MS scanning functions to aid the identification of specific functionalities and immunodeterminants of LPS, such as pyrophosphoethanolamine, phosphocholine, and N-acetyl neuraminic acid (Neu5Ac), which represent less than 2% of the overall LPS population. The combination of high-resolution capillary electrophoresis with sensitive tandem mass spectrometry (MS/MS) provides a unique analytical tool to probe the subtle structural changes resulting from oligosaccharide branching and location of substituted LPS isoforms. The ability to detect a diverse LPS population over a wide dynamic range of expression using CE-MS enables the correlation of structural changes between bacterial strains and isogenic mutants to assign functional gene relationship.

Implications of sequencing bacterial genomes for pathogenesis and vaccine development.

Improvements in homology search methodology and functional predictions are being complemented by the increase in the volume of sequence data with which comparative analyses can be performed. The experimental methods needed for investigation of gene function and expression in a variety of model systems of infection continue to develop. The identification of surface-exposed microbial structures and their conservation in natural populations of pathogenic species offers prospects for developing novel vaccines. A major challenge is the development of efficient screening methods to select the most promising candidates, such as immunisation with DNA.

Enzyme linked immunosorbent assay (ELISA) for the detection of serum antibodies to the inner core lipopolysaccharide of Neisseria meningitidis group B.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in approximately 70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5-6% and inter-assay CV was 19-22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occurring serum antibodies (specific to inner core LPS) between healthy adults (18-65 years, N=54) and healthy infants (3-4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5. 0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5. 7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26-113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3. 1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5-181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.

Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin.

Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which may be applicable to both epidemiological research and clinical studies.

Are surveillance of resistant enteric bacilli and antimicrobial usage among neonates in a newborn intensive care unit useful?

From March 1976 through December 1978, the prevalence of ampicillin- and gentamicin-resistant enteric bacilli was monitored in fecal cultures of neonates in an intensive care unit. Substantial fluctuations in colonization rates were observed which did not correlate with the occurrence of sepsis due to these organisms nor with variations in antibiotic use. This experience suggests that the availability of these surveillance data did not result in more effective control of neonatal sepsis due to enteric bacilli.

Phase variation of lipopolysaccharide in Haemophilus influenzae.

Lipopolysaccharide (LPS) structures on Haemophilus influenzae, defined by monoclonal antibodies, can show phase variation from generation to generation. Several genetic loci are involved in LPS biosynthesis by H. influenzae. In this paper, we describe three loci which play a role in LPS phase variation: the lic loci; lic1 and lic3 have been sequenced and lic2 has been partially sequenced. Each locus consists of multiple open reading frames (ORF), and each contains a repetitive sequence within the 5' end of the first ORF which may be involved in the phase variability. Genes within lic1 and lic2 are directly involved in the expression of phase-variable epitopes, but the role of genes within lic3 is at a more complex level.

Haemophilus influenzae type b carriage and immunity four years after receiving the Haemophilus influenzae oligosaccharide-CRM197 (HbOC) conjugate vaccine.

Late in 1991, before the implementation of a national immunization program against Haemophilus influenzae type b (Hib) in the United Kingdom, we performed a 4-year follow-up of 120 children who in 1987 had been enrolled in an immunogenicity trial in which 60 of them (vaccinees) received an Hib conjugate vaccine (HbOC) at the same time as diphtheria-tetanus toxoid-pertussis vaccine at the ages of 3, 5 and 9 months. Sixty others (controls) received only diphtheria-tetanus toxoid-pertussis vaccine at the same ages and were not subsequently immunized against Hib. We investigated Hib pharyngeal colonization using the antiserum agar method and the concentrations of serum IgG antibody to the type b capsule by enzyme-linked immunosorbent assay. At 4 years of age the Hib colonization rates in vaccinees and controls were 8% (5 of 60) and 5% (3 of 60), respectively. The children colonized with Hib had greater serum anti-capsular IgG concentrations than did noncolonized children (P < 0.001), and colonized vaccinees tended to have higher concentrations than colonized controls (P = 0.053). Regardless of Hib colonization status vaccinees had greater antibody concentrations than controls (P < 0.001). Forty-nine percent of vaccinees had an antibody concentration > 1 microgram/ml. There was an inverse relationship between the Hib colony count on culture and the serum IgG concentration. These data indicate that the increase in serum antibody concentration after immunization with an Hib conjugate vaccine is sustained and that immunization primes for a booster response on exposure to Hib. There may be a relationship between previous Hib conjugate immunization and the density of Hib colonization in children.