Alice L. Erwin

Use of a Whole Genome Approach To Identify Vaccine Molecules Affording Protection against Streptococcus pneumoniae Infection

ABSTRACT Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniaeinfection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.

Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis

ABSTRACT Four pneumococcal genes (phtA, phtB, phtD, andphtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed inEscherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae

ABSTRACT The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing.

The gram-negative bacterium Haemophilus influenzae is a human-restricted commensal of the nasopharynx that can also be associated with disease. The majority of H. influenzae respiratory isolates lack the genes for capsule production and are nontypeable (NTHI). Whereas encapsulated strains are known to belong to serotype-specific phylogenetic groups, the structure of the NTHI population has not been previously described. A total of 656 H. influenzae strains, including 322 NTHI strains, have been typed by multilocus sequence typing and found to have 359 sequence types (ST). We performed maximum-parsimony analysis of the 359 sequences and calculated the majority-rule consensus of 4,545 resulting equally most parsimonious trees. Eleven clades were identified, consisting of six or more ST on a branch that was present in 100% of trees. Two additional clades were defined by branches present in 91% and 82% of trees, respectively. Of these 13 clades, 8 consisted predominantly of NTHI strains, three were serotype specific, and 2 contained distinct NTHI-specific and serotype-specific clusters of strains. Sixty percent of NTHI strains have ST within one of the 13 clades, and eBURST analysis identified an additional phylogenetic group that contained 20% of NTHI strains. There was concordant clustering of certain metabolic reactions and putative virulence loci but not of disease source or geographic origin. We conclude that well-defined phylogenetic groups of NTHI strains exist and that these groups differ in genetic content. These observations will provide a framework for further study of the effect of genetic diversity on the interaction of NTHI with the host.

Molecular Validation of LpxC as an Antibacterial Drug Target in Pseudomonas aeruginosa

ABSTRACT LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC inhibitors for P. aeruginosa raised the possibility that LpxC activity might not be essential for all gram-negative bacteria. By placing the lpxC gene of P. aeruginosa under tight control of an arabinose-inducible promoter, we demonstrated the essentiality of LpxC activity for P. aeruginosa. It was found that compound L-161,240, the most potent inhibitor from the previous study, was active against a P. aeruginosa construct in which the endogenous lpxC gene was inactivated and in which LpxC activity was supplied by the lpxC gene from E. coli. Conversely, an E. coli construct in which growth was dependent on the P. aeruginosa lpxC gene was resistant to the compound. The differential activities of L-161,240 against the two bacterial species are thus the result primarily of greater potency toward the E. coli enzyme rather than of differences in the intrinsic resistance of the bacteria toward antibacterial compounds due to permeability or efflux. These data validate P. aeruginosa LpxC as a target for novel antibiotic drugs and should help direct the design of inhibitors against clinically important gram-negative bacteria.

Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon—‘phasevarion’. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5′-CGAAT-3′. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.

Role of lgtC in Resistance of Nontypeable Haemophilus influenzae Strain R2866 to Human Serum

ABSTRACT We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame (“off”) in most colonies of R3392 but in frame with its start codon (“on”) in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galα1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Peptides Selected for Binding to a Virulent Strain of Haemophilus influenzae by Phage Display Are Bactericidal

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is an obligate parasite of the oropharynx of humans, in whom it commonly causes mucosal infections such as otitis media, sinusitis, and bronchitis. We used a subtractive phage display approach to affinity select for peptides binding to the cell surface of a novel invasive NTHi strain R2866 (also called Int1). Over half of the selected phage peptides tested were bactericidal toward R2866 in a dose-dependent manner. Five of the clones encoded the same peptide sequence (KQRTSIRATEGCLPS; clone hi3/17), while the remaining four clones encoded unique peptides. All of the bactericidal phage peptides but one were cationic and had similar physical-chemical properties. Clone hi3/17 possessed a similar level of activity toward a panel of clinical NTHi isolates and H. influenzae type b strains but lacked bactericidal activity toward gram-positive (Enterococcus faecalis, Staphylococcus aureus) and gram-negative (Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica) bacteria. These data indicate that peptides binding to bacterial surface structures isolated by phage display may prove of value in developing new antibiotics.

Heterogeneity in Tandem Octanucleotides within Haemophilus influenzae Lipopolysaccharide Biosynthetic Gene losA Affects Serum Resistance

ABSTRACT Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA. Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies (losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA-on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA, a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.

A fluorescent microplate assay quantifies bacterial efflux and demonstrates two distinct compound binding sites in AcrB

ABSTRACT A direct assay of efflux by Escherichia coli AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type E. coli. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC4-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in E. coli. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC4-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the acrB deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC4-(3) competes with macrolides but not with Nile Red, we propose that DiBAC4-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.