William C. Ray

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae: Comparative Study with H. influenzae Serotype d, Strain KW20

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NPs genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.

Structure and function of respiratory syncytial virus surface glycoproteins.

The two major glycoproteins on the surface of the respiratory syncytial virus (RSV) virion, the attachment glycoprotein (G) and the fusion glycoprotein (F), control the initial phases of infection. G targets the ciliated cells of the airways, and F causes the virion membrane to fuse with the target cell membrane. The F protein is the major target for antiviral drug development, and both G and F glycoproteins are the antigens targeted by neutralizing antibodies induced by infection. In this chapter, we review the structure and function of the RSV surface glycoproteins, including recent X-ray crystallographic data of the F glycoprotein in its pre- and postfusion conformations, and discuss how this information informs antigen selection and vaccine development.

A time series analysis of microarray data

As the capture and analysis of single-time-point microarray expression data becomes routine, investigators are turning to time-series expression data to investigate complex gene regulation schemes and metabolic pathways. These investigations are facilitated by algorithms that can extract and cluster related behaviors from the full population of time-series behaviors observed. Although traditional clustering techniques have shown to be effective for certain types of expression analysis, they do not take the biological nature of the process into account, and therefore are clearly not optimized for this purpose. Moreover, the current approaches provide internal comparisons for the experiments utilized for clustering, but cross-comparisons between clustered results are qualitative and subjective. We present a combination of current and novel methods for the analysis of time series gene expression data. We focus on an actual study we have performed for Haemophilus influenzae which is a major cause of otitis media in children. We first perform a discretization of the gene expression data that takes both positive and negative correlations into consideration and then develop a clustering algorithm optimized for such data that allows elucidation and searching of time-series patterns. The resulting approach allows time-series data to be usefully compared across multiple experiments. We demonstrate the success of our algorithm by showing some of the genes that it finds to be co-regulated are not detected by current methods. As a result we are able to identify several signal pathways that initiate competence development, and to characterize the transcriptomes of wild-type and an adenylate cyclase mutant (cya) strains under both nutrient-limiting and nutrient-complete growth conditions.

The OxyR Regulon in Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic oxyR mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.

Partial Analysis of the Genomes of Two Nontypeable Haemophilus influenzae Otitis Media Isolates

ABSTRACT In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.

Visibility computation for efficient walkthrough of complex environments

In many virtual reality applications as well as general computer graphics we need to consider large numbers of objects to render one image. In many cases rendering can be preceded by a culling phase that employs simple mechanisms to reject most of the objects. As a result, only a very small portion of the model has to go through the time-consuming process of hidden object removal. We report on such a culling mechanism that is based on regular space subdivision into cells followed by cell classification into interior, exterior, and wall cells. A special cell-to-cell visibility algorithm is then activated between every two nonexterior cells. Only the objects in the potentially visible set of cells are actually submitted to the hidden object removal algorithm. We report on the implementation of the algorithm and its performance for walkthrough of various environments.

Targeting RSV with Vaccines and Small Molecule Drugs

Respiratory syncytial virus (RSV) is the most significant cause of pediatric respiratory infections. Palivizumab (Synagis®), a humanized monoclonal antibody, has been used successfully for a number of years to prevent severe RSV disease in at-risk infants. However, despite intense efforts, there is no approved vaccine or small molecule drug for RSV. As an enveloped virus, RSV must fuse its envelope with the host cell membrane, which is accomplished through the actions of the fusion (F) glycoprotein, with attachment help from the G glycoprotein. Because of their integral role in initiation of infection and their accessibility outside the lipid bilayer, these proteins have been popular targets in the discovery and development of antiviral compounds and vaccines against RSV. This review examines advances in the development of antiviral compounds and vaccine candidates.

Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits.

Uropathogenic Escherichia coli (UPEC) utilizes a complex community-based developmental pathway for growth within superficial epithelial cells of the bladder during cystitis. Extracellular DNA (eDNA) is a common matrix component of organized bacterial communities. Integration host factor (IHF) is a heterodimeric protein that binds to double-stranded DNA and produces a hairpin bend. IHF-dependent DNA architectural changes act both intrabacterially and extrabacterially to regulate gene expression and community stability, respectively. We demonstrate that both IHF subunits are required for efficient colonization of the bladder, but are dispensable for early colonization of the kidney. The community architecture of the intracellular bacterial communities (IBCs) is quantitatively different in the absence of either IhfA or IhfB in the murine model for human urinary tract infection (UTI). Restoration of Type 1 pili by ectopic production does not restore colonization in the absence of IhfA, but partially compensates in the absence of IhfB. Furthermore, we describe a binding site for IHF that is upstream of the operon that encodes for the P-pilus. Taken together, these data suggest that both IHF and its constituent subunits (independent of the heterodimer), are able to participate in multiple aspects of the UPEC pathogenic lifestyle, and may have utility as a target for treatment of bacterial cystitis.

Haemophilus ducreyi Strain ATCC 27722 Contains a Genetic Element with Homology to the Vibrio RS1 Element That Can Replicate as a Plasmid and Confer NAD Independence on Haemophilus influenzae

ABSTRACT The nucleotide sequence of pNAD1, a plasmid from Haemophilus ducreyi identified on the basis of its ability to confer NAD independence on Actinobacillus pleuropneumoniae and H. influenzae, has been determined. In addition to containing the nadV gene, the plasmid contains homologues of the rstR and rstA genes, genes encoding repressor and replication proteins, respectively, in the Vibrio CTXφ and the Vibrio RS1 element, suggesting a single-stranded bacteriophage origin for pNAD1. Tandem copies of the plasmid are integrated into the H. ducreyi 35000HP genome.

MAVL and StickWRLD: visually exploring relationships in nucleic acid sequence alignments

Many powerful tools have been created to detect and describe the similarities between nucleic acid or protein sequences. Frequently these take the form of a sequence consensus, expressing simple most popular positional identities, positional identities with allowances for varying positions or some type of statistical description of the positional frequency characteristics of the defining sequence family. Despite the fact that some provide intuitively interpretable descriptions of the consensuses themselves, they typically do not give the viewer any information about regions of the sequence that might have inter-positional dependencies, and that therefore do not obey a strict consensus behavior. Herein, we present MAVL (Multiple Alignment Variation Linker) and StickWRLD. MAVL is our web-based application for detecting and displaying both positive and negative inter-positional correlations in nucleic acid sequences. MAVL examines all positional pairs in each of a collection of pre-aligned sequences and determines any pairs that occur with either greater or lesser frequency than a positional frequency matrix would predict. These data are then composited into a StickWRLD representation and supplied back to the user as a VRML (virtual reality modeling language) file. MAVL and StickWRLD can be accessed at http://www.microbial-pathogenesis.org/stickwrld/. A tutorial that explains MAVL features and demonstrates typical user interactions with StickWRLD graphs is available at http://www.microbial-pathogenesis.org/stickwrld/tutorial/sticktut2.html. This tutorial is quite large; please be patient while it loads.