Haemophilus ducreyi is the cause of the sexually transmitted disease chancroid, whereas Haemophilus influenzae is a cause of meningitis, otitis media and other human infections. Although both are classified in the genus Haemophilus, 16S ribosomal sequence data indicates that H. ducreyi and H. influenzae are distantly related members of the Pasteurellaceae family.

We have recently completed the genome sequence of the human passaged derivative of H. ducreyi strain 35000. Although H. ducreyi produces a number of unique putative virulence determinants, we found that a large number of the housekeeping genes were highly conserved between H. ducreyi and H. influenzae.

In order to increase our understanding of the relative chromosomal organization of H. ducreyi and H. influenzae, we performed an orf by orf dot plot analysis of the 2 genomes. A single cluster of ribosomal protein genes and 3 Mu-like phage elements in the H. ducreyi genome had a similar gene order compared to homologues in the H. influenzae genome; but surprisingly, there was little additional conservation of gene order.

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.

We are studying a potential conformational change undergone by the AAV2 capsid (external shell, or packaging) protein, on experiencing a pH shift after endocytosis and acidification of the endosome vescicle. The AAV2 capsid is composed of 60 identical protein subunits arranged in 20 symmetric trimeric groups of 3, aligned to the faces of an icosahedron. The 5-fold axes of symmetry possess an apparent pore, while the 3-fold axes of symmetry are comparatively tightly interwoven.

While not yet completely characterized at the molecular level, AAV2 escape and targetting of the nucleus requires passage through an acidified endosome. We hypothesize therefore that the capsid undergoes a conformational change at low pH, exposing a protected functional domain, and that the conformational change displays hysteresis or anisotropism upon release to normal neutral cytosol pH levels.

Otitis media is a highly prevalent pediatric disease with an estimated 6 billion dollar yearly economic impact in the US alone. Interestingly, this disease is often caused by a bacteria that is a normal component of nasal flora, and that only opportunistically chooses to invade the normally sterile middle-ear space. Clearly, understanding the physical interactions of bacteria and host that result in the normally commensal Haemophilus influenzae bacteria ascending the eustacian tube and colonizing the middle ear, is an important component of understanding the pathogenesis of this disease. This process is commonly modeled using the chinchilla as a model host, classically by infecting the host, and monitoring the process of the disease by sacrificing and dissecting the animal after some period. A large number of animals infected and surveyed in this manner gives a picture of the progress of the infection over time. It is however, costly, wasteful of chinchillas, and inaccurate in that any given chinchilla can only be surveyed once, reducing the resulting conclusions to only aggregate descriptions of behavior.

Researchers have recently developed a method by which bacteria engaged in pathogenic processes can be tagged with a luminescent protein (luciferase) causing them to emit light during active pathogenesis. Sufficient light is emitted, and sufficiently sensitive cameras are available, that the emission can be detected and measured outside the animal's head. This promises to enable the researcher to track the progress of disease in a single animal over multiple timepoints, leading to both a more accurate understanding of specific disease timecourse events, as well as a reduction in the unnecessary use of single-timepoint chinchillas. Impeding this process is a lack of a complete anatomic atlas of chinchilla nasal and ear anatomy. A researcher observing a pattern of light emission thus has no way to definitively assign the emission to a particular anatomic origin. Further complicating the matter, the anatomy is decidely non-homogeneous, and light originating at any point in the nasal cavity will pass through a number of air spaces and tissue layers, before being measured by the camera. We therefore have undertaken to produce a high-resolution volumetric model of the chinchilla head, from which a better understanding of chinchilla anatomy may be derived, and from which we will model an inverse transform of the "fluffy lens" chinchilla head, enabling researchers to more accurately pinpoint the pathogenic bacteria in the host.