Allison F. Gillaspy

To examine the role of the accessory gene regulator (agr) in staphylococcal osteomyelitis, we compared a Staphylococcus aureus osteomyelitis isolate (UAMS-1) with a derivative of the same strain (UAMS-4) carrying an inactivated agr locus. Virulence was assessed with a rabbit model of acute, exogenous osteomyelitis. Bacteria were delivered by microinjection into the midradial region of the forelimb. After 4 weeks, UAMS-1 was identified in the bone of 12 of 13 rabbits infected with > or = 2 x 10(6) CFU and 5 of 6 infected with < or = 2 x 10(5) CFU. In contrast, UAMS-4 was found in 6 of 13 infected with the higher dose and 1 of 6 infected with the lower dose. Additionally, on the basis of a five-point scale assessing radiographic evidence of disease, rabbits infected with UAMS-1 had average scores of 2.64 +/- 0.30 (high dose) and 1.43 +/- 0.39 (low dose) while rabbits infected with UAMS-4 had average scores of 0.95 +/- 0.23 (high dose) and 0.63 +/- 0.20 (low dose). Uninfected controls had an average score of 0.53 +/- 0.08. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.047). The results obtained with UAMS-4 were not significantly different from those obtained with the controls at either dose of UAMS-4 (P > or = 0.150). On the basis of a similar five-point scale assessing histopathological evidence of disease, rabbits infected with UAMS-1 had average scores of 2.31 +/- 0.22 (high dose) and 1.96 +/- 0.36 (low dose) while rabbits infected with UAMS-4 had average scores of 1.58 +/- 0.29 (high dose) and 0.83 +/- 0.32 (low dose). Controls had an average score of 0.33 +/- 0.05. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.040). However, the results obtained with UAMS-4 were significantly different from the controls only at the high dose of UAMS-4 (P = 0.025). We conclude that mutation of agr reduces the incidence and severity of disease but does not eliminate the ability to colonize bone and cause histopathological evidence of osteomyelitis.

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 86-028NP's 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.

Manipulation of the fungal epigenome is hypothesized to be an effective method for accessing natural products from silent biosynthetic pathways. A library of epigenetic modifiers was tested using the fungus Aspergillus niger to determine the impact of small-molecule inhibitors on reversing the transcriptional suppression of biosynthetic genes involved in polyketide (PKS), non-ribosomal peptide (NRPS), and hybrid PKS-NRPS (HPN) production. Examination of expressed sequence tag libraries from A. niger demonstrated that >70% of its PKS-, NRPS-, and HPN-encoding gene clusters were transcriptionally suppressed under standard laboratory culture conditions. Using a chemical epigenetic methodology, we showed that treatment of A. niger with suberoylanilide hydroxamic acid and 5-azacytidine led to the transcriptional upregulation of many secondary-metabolite-encoding biosynthetic gene clusters. Chemical epigenetic modifiers exhibited positional biases for upregulating chromosomally distal gene clusters. In addition, a phylogenetic-based preference was noted in the upregulation of reducing clade I PKS gene clusters, while reducing clade IV PKS gene clusters were largely unaffected. Manipulating epigenetic features in fungi is a powerful method for accessing the products of silent biosynthetic pathways. Moreover, this approach can be readily incorporated into modern microbial screening operations.

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum‐sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA‐binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of α‐helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high‐affinity binding sites composed of two half‐sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (K D ) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half‐sites and may involve other sites located downstream of the promoters.

Comparison of Staphylococcus aureus strains carrying mutations inactivating the staphylococcal accessory regulator (sar ) and/or the accessory gene regulator (agr ) suggests that sar is the primary regulatory element controlling transcription of the collagen adhesin gene (cna ) and that the regulatory effect of sar is independent of the interaction between SarA and agr. To test this hypothesis, we cloned the regions encoding each of the overlapping sar transcripts, all of which include the sarA open reading frame (ORF), and introduced each clone into cna-positive sar and agr mutants. The introduction of each clone restored the expected sar transcripts and the temporal pattern of sar transcription. The introduction of each clone also complemented the defect in cna transcription and restored collagen binding to wild-type levels. This was true even when the clones were introduced into a sar/agr double mutant. These results confirm the hypothesis that the sar-mediated regulation of cna transcription occurs via an agr-independent pathway. Direct evidence supporting this hypothesis comes from electrophoretic mobility shift assays demonstrating that SarA exhibits high-affinity binding to cis elements upstream of the cna structural gene. We also examined the correlation between sar transcription and the production of SarA. Western blot analysis of two wild-type strains indicated that SarA was produced in indistinguishable amounts during both the exponential and the post-exponential growth phases.