Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

Control of {\it recA\/} expression in {\it Escherichia coli}

The recA gene is essential for SOS response induction, for inducible DNA repair and for homologous recombination in E. coli. The level of recA expression is significant for these functions. A basal level of about 1000 molecules of RecA protein is sufficient for homologous recombination of the cell and is essential for the induction of the SOS response. Based on previous observations, two models regarding the origin of the basal RecA protein were postulated. One was that it comes from the leaky expression of the LexA repressed promoter. The other was that it is from another weak but constitutive promoter. The first part of this thesis is to study these possibilities. An $\Omega$ cartridge containing the transcription terminator of gene 32 of T4 phage was exploited to define a second promoter for recA expression. Insertion of this $\Omega$ cartridge downstream of the known promoter gave rise to only minor expression. Purification and N-terminus sequencing of the RecA protein from the insertion mutant did not support the existence of a second promoter. To determine whether the basal RecA is due to the leaky expression of the known LexA repressed promoter, recA expression of a SOS induction minus strain (basal level expression of recA) was compared with that of a recA promoter down mutation recA1270. The result demonstrated that there is leaky expression from the LexA repressed promoter. All the evidence supports the conclusion that there is only one promoter for both basal and induced expression levels of recA.^ Several translation enhancer sequences which are complementary to different regions of the 16S rRNA were found to exist in recA mRNA. The leader sequence of recA mRNA is highly complementary to a region of the 16S rRNA. Thus it appeared that recA expression could be regulated at post-transcriptional levels. The second part of this thesis is focused on the study of the post-transcriptional control of recA expression. Deletions of the complementary regions were created to examine their effect on recA expression. The results indicated that all of the complementary regions were important for the normal expression of recA and their effects were post-transcriptional. RNA secondary structures of wild type recA mRNA was inspected and a stem-loop structure was revealed. The expression down mutations at codon 10 and 11 were found to stabilize this structure. The conclusions of the second part of this thesis are that there is post-transcriptional control for recA expression and the leader sequence of recA mRNA plays more than one role in the control of recA expression. ^

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^