Periodontal diseases are a group of prevalent inflammatory diseases affecting the tissues supporting and surrounding the teeth. In periodontal diseases, the inflammatory response of the periodontal tissues is induced by a polymicrobial biofilm formed at or below the gingival margin. Aggregatibacter actinomycetemcomitans, a Gram-negative oral pathogen identified within this biofilm, is a causative agent of both chronic and localized aggressive periodontitis. Dissemination of A. actinomycetemcomitans from the oral cavity also initiates multiple systemic infections, including soft tissues abscesses, pneumonia and endocarditis. The innate immune response is the first line of defense against bacterial infections. Dendritic cells, a group of professional antigen-presenting cells of the innate immune system, express multiple surface C-type lectin receptors for the recognition of glycoproteins associated with bacterial cells. Internalization of the organism by receptor-mediated endocytosis results in phagolysosome formation and degradation of the bacteria for antigen presentation. Surface displayed antigens presented in an MHC complex to antigen-specific T lymphocytes initiate an adaptive immune response. A. actinomycetemcomitans expresses surface structures Extracellular matrix protein adhesin A (EmaA), which extends from the cell envelope, and is composed of three identical glycoproteins. The role of these structures in the interaction of A. actinomycetemcomitans with dendritic cells is understudied. A series of experiments presented here investigated the interaction of A. actinomycetemcomitans with dendritic cells and the role of EmaA in this process. A. actinomycetemcomitans was internalized and found to survive within dendritic cells. The internalization of bacteria was observed to be associated with the presence of EmaA. Fewer emaA mutant bacteria were recovered from dendritic cells when compared with the parent strain. In trans complementation of the emaA mutant strain restored the interactive and survival capability of the bacteria. These data suggest that EmaA mediates the interaction of A. actinomycetemcomitans with dendritic cells. The migration of dendritic cells to draining lymph nodes suggests that the EmaA-mediated internalization and intracellular survival is a potential mechanism for the immune evasion and in vivo dissemination of A. actinomycetemcomitans.

Clostridium difficile is a Gram-positive spore-forming strict anaerobe that can cause severe colitis in humans. C. difficile is best known as the leading cause of nosocomial-acquired diarrhea, particularly in people undergoing antibiotic therapies, since it is naturally resistant to most antibiotics. A clinical feature that makes C. difficile infection, or CDI, particularly difficult to treat is the organism's inherent ability to resist antibiotic therapies while in its spore form. Since oxygen is toxic to C. difficile, spores are the major transmissive form; they are also resilient to most disinfectants, which makes them extremely difficult to eliminate to prevent additional infections. While over fifty years of studies on the spore-forming model organism Bacillus subtilis laid the foundation of how sporulation and germination occurs, little was known about how C. difficile regulates spore formation and/or what proteins are necessary for sporulation and germination processes. The work presented in this dissertation addresses how C. difficile regulates sporulation, identifies genes that are regulated during sporulation, and characterizes some key proteins that are required for either sporulation or germination. During the developmental process of sporulation, a cell divides into two asymmetrical compartments. In each compartment, specific transcriptional programs controlled by sporulation-specific sigma factors, drive the cell through a series of morphological events, culminating in the formation of a spore. Using genetic and cell biological techniques, we show that mutations in the genes encoding the master transcriptional regulator Spo0A and the sporulation-specific sigma factors σF, σE, σG, and σK block sporulation at various stages. Analysis of the mutants and wild type C. difficile strain using RNA-Sequencing identified genes regulated by a given sigma factor and revealed that the sigma factors control sporulation in a manner that differs from B. subtilis. Whereas the sporulation-specific sigma factor activity is regulated in a sequential manner involving cross talk between the different compartments in B. subtilis, C. difficile regulates these factors in a bifurcated manner, with less cross-compartment regulation. Guided by our RNA-Sequencing results, we constructed targeted gene mutations in spoIIQ and spoIIIA-H, which are important for forming a channel known as the 'feeding tube' in B. subtilis. We demonstrated that these proteins are necessary for maintaining forespore integrity, tethering the coat to the forespore, and engulfment. Using metabolic labeling, we show that while spoIIQ and spoIIIA mutants cannot finish the phagocytic-like process of engulfment, they are capable of transforming peptidoglycan, which is a necessary step for engulfment to occur. We also constructed a targeted gene mutation in a gene that is highly transcribed during sporulation, now known as gerS. We show that a gerS mutant cannot degrade cortex during germination and is required for SleC-mediated cortex hydrolysis, making GerS a novel regulator of C. difficile spore germination. Altogether, this research provides a framework for understanding how the pathogen C. difficile undergoes sporulation and is therefore capable of infecting humans. Further, our studies reveal important factors that mediate the essential process of engulfment during sporulation and an important factor that mediates cortex hydrolysis during germination. This work has demonstrated that C. difficile regulates sporulation and germination differently than what has previously been described in other Firmicutes.

Deoxyribonucleic acid (DNA) analyses were used to assess on a molecular level, the relationships among representatives of the genera Streptomyces, Nocardia and Mycobacterium. The methods developed in this study have been used for routine analysis of DNA from a large number of actinomycetes and have given reliable and reproducible data. DNA isolated from various actinomycetes was characterized by buoyant density determinations in CsCl from which the mole fraction guanine plus cytosine (GC) content was calculated. All the streptomycete DNA preparations studied had buoyant densities in the range of 1.7287 to 1.7312 g cm-3 which corresponded to GC compositions of 70% to 73% GC respectively. The nocardial DNA preparations tested fell in two groups, one with a GC content in the range of 62 to 64% GC and another in a 68 to 70% GC group. The mycobacterial DNA tested had GC values overlapping those of the nocardial DNA specimens; moreover, mycobacterial DNA exhibited a bimodal clustering of GC values, 64 to 65% GC and 67 to 70% GC. All DNA preparations examined by equilibrium buoyant density centrifugation in CsCl contained a single component with no satellite bands. The method of Warnaar and Cohen for assay of DNA/DNA reassociation on membrane filters was modified for studying reassociation of DNA in high GC organisms, DNA isolated from selected actinomycetes was tested for homology with Streptomyces venezuelae S13 mycelial DNA by direct reassociation experiments . Unlabeled DNA from the various actinomycetes was immobilized on Schleicher and Scheull nitrocellulose B-6 membrane filters and then incubated for 15 to 20 hr at 70 C with 14c-labeled DNA. The measure of relatedness was the relative percentage of renaturation of a denatured test DHA with labeled, dentured homolocous DNA. Unrelated DNA having GC contents of 50 and 70% were included as controls. The streptomycetes studied were relatively homogeneous in that measurable interspecific duplexes were formed between the reference DNA and all streptomycete DNA examined. Significantly, the results also suggested that S. venezuelae S13 was related to the nocardial specimens examined but was not related to the mycobacterial cultures studied. The results agreed generally with prior agar-gel studies on DNA reassociation and with previous classifications. Nucleotide sequence divergence in DNA extracted from streptomycetes and nocardiae was determined by measuring the extent of renaturation at 60 C and 70 C. The use of thermal elution of labeled, renatured duplexes from filters substantiated the existence of a class of nucleotide sesquences which can reassociate at 60 C but cannot reassociate at the more exacting 70 C incubation temperature. The use of exacting incubation conditions (70 C) permitted the formation only of t hose DNA duplexes that exhibited a high degree of thermal stability and hence, closely related to the reference DNA. The non-exacting 60 C incubation allowed those sequences to associate which were distantly related. The ratio of binding at 70 C to the binding at 60 C was designated the Divergence Index (DI). The DI was useful for gauging the presence or absence of closely related genetic material and for determining divergence patterns. The conclusions obtained from this method were corroborated by the much more time consuming thermal elution method. The divergence studies suggested that the streptomycetes contain a wide spectrum of related sequences compared to the reference DNA. Interestingly, the nocardiae examined seemed to have a small but significant amount of conserved nucleotide sequence compared to the S. venezuelae Sl3 reference. During these studies on actinomycete DNA it was realized that DNA from S. venezuelae S13 spores had novel properties. As spores aged the buoyant density in CsCl decreased from 1.727 to 1.707 g cm-3, the midpoint of thermal denaturation (Tm) in 0.1 x SSC increased from 85 to 88.5 C, and the apparent reassociation with mycelial DNA decreased from 100 to 30% . Spore DNA in 5 M NaCl04 had the same Tm as mycelial DNA. Spore DNA (1.707 g cm-3 ) after heat denaturation showed a single band in CsCl (1.722 g cm-3). Spore DNA was resistant to pancreatic deoxyribonuclease I, but became progressively sensitive after treatment with 0.5 M sodium acetate. Chemical nucleotide analysis of spore and mycelial DNA showed no detectable difference in GC content. The aberrant nature of spore DNA was not affected by pronase or ribonuclease. Washing the spores with ethanol and acetone prior to DNA extraction restored the isolated DNA to normal buoyant density and Tm values. When alcohol and chloroform extracts of spores were dried and mixed with authentic DNA preparations, no change in Tm or buoyant density was found. Spore DNA was yellow in color at pH values below 12 and pink in color at values above 12. Spore DNA heated in high salt showed three characteristic peaks in Sephadex G-100 column chromatography, but only one peak was found with comparably treated mycelial DNA . Attempts to characterize these peaks have been inconclusive to date. Chemical analyses of spore DNA showed 15 to 20% Folin positive material, 40 to 50% more phosphorus than mycelial DNA and no detectable sugars. It appeared that whatever was bound to the spore DNA could be partitioned to added DNA. Experiments of this type were successful only if the test DNA was added to freshly disrupted, dehydrated spores with crushed dry ice. Rehydration of spores showed loss of this binding activity. No chromatographically identifiable compounds or characteristic colors were extracted from spore DNA by a number of solvents and conditions including: acetone, ethanol, ethanol-ether, ethyl acetate, butanol, 8 M urea, 5% cold trichloroacetic acid and 1 N KOH at 100 C for 30 min. A crude pigmented fraction was isolated from spores which had similar chemical characteristics as aberrant spore DNA. This pigment could not be demonstrated in mycelia; moreover, pigment production seemed to be directly correlated with the age of the spores. The data suggested that this pigment is probably bound to spore DNA and is responsible for the aberrant characteristics of S. venezuelae S13 spore DNA.

The molecular nature and replicative behavior of R factor 222 was examined in Proteus mirabilis . In deoxyribonucleic acid (DNA} from R+ P. mirabilis , R factor 222 was identified by CsCl density gradient centrifugation as 2 satellite DNA bands at densities corresponding to 50 and 58 moles percent guanine plus cytosine (% GC) . Replication of the 50 and 58% GC components of R factor 222 in P. mirabilis was analyzed during growth in the presence and absence of chloramphenicol (CAM} and after shifting exponentialand stationary- phase cells to conditions which inhibit host protein or DNA synthesis . CAM reduced the cellular growth rate but increased the amount of both R factor components relative to host chromosomal DNA . However, the 58% GC component showed a larger proportionate increase. This was inferred to indicate reduced synthesis of an inhibitor that acts on both R factor components and an initiator required for replication of the 50% GC component . Replicative patterns observed after shifting exponential- and stationary-phase cel ls grown with or without CAM to minimal medium or CAM for one generation, or puromycin for 3 hr , corroborated this interpretation. After shifts of exponential- phase cells from either medium, replication of the 50% GC components parallel ed host replication, thus indicating a requirement for protein synthesis . Under these conditions , replication of the 58% GC replicon increased due to reduced inhibitor synthesis . R factor DNA content remained constant after shifting stationary- phase cells from drug-free medium, whereas increased replication of the 58% GC component occurred after identical shifts of CAM- grown cells of the same chronological age. This indicated that effective concentrations of the regulatory inhibitor were attained in the stationary-phase cells grown in drug-free medium . Similar responses were observed after shifts to 5 C or to medium containing streptomycin or tetracycline . Absence of replication of the 50% GC component after shifting to medium containing nalidixic acid or phenethyl alcohol and the hereditary persistence of this replicon during growth indicated that the 50% GC replicon was attached to the membrane . Thus , in P . mirabilis the three replicons of R factor 222 are regulated as follows : the composite R factor and transfer portion (RTF) replicons , represented by the 50% GC component, require protein synthesis and membrane attachment for replication and are negatively regulated by an inhibitor ; the 58% GC or resistance determinant s replicon exists cytoplasmically and is subject only to negative replicative control . The unusually low hyper chromic shift and the abnormal buoyant density shifts in CsCl observed after thermal denaturation of R f actor DNA indicated an abnormal chemical composition . DNA from P. mirabilis harboring R factor 222 was examined chromatographically after enzymatic and chemical hydrolyses . Preliminary results indicated the presence of an unusual chemical component in R factor DNA which reacts positively to carbohydrate development and possesses chromatographic and spectrophotometric properties similar to 5-hydroxymethyl cytosine.

Many lines of evidence exist associating herpes simplex virus (HSV) with the development of carcinoma. Data from human studies includes seroepidemiologic studies of carcinoma patients and the localization of viral markers in human cancers. Experimental studies include in vitro transformation of cultured cells and viral induced alterations of host DNA. Much of this evidence is anecdotal or associative in nature and does not prove a cause and effect. The purpose of this research was to investigate the oncogenic potential of HSV type 2 (HSV-2) in vivo and in vitro. An in vivo mouse model for lib carcinogenesis was designed to combine HSV-2 infection, ultraviolet (UV) exposure and tetradecanyl-phorbolacetate (TPA) application. Preliminary studies showed that HSV-2 inoculation onto abraded mouse lips was capable of causing vesicular ulcerative lesions. These lesions healed completely after 10-14 days. Repeated herpetic lip infections failed to induce tumors. UV-irradiation delivered to the lesion site daily for 6 minutes at 42 ergs/mm2/s on days 3 through 6 postinfection caused hyperkeratosis, acanthosis and dysplasia to develop in several lips; while the same UV exposure delivered by itself failed to alter the histologic appearance. The addition of related TPA application to the HSV + UV regimen promoted tumor emergence. Thirty-two of 156 BALB/c mice developed tumors. Although the majority were papillomas, six were squamous cell carcinomas. These tumor bearing mice had increased HSV specific antibody titers. HSV antigens were shown to be present in outgrowths from explanted tumors as well as in tumor biopsies by immunoperoxidase staining with HSV specific antiseras. Another series of in vivo studies showed that HSV lip infection initiated prior to or during repeated chemical carcinogen (dimethylbenzanthracene/DMBA) application was capable of reducing the incidence of tumors as compared to DMBA application without superimposed HSV infection. Comparison of results from this system and those from the HSV + UV + TPA system emphasized the fact that the lytic capability of HSV must be inhibited for the virus to express its oncogenic potential. It was hypothesized that the in vivo UV-irradiated HSV acted as the inducer and TPA as the promoter, analogous to the classical two-state carcinogenesis model. Since neither the HSV infection by itself, the UV exposure by itself, the TPA application by itself nor any combination of two induced the development of squamous cell carcinomas, HSV was considered a carcinogen with UV-radiation. An analogous tripartite system was devised in vitro. Hamster embryo cells were infected with HSV-2 irradiated for 6, 7, or 8 minutes at 42 ergs/mm2/s. Twenty-one days postinoculation transformed foci had developed with frequencies (FFU/PFU) of 2.9 x 10 -7, and 3.9 x 10 -7, respectively. This represents 0.9, 3.0, and 1.3 FFU/10 6 cells. The 7 minute transformation frequencies were further increased to 1.7 x 10 -6 by the addition of TPA to the growth media (0.1 ug/ml) 48 hours postinoculation. This represents 5.1 FFU/10 6 cells. Thus, TPA was capable of tumor promotion in vivo and increasing transformation efficiency in vitro. Furthermore, TPA was shown by two dimensional gel electrophoresis to increase protein synthesis in transformed cells and by cell sorter analysis to increase cellular replicative activity. A transformed cell line, 3-26-7#1, derived from a UV-inactivated HSV transformation experiment, was shown to possess HSV antigens by immunoperoxidase staining with specific antisera and to be oncogenic when injected into newborn hamsters. Sera from the tumor bearing animals had HSV neutralizing antibodies and when used as the primary antisera in the immunoperoxidase stain could detect antigens in HSV infected cells and outgrowths from explanted hamster tumors. Another transformed cell line, 333-8-9, was shown by in situ hybridization to have HSV specific mRNA in its cytoplasm. These data reveal UV-irradiated HSV capable of tumor induction in vivo and of transforming hamster cell in vitro. In these systems, HSV can be considered a cocarcinogen.

Complement is one of the major innate nonspecific mechanisms for host defense in serum. More than a dozen proteins have been isolated from serum which have a function in either the activation or regulation of complement action. Complement is similar to the coagulation sequence in that it comprises a number of precursor proenzymes in plasma that are sequentially cleaved to generate biologically and enzymatically active proteins.

Trimethoprim resistance(Tp r) is encoded by conjugative plasmids in clinically significant staphylococcal isolates. Two genetically and physically similar plasmids from S. aureus, pG01 and pGOS, have Tp r genes that map in different locations on these plasmids. In order to study the relatedness of the Tp r genes and their products to other known Tp r genes, a 1.2 kb fragment of pG01 and a 4.2 kb fragment of pGOS were cloned in E. coli and used as probes for in situ filter hybridization experiments. A 500 base pair subclone of the original 1.2 kb fragment containing only the staphylococcal Tp r structural gene, showed no homology with genes from E. coli encoding a dihydrofolate reductase(DHFR) with an altered Tp r binding affinity or the B. subtilis gene for DHFR. Positive hybridization signals were seen with restriction fragments from pG01, pGOS, and plasmid DNA from five other Tp r staphylococci. A 700 bp portion of the original fragment showed homology with several different restriction fragments of EcoRI-digested pGOl and pGOS, suggesting the presence of repeated sequences on both plasmids. These sequences corresponded to areas of the plasmids known to be involoved in deletions which occur during viral transductions. Lysates of bacteria containing the cloned and native Tp r genes were assayed spectrophotometrically for DHFR activity and compared with activity of E. coli containing genes for DHFR type I and type II. In addition, the Tp Ic 50 (the concentration of Tp required to reduce DHFR activity by 50%) was determined. Tp r staphylococci containing the plasmid-encoded Tp r gene had twenty times higher specific activity than Tp sensitive staphylococci. E. coli containing the cloned staphylococcal gene had DHFR activity equal to that of staphylococcal strains from which the clones were derived and 300 times higher activity than Tp sensitive E. coli. Determination of the Tp Ic 50 showed the staphylococcal protein to be 7000 times more resistant to Tp than the normal cellular DHFR, but four times less resistant to Tp than the DHFR type I and 450 times less resistant than DHFR type II. The staphylococcal Tp r gene product is a protein with DHFR activity that is resistant to Tp inhibition. The gene is expressed in E. coli, but is dissimilar to several previously characterized E. coli Tp r genes.

Macrophage phenotype/function can be modulated by various T-cell lymphokines (LK). The alteration of macrophage phenotype is a result of LK concentration, duration of exposure, and the level of macrophage activation when obtained from in vivo sources through elicitation by either sterile irritants or immune cellular mechanisms. In order to dissect macrophage activation into discrete signals T cell hybridomas were constructed by fusing HAT sensitive BW5147 cells with nylon-wool purified, con A stimulated T cells. The resulting T cell hybrids were screened for their ability to: (a) protect macrophages from the cytopathic effect of Naegleria; (b) induce class II MHC gene product (Ia antigen) expression; (c) increase cytostasis and tumoricidal activity; and (d) alter ectoenzyme profiles on either resident or thioglycollate (TG) elicited macrophages. Two hybridomas (T-3 and T-9) were selected for further evaluation because of their activity patterns. Supernatants from T-3 and T-9 were compared with cloned Y-interferon (γ-IFN) for alteration of biological activities. Both T-3 and T-9 were able to protect resident macrophage cells from Naegleria but had no protective effect on TG-macrophages. T-9 supernatant had patterns of activity similar to γ-IFN while T-3 patterns were different. The addition of anti-Y-IFN removed T-9 cytostatic activity while not affecting T-3 induced activity. The LK inducing protection from the cytopathic effect of Naegleria lysate is not γ-IFN but another molecular moiety. It was also shown that γ-IFN does not protect TG-macrophages from the destructive effects of adenylate cyclase produced by Bordetella pertussis. We conclude that activation of macrophages for the destruction of tumor cells and activation for protection against amoeba and bacteria occur via different biological pathways. Furthermore, we have proposed an association between the cell cycle and the responsiveness of resident and TG-elicited macrophages to specific LK.

Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0°C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared to 37°C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with Bis (sulfosuccinimidyl) suberate (BS3) covalently crosslinked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC using the 1gG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. Intact RMC were pretreated with serine esterase inhibitors prior to 125I-C3a and BS3 exposure. The cells to which 125I-C3a had been crosslinked to were solublized and analyzed by SDS PAGE and autoradiography. There were three bands visualized, a 35,000 dalton band which was defined as chymase, and two undefined 45,000 and 55,000 dalton bands. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of 125I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC.

<p> The amino acid sequence of a protein determines its three-dimensional structure, which in turn determines its functional properties. An intensively studied but still partially unresolved question is how the structure of a protein relates to its functional properties. Here we use photoactive yellow protein (PYP) as a model system to examine questions on protein structure-function relationships. PYP is a bacterial blue light photoreceptor, a prototype of the diverse PAS domain superfamily, and a model system for functional protein dynamics. The work in this thesis was directed at three aims: (1) developing tools to identify the structural change that triggers intramolecular proton transfer during the PYP photocycle; (2) the functional role of PAS-conserved residue Ile39 in PYP; and (3) determining to what extent the extensively studied structure-function relations in the PYP from <i>Halorhodospira halophila </i> apply to the PYP from <i>Rhodospirillum centenum.</i> (1) The molecular events that cause directional proton transfer in proteins are largely unknown. We develop tools to allow the testing of the specific hypothesis that the disruption of the Tyr42-<i>p</i>CA hydrogen bond during the PYP photocycle causes proton transfer. We developed an effective approach for obtaining Tyr-D₄-labeled PYP that can be used in infrared studies to identify Tyr side chain signals. (2) The PAS domain superfamily is defined by weak but characteristic amino acid sequence conservation, but the functional role of PAS-conserved residues remains poorly understood. We examined PAS-conserved residue Ile39 through biophysical characterization of the I39A PYP mutant. This work revealed that Ile39 is at the core of a set of hydrophobic interactions conserved in PAS domains, is not an essential part in the transmission mechanism of allosteric structural changes during PYP signaling and affects both signaling kinetics and folding cooperativity. (3) We found that structure-function rules for Hhal PYP qualitatively transfer to Rcen PYP, including the role of Glu46 as the electrostatic epicenter for driving conformational changes. The resulting set of Rcen PYP mutants with altered photocycle rate and reduced conformational changes provides a powerful tool for future studies on the photocycle events that are needed for in vivo signaling by PYP.</p>