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Many spore forming bacteria are the causative agents of severe disease, such as Bacillus anthracis and anthrax. In these cases, the spore often acts as the infectious agent. Spores boast extreme resistance to chemical and UV damage among other bactericidal conditions. This is problematic due to the difficulty and economic costs of decontaminating exposure sites. The present work focuses on identifying and characterizing proteins active within spore germination, with a focus towards understanding the triggering of the major stages of germination. Understanding how each stage is initiated could allow for development of methods that induce these processes to efficiently germinate spores, thus facilitating cheap and effective decontamination. Sequencing of a spore transposon insertion library after exposure to germinants led to the identification of 42 genes with previously uncharacterized roles in spore germination. Fourteen of the genes, encoding proteins associated with the inner spore membrane, were further characterized. Mutants lacking these genes portrayed phenotypes consistent with failure of a GerA receptor-mediated germination response, and these genes affect the earliest stages of germination. Chemical cross-linking was used to characterize protein interactions important for stage II of spore germination. Site-directed in vivo crosslinking indicated that YpeB may exist as a multimer within the dormant spore. Further investigation of individual protein domains using bacterial two-hybrid analysis suggested that both N- and C-terminal domains of YpeB contribute to the formation of a multimer. In addition, the uncharacterized YpeB N-terminal domain was demonstrated to have strong self-association and may mediate self-association within the dormant spore. Additional genes that contribute to efficient initiation of spore germination in a GerA-dependent manner were identified via TnSeq. Chemical cross-linking of dormant spores was implemented to characterize protein interactions leading to stabilization and activation of an important enzyme that contributes to cortex degradation in stage II of germination. The presented studies employed a variety of techniques to provide additional insight into both stages of spore germination with a goal of furthering understanding of specific events that contribute to a loss of spore dormancy. / Doctor of Philosophy / Few bacterial species can undergo a specialized division process leading to the generation of a bacterial endospore. Endospores are dormant cells that boast resistance to a variety of environmental conditions that would otherwise cause bacterial cell death. These resistance traits make endospores immune to traditional bactericidal methods, making decontamination a nontrivial task. Further complicating the matter, spores are often the infectious particle of the associated disease, including hospital acquired diarrhea, infant botulism, anthrax, and many others. Presented work focuses on furthering understanding the process by which a dormant spore returns to a typical growing bacteria cell. Comprehension of major steps in this process may lead to novel methods for spore cleanup in which mechanisms within the spore are subverted to force a return to a typical bacterial cell state.

Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs), resulting in cells that are easily killed. This work investigated the regulation of the GSLE SleB by other proteins in the spore. A full understanding of how GSLEs are held inactive in the dormant spore and are activated during germination could lead to development of simplified spore decontamination strategies in which spore germination is the first step. It was found that SleB and YpeB are co-dependent. In the absence of one protein, the other is degraded during sporulation by an unidentified protease(s), although HtrC and SpoIVB are not likely responsible. Specific regions and residues of YpeB were also identified as being important to its relationship with SleB. While some evidence suggests that SleB and YpeB physically interact, a direct interaction was not observed in vivo or in vitro. YpeB was demonstrated to be proteolytically processed by HtrC during germination, resulting in stable products containing the YpeB C-terminus. The presence of inhibitory PepSY domains at the C-terminus of YpeB, coupled with YpeB degradation during germination, may suggest that YpeB processing results in SleB activation. Modification of the predominant YpeB cleavage sites or deletion of htrC reduced proteolysis, but cleavage at other sites still resulted in YpeB instability. Additionally, these changes did not have a significant impact on SleB activity. SleB regulation by other spore proteins was also examined. To test if SleB activation is Ger receptor-dependent, Bacillus subtilis strains lacking Ger receptors and/or GSLEs were germinated via non-nutrient means. Results indicated SleB can be activated independent of these proteins. B. anthracis homologs of the B. subtilis lipoproteins YlaJ and YhcN were also studied, but deletion of these genes did not result in significant changes in SleB stability or activity. / Ph. D.

Bacillus anthracis, l'agent infectieux responsable de la maladie du charbon, est un agent pathogène majeur du risque biologique provoqué, notamment en raison de la sévérité de la forme respiratoire de la maladie. Celle-ci résulte de l'inhalation de spores dont les mécanismes de pénétration au niveau pulmonaire sont mal connus à l'heure actuelle. Cette thèse présente les apports des microscopies confocale et biphotonique à l'étude de ces mécanismes de pénétration des spores inhalées. Le modèle murin CX3CR1+/gfp, dont la sous-population CD11b+ de cellules dendritiques (DCs) exprime constitutivement la protéine de fluorescence verte (GFP), a été utilisé dans ces travaux. Une première partie présente le développement d'une méthode automatisée de discrimination des DCs parmi d'autres populations cellulaires exprimant le même fluorophore, en se basant sur le calcul d'un coefficient morphologique. Cette méthode a permis d'étudier dans un deuxième temps le comportement spécifique de la sous-population de DCs CD11b, après infection par des spores de B. anthracis. L'étude microscopique a été d'abord effectuée in situ, c'est-à-dire sur des explants pulmonaires maintenus dans des conditions favorables à la préservation de l'activité cellulaire, puis in vivo, sur des souris anesthésiées et ventilées. Le protocole d'imagerie tire profit d'une stratégie d'acquisition et de traitement a posteriori des données permettant de surmonter, sans contrainte mécanique appliquée à l'organe, les problèmes de focalisation liés aux mouvements thoraciques durant la ventilation de l'animal. Cette stratégie originale utilise un sur-échantillonnage de l'acquisition et profite du signal de seconde harmonique généré par le collagène comme référence spatiale ; elle a permis l'observation in vivo d'interactions entre DCs et macrophages au niveau pulmonaire. Ces interactions, de type synapse immunologique, sont favorisées par l'infection et présentent donc un rôle fonctionnel qui reste à définir. La formation de synapses immunologiques entre macrophages et DCs pourrait non seulement représenter un chaînon manquant à l'explication de la pénétration des spores de B. anthracis au niveau pulmonaire, mais pourrait aussi constituer un enjeu crucial dans la compréhension de la réponse immunitaire associée aux infections pulmonaires. / Bacillus anthracis, the causative agent of anthrax, is a major bioterrorism pathogen mainly because it can lead to a severe respiratory form of the disease. This form results from inhalation of spores, whose ways of entry into the lungs are not fully understood. This thesis reports the contribution of confocal and two-photon microscopy to the study of the penetration mechanisms of inhaled spores. The animal model utilized was CX3CR1+/gfp mouse, which constitutively expresses the green fluorescent protein (GFP) on CD11b+ dendritic cells (DCs). First, we present an automated method allowing discrimination of DCs among other GFP expressing cells, based on a morphologic coefficient. This method was then applied to the study of the specific behavior of CD11b DCs, after infection by B. anthracis spores. The microscopic study was first performed in situ, i.e. on explanted organs kept in conditions favorable to cell dynamics, then in vivo, i.e. on anesthetized and ventilated mice. In this case the imaging protocol profits from both acquisition and post-processing strategies, and allowed overcoming the focalization pitfalls coming from chest movements during ventilation. This novel strategy is based on an over-sampling of frame acquisition and utilizes second harmonic generation signal from alveolar collagen as a spatial reference. It led to the first ever in vivo observation of interactions between DCs and macrophages at the lung level. These immunological synapse-like structures are promoted by infection and thus display a functional role unknown until now. The formation of macrophages-DCs immunological synapses not only could represent a missing-link in figuring out the B. anthracis spore penetration mechanisms at the lung level, but more importantly could lead to a better understanding of the immune response associated with pulmonary infections.

Acquisition and retention of spores of an anthrax surrogate, Bacillus atrophaeus Nakamura ("BG") were evaluated in eight insect species. Species included: house cricket (Acheta domesticus L.), German cockroach (Blatella germanica L.), common house fly (Musca domestics L.), blue bottle fly (Calliphora vomitoria L.), hairy rove beetle Creophilus maxillosus L.), yellow mealworm (Tenebrio molitor L.), common paper wasps (Polistes exclamans exclamans Viereck), red paper wasps (Polistes Carolina L.), red harvester ant (Pogonymyrmex barbatus Smith). Individual insects were offered BG-treated food and sacrificed at specified time intervals following one, two or three meals. Resulting samples were surface-washed five consecutive times then homogenized to release gut contents, and the homogenate and first and fifth washes were cultured on Trypticase Soy Agar to determine recovery of BG spores. All species delivered spores but BG retention among species varied over time. Results demonstrate the potential of insects to serve as biosentinels for detecting the presence of spore-forming bacteria in the environment.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

Control of mRNA stability is an important aspect in the regulation of gene expression. A well studied signal for rapid transcript decay in mammalian cells is the AU-rich element (ARE), which is found in the 3’ untranslated region (UTR) of many labile transcripts. These sequence elements confer destabilization of transcripts by binding to AU-binding proteins (AUBPs) that can recruit cellular decay enzymes. The stability of ARE-containing mRNAs can be regulated by extracellular stimuli, which allows for cells to adapt to the changing environment. AREs are found in many transcripts that encode for inflammatory genes, including TNF, GM-CSF, and IL-8. Pathogens evolve and devise mechanisms to subvert the immune response of the host to aid in its infection. Bacillus anthracis is one such infectious agent that can disable numerous arms of the host immune response. Its secreted toxin, anthrax lethal toxin (LeTx), causes the accelerated decay of the IL-8 mRNA. IL-8 is a dual function cytokine and chemokine that can recruit and activate neutrophils at the site of infection. Through the inactivation of MAPK pathways, LeTx activity causes the destabilization of IL-8 transcripts through its ARE. In this thesis, I show that an AUBP, TTP, is dephosphorylated by LeTx and MAPK inhibitors, and knock-down of its expression stabilized IL-8 transcripts. LeTx activity also increased the colocalization of TTP to P-bodies, cytoplasmic sites concentrated with RNA decay enzymes. This suggests that the post-translational modification of TTP induced by LeTx led to its enhanced destabilization function. Identified TTP-associated proteins, non-muscle myosin heavy chain 9 (myosin-9) and HSC-70, were examined for their role in IL-8 transcript decay. Knock-down of each protein led to a slower rate of IL-8 mRNA destabilization. However, treatment of LeTx continued to mediate accelerated destabilization of IL-8 in these siRNA-transfected cells. This suggests that LeTx, myosin-9, and HSC-70 modulate the destabilization function of TTP independently.

In this study, a system for the collection of bioaerosols emitted from the mail sorting process was designed and characterized. Two different wetted-wall cyclones, the JBPDS cyclone and the glass cyclone sampler (GCS), were evaluated as system collection devices. These devices operate at 780 L/min and have a D50 of ~ 1 μm. A trimming impactor with a D50 of 10 μm was used upstream of the collection devices. Using two reference probes, the cyclone liquid outputs were compared with aerosol collected on filters and the output of an Aerosol-to-Hydrosol Transfer Stage (AHTS). The mass emission rate of the postal sorting machine was 3.15 mg/min and found not to vary significantly with flow rates above 700 L/min. On average, greater than 66% of the mass collected had a Da < 10 μm. Using a Coulter Counter, the volume median diameter (volume equivalent) for both device hydrosol outputs was 4.18 μm. For the effluent aerosol, the volume median diameter was 12.5 μm. For a bioaerosol release, this study found that greater than 65% (by volume) of the material released had a Da greater than 7.2 μm. Using filters, it was found that on average, 95% of the bioaerosol particles emitted had a Da less than 10 μm. According to the reference data, the expected number of bioaerosol particles in 1.5 times that collected by the GCS and 5.5 times that collected by the JBPDS cyclone for a one milligram release. The time constant for the system in response to a letter release was found to be 1.3 minutes for the GCS and 1.75 minutes for the JBPDS cyclone. A final note to this study states that the probe dimensions were incorrectly developed, therefore affecting the aspiration efficiency of the probes. In turn, this may have affected the outcome of some of the results. A plot is given at the end of the paper showing to what extent the results may have been affected. It is recommended that further experimental studies be performed to verify the results in this study.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed Feb. 4, 2010). Additional advisors: Subramaniam Ananthan, David E. Graves, Craig D. Smith, Sadanandan E. Velu. Includes bibliographical references.

Bacillus anthracis, the etiological agent of anthrax, is a spore- forming, toxin- producing bacterium. Currently, treatment of B. anthracis infections requires a 60- day antibiotic regimen. However, better therapeutics are required. Therefore, this study looked at the effect of levofloxacin and linezolid on B. anthracis cell viability, toxin production and spore formation using in vitro static models and a pharmacodynamic model. It was hypothesized that the combination would be the most effective at preventing toxin and spore production resulting in greater bacterial killing. However, these studies suggest otherwise. Nevertheless, clinically, the combination therapy may be more effective in rapid killing of vegetative B. anthracis and may be able to reduce the duration of therapy (by reducing the likelihood of spore survival). Therefore, the clinical benefit of combined therapy on long-term recurrence cannot be determined from these in vitro models. Further investigation with combination therapy is warranted. / October 2015