Humidity responsive materials sense, respond and adapt to the environment in response to changes in humidity. An important potential application of this material technology is the creation of “smart textiles” that facilitate moisture management in clothing. Materials used for clothing must have characteristics such as elasticity, washability and abrasion resistance, but smart textiles that have been demonstrated to date lack these characteristics. It is the need for improved materials that motivated the present study. Here, we developed spore-cellulose nanofiber composites (CNF) and spore-polyurethane (PU) composites, which are two biologically-based humidity-responsive materials that derive their high energy density humidity responsiveness from spores. We demonstrate the use of these hygromorphing materials for smart textiles by coupling the responsive materials to fabrics to create a textile that vents in humid environments and closes in dry environments. This material can be used in clothing to enable fast evaporation of sweat from the skin and improved comfort. Because the spore-CNF composite is not elastic stretchy or water resistant and therefore is undesirable for real world clothing applications, we also developed a stretchy spore-PU composite that is simultaneously humidity responsive, stretchy and water and abrasion resistant. In addition, we fabricated spore-PU based hygromorphing fabric bilayer actuators to create venting smart textiles with adaptive permeability properties that are compatible with clothing applications. These smart fabrics have the potential to improve the functionality and utility of garments, especially those intended for athleticwear, workwear and protective garments.

Spores of foodborne pathogens such as Clostridium botulinum, Clostridium perfringens and Bacillus cereus are widely distributed in nature. Presence of those spores in food products, particularly C. botulinum spores in vacuum packed, ready-to-eat low-acid products, is a great safety concern. The research here described is a first effort towards understanding the role of the spore coat proteins in the inactivation of bacterial spore using high pressure processing. This study proposes a coat protein solubilization methodology using non-ionic detergents minimizing protein damage and compatible with spectroscopy methods. The methodology developed here was compared with approaches proposed in the literature with respect to protein yield, protein fractions identified, amino acid composition and suitability with spectroscopy techniques for the further analysis of coat proteins. Bacillus subtilis ATCC 6633 spore coat proteins were solubilized (n=3) using octyl-β-D-glucopyranoside (OGP) at room temperature and urea/sodium dodecyl sulphate (UDS) at 37C and 70C. Analysis of variance (ANOVA) showed no significant (95% confidence) differences between the three repetitions of the three spore coat protein solubilization methods. Protein yield was significantly larger (95% confidence) when using UDS at 70C as compared to UDS at 37C. OGP gave the lowest protein yield but allowed circular dichroism (CD) analysis of the spore coat protein solution with minimum blank signal. SDS-PAGE revealed that the UDS-70C coat protein solutions consisted of five major and six minor proteins ranging 6 to 65 kD while the OGP solution appear to consist of four major and nine minor bands in the same mw range. Amino acid analysis of the protein extracted by the OGP method was conducted using reverse phase HPLC (RP-HPLC) and compared with published information. The OGP spore coat protein solution showed a higher proportion of aspartate, glutamate, alanine and tyrosine. Pressure, heat and time effects were studied on spore coat proteins obtained from untreated and pressure-treated B. subtilis ATCC 6633 spores. Pressure treatments of spores, and of extracted spore coat protein solutions, at 50 kpsi (345 mPa) and 85 kpsi (586 mPa) for 10 and 30 min at constant 85C along with appropriate heat- and pressure-only controls and untreated sample, were used to study the effect of pressure, heat and time on spore coat proteins. Both spore coat protein solubilization procedures showed a significant reduction in protein yield for pressure-only, heat-only and pressure/heat treated spores when compared with untreated spores. When OGP-solubilized proteins from untreated spores were pressure treated, SDS-PAGE profile showed an increasing overall band intensity with increasing pressure and time. In the case of protein solution obtained from pressure-treated spores the electrophoretic pattern showed the loss of higher molecular weight proteins. The significance of this study is that for the first time we have observed extensive changes on spore coat proteins caused by pressure, as well as heat treatments. Future studies will examine what is the probable physiological role of the proteins damaged by these physical treatments. An advantage of the protein solubilization here developed will allow the application of spectroscopy techniques to characterize changes in spore coat proteins. / Graduation date: 2003

Esporos de Bacillus subtilis podem ser utilizados como veículos vacinais capazes de expressar ou adsorver proteínas heterólogas em sua superfície. Estudos recentes indicaram que esporos de B. subtilis também conferem um forte efeito adjuvante para vacinas baseadas em proteínas purificadas administradas por via parenteral. Nesse cenário, o objetivo do presente projeto foi avaliar o papel adjuvante dos esporos de B. subtilis e viabilizar sua utilização como carregadores de vacinas de DNA pelo método da biobalística (gene gun). Nesta metodologia, os esporos foram utilizados como substitutos das micropartículas de ouro usualmente empregadas, mantendo a capacidade de transfecção de células eucarióticas in vitro, e produção de anticorpos específicos em camundongos C57BL/6 imunizados com o plasmídeo vacinal pgDE7h (que codifica para uma forma atóxica da oncoproteína E7 do HPV-16). Além disso, esporos de B. subtilis foram capazes de ativar células dendríticas in vitro e, quando coadministrados a pgDE7h pela via intramuscular, conferiram um aumento de 50% na proteção anti-tumoral de animais previamente transplantados com a linhagem tumoral TC-1 (que expressa a proteína E7 do HPV-16), em uma relação dose-dependente. / Bacillus subtilis spores can be used as vaccine vehicles capable of to display or adsorb heterologous proteins in its surface. Recent studies have indicated that spores of B. subtilis confer a strong adjuvant effect for vaccines based on purified proteins administered by the parenteral route. In this scenario, the objective of this project is to evaluate the adjuvant role of B. subtilis spores e viabilize its use as DNA vaccine carriers of biolistic method (gene gun). In this methodology, the spores are used as gold microparticles substitutes usually employed, maintaining the ability to transfect eukaryotic cells in vitro and production of specific antibodies in C57BL / 6 mice immunized with the vaccine plasmid pgDE7h (encoding a non-toxic form of the E7oncoprotein of HPV-16). Additionally, spores of B. subtilis were able to activate dendritic cells in vitro when co-administered intramuscularly to pgDE7h, conferring an increase of 50% of anti-tumor protection in animals previously transplanted with the TC-1 tumor cell line (expressing the E7 protein of HPV-16) in a dose-dependent manner.

O S. mutans é o agente etiológico da cárie dental humana, uma doença com ampla distribuição mundial. A adesão à superfície dental depende da interação entre a proteína de superfície P1 e a aglutinina salivar (SAG) adsorvida ao dente. A região N-terminal da P1 é um alvo vacinal importante que está diretamente associada às funções de adesão e agregação. Este trabalho teve o objetivo de avaliar estratégias vacinais contra o S. mutans baseadas na proteína P1 usando linhagens recombinantes de B. subtilis. O B. subtilis é uma bactéria gram positiva, formadora de esporos, não patogênica, empregada como sistema de expressão de proteínas heterólogas e como veículo vacinal administrado por vias de mucosas. Empregamos o B. subtilis para expressar e purificar a proteína P139-512 derivada da proteína P1 de S. mutans UA159. O antígeno P139-512 apresentou epítopos lineares e conformacionais semelhantes aos presentes na proteína P1 nativa. O sítio de ligação à SAG está preservado nessa proteína assim como suas propriedades imunogênicas. A coadministração parenteral do antígeno com adjuvantes vacinais promoveu resposta sistêmica específica com anticorpos eficazes no bloqueio da adesão de S. mutans. Por fim, usamos esporos de B. subtilis como veículo de entrega de mucosa para o antígeno alvo de S. mutans. Esporos de B. subtilis foram modificados para expressar na superfície adesinas bacterianas (SlpA, InvA ou Inv600) com capacidade de ligação ao epitélio intestinal e, quando no estágio de célula vegetativa, expressar intracelularmente o antígeno P139-512. A imunização oral com os esporos adesivos induziu altas concentrações de anticorpos sistêmicos e de mucosa. A imunização nasal ou sublingual com os esporos recombinantes induziu níveis de anticorpos sistêmicos maiores do que aqueles obtidos após a imunização oral. Além disso, esses anticorpos foram mais eficientes em bloquear a adesão de S. mutans à SAG imobilizada, sem interferir com a agregação. Em conclusão, os resultados obtidos abrem perspectivas interessantes para o desenvolvimento de vacinas anti-cárie baseadas em linhagens de B. subtilis. / S. mutans is the major etiologic agent of human dental caries, a disease with worldwide distribution. The adhesion to the tooth surface is dependent on the interaction of the P1 surface protein and salivary agglutinin (SAG) adsorbed to the tooth. The N-terminal region of P1 is an important vaccine target that is directly associated with adhesion and aggregation functions. This study aimed to evaluate vaccination strategies against S. mutans based on the P1 protein using recombinant B. subtilis strains. B. subtilis is a gram positive, spore-forming, non-pathogenic bacterium used as expression system for heterologous proteins and as a vaccine vehicle administered by mucosal routes. Inicially, we employed a recombinant B. subtilis strain to express and purify the P139-512 protein derived from the S. mutans UA159 P1 protein. The P139-512 antigen showed important conformational and linear epitopes similar to those present in the native P1 protein. The SAG-binding site is preserved in P139-512 as well as immunological properties. The parenteral co-administration of antigen with vaccine adjuvants stimulated systemic antibodies effective in blocking adhesion of S. mutans to SAG. Lastly, we used B. subtilis spores as a mucosal delivery vehicle for antigen targeting. B. subtilis endospores were modified to display bacterial adhesins (SlpA, InvA or Inv600), capable to bind to the intestinal epithelium, on the spore surface and to express intracellularly the P139-512 antigen during the vegetative cell stage. Oral immunization with adhesives spores induced high systemic and mucosal specific antibodies levels. The nasal or sublingual immunization with B. subtilis recombinant spores induced higher amounts of systemic antibodies than the oral immunization. Furthermore, the specific antibodies were highly effective in blocking the adherence of S. mutans to immobilized SAG, without interfering with aggregation. In conclusion, the results open interesting perspectives for the development of anti-caries vaccines based on B. subtilis strains.

Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

The full exploitation of bacterial whole-cell biosensing systems in field applications requires the survival of bacterial cells and long term-preservation of their sensing ability during transportation and on-site storage of such analytical systems. Specifically, there is a need for rapid, simple and inexpensive biosensing systems for monitoring human health and the environment in remote areas which often suffer from harsh atmospheric conditions and inadequate commercial distribution and storage facilities. Our laboratory has previously reported the successful use of bacterial spores as vehicles for the long-term preservation and storage of whole-cell biosensing systems at room temperature. In the present research, we have accomplished a year-long study to investigate the effect of extreme climatic conditions on the stability of spores-based whole-cell biosensing systems. The spores were stored in laboratory conditions that simulated those found in real harsh environments and germination ability and analytical performance of the spore-based sensing systems upon storage in such conditions was monitored. Our results proved that the intrinsic resistance of spores to harsh environmental conditions helped maintain the integrity of the sensor bacteria. The revived active cells actually retained their analytical performance during the course of the twelve-month storage study.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, germinate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product. In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris. The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KC1). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides. The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains. A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KC1) attached in greater numbers (1 Log₁₀ CFU cm⁻�) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log₁₀ CFU cm⁻�, respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log₁₀ CFU cm⁻�), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log₁₀ CFU cm⁻� reduction) compared with spores that attached in simple 1:1 electrolyte media. The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log₁₀ CFU mL⁻�) after a 30 min caustic wash at 65 �C in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run. In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.

Fluorescence techniques are being considered for the detection and identification of bacterial spores. This thesis sets out to empirically characterize the detailed autofluorescence spectroscopic properties of spores and their target molecules. The multiwavelength fluorescence studies from a unique endogenous biomarker, dipicolinic acid (DPA) and its calcium salt (CaDPA) in bacterial spores are found to be useful for fluorescence characterization of spores. A systematic determination of the fluorescence profile of the major chemical components of Bacillus spores and the effect of UV irradiation on them has been performed in dry samples, wet paste and in aqueous solution. The thesis applies reliable tools for accurately describing complex nature of spectral profile from bacterial spores, and for interpreting and identifying their spectral properties. We show that multiwavelength fluorescence technique combined with Principal Component Analysis (PCA) clearly indicates identifiable grouping among dry and wet Bacillus spore species. Differences are also observed between dried, wet and redried spores, indicating the stark effect of hydration on fluorescence fingerprints. The study revealed that changes in fluorescence of spores due to hydration/drying were reversible and supports a recent model of a dynamic and dormant spore structure. The spectra were analysed with PCA, revealing several spectroscopically characteristic features enabling spore species separation. The identified spectral features could be attributed to specific spore chemical components by comparing the spore sample signals with spectra obtained from the target molecules. PCA indicated underlying spectral patterns strongly related to species and the derived components were correlated with the chemical composition of the spore samples. More importantly, we examined and compared the fluorescence of normal spores with a mutant of the same strain whose spores lack DPA. We discovered that the dramatic fluorescence enhancement of Bacillus spores can be caused by UV irradiation in the spectral region of this unique biomarker without any pre treatment. Differences between spectra of spores, spore strains and other biological samples are very marked and are due to the dominance of the dipicolinate features in the spore spectra. This could lead to a cheap, more sensitive, faster and reagentless bacterial spore detector.

Thesis (M.S.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2007. / Includes bibliographical references (p. 29-31).