Clostridium perfringens is a Gram-positive bacterium that secretes a biofilm matrix material. The goal of these experiments was to identify pilin mutants that are needed for secretion of the biofilm matrix and develop a functional model for a type II secretion system (T2SS) in C. perfringens. Protein tagging, western blot, and slot blot experiments were done to quantify protein secretion. After performing experiments using a CPE0515-FLAG construct, it was concluded from immunoblot densitometry data that, except for the pilA1 deletion mutant, none of the 18 tested pilin mutants had a statistically significant difference from the wild type (WT) with regard to protein secretion. From slot blot densitometry assays, it was concluded that the pilA1 and CPE2280 mutants showed statistically significant lower values than the WT but the pilA2 and CPE1841 mutants had values that were higher than the wild type. Testing the construct containing only CPE0514 and CPE0515-FLAG showed that CPE0516 and CPE0517 are not needed for secretion of the protein CPE0515. HA-tagged CPE0516 qualitative immunoblots showed that, unlike CPE0515, oligomerization of CPE0516 is not occurring, and that this protein likely forms a heat stable dimer. Overall, the data did not allow us to construct a T2SS model, since there were not enough proteins revealed to be involved to create a complete Type II secretion system. / Master of Science / The methods by which C. perfringens can persist and survive in environmental conditions is something that would be useful to learn more about. One of the methods that many bacteria use to survive is by creating a biofilm matrix material, which provides protection for the bacteria from environmental stresses. In this study, the goal was to determine which specific proteins are needed for the secretion of the biofilm matrix material. Using molecular biology techniques, the proteins thought to be involved in biofilm formation quantified. The results showed that while two proteins ultimately appeared to be needed for secretion, there were not enough proteins involved to create a complete model for a functional secretion system in C. perfringens.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic, moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the same direction. An arsC (codes for an arsenate reductase), usually associated with ars operons, was absent from this ars operon. PCR and Southern-hybridization experiments revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located upstream of the arsRB operon. The intergenic region between the termination end of the kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp, suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As precursor gene in its native Sulfobacillus host. The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that processes involved in the production of functional proteins from the ars operon transcript were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to confer resistance to arsenic in the heterologous E. coli host. Eight Sulfobacillus strains isolated from different geographical areas were subjected to amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende, asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van ‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa) kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs 77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie. Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te verleen. Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al., 2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van ‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is. Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van restriksie ensiem herkenning setels, geïdentifiseer kan word.

Bacteria adhere despite severe mechanical perturbations. In Gram-positive bacteria, this adhesion is dependent upon a set of extracellular proteins, most notably pili, that have a unique abundance of internal disulfide, isopeptide, and thioester bonds. How these cell adhesion proteins manage to withstand such mechanical assaults, and what role these internal covalent bonds play to that end, remain open questions. Herein, we apply single-molecule force spectroscopy to delve into the mechanical behavior of three Gram-positive pilus proteins. We find that structural components of the Actinomyces oris and Corynebacterium diphtheriae pili have isopeptide-delimited extensions at extreme mechanical forces. This behavior enables efficient energy dissipation under high mechanical loads. Meanwhile, the pilus tip adhesin of Streptococcus pyogenes can covalently bind to targets via its internal thioester bond. We find that reactions with this internal thioester bond are reversible, and that both the nucleophilic bond cleavage and its spontaneous reformation are negatively force-dependent, inhibited at forces above ~30 pN and above ~7 pN, respectively. Based on these observations, we propose a model of shear-enhanced covalent adhesion for Gram-positive bacteria. Finally, we move from single-molecule characterization to application as we explore the potential of a peptide competitors to modulate the folding and function of bacterial virulence factors.

Bacillus subtilis é uma bactéria gram-positiva, não patogênica, formadora de esporos e com um grande conhecimento disponível a cerca de sua genética e fisiologia, comparável apenas à Escherichia coli K12. Recentemente, linhagens geneticamente modificadas de B. subtilis foram utilizadas como veículos vacinais mas, até o momento, não se havia avaliado a indução de respostas imunológicas citotóxicas (linfócitos T CD8+) específicas em camundongos imunizados com esporos ou células vegetativas. No presente trabalho, avaliouse a indução de linfócitos T CD8+ em animais imunizados com linhagens vacinais de B. subtilis. Inicialmente empregou-se como alvo a proteína E7 de vírus papiloma humano tipo 16 (HPV-16) fusionada ou não à proteína gD do vírus herpes simples tipo 1 (HSV-1). Em uma segunda estratégia, empregou-se como alvo a subunidade B da toxina termolábil (LTB) de E. coli enterotoxicogênica (ETEC) co-expressa ou fusionada à proteína GroEL2 de Mycobacterium bovis. As quantidades de E7 e gDE7 expressas pelas linhagens recombinantes de B.subtilis ficaram abaixo do limite de detecção e não foram suficientes para ativação de células T CD8+ específicas. Por outro lado, linhagens de B. subtilis capazes de expressar LTB e GroEL2 promoveram a ativação de linfócitos T CD8+ específicos. De um modo geral, o presente trabalho representa uma contribuição inédita sobre a ativação de respostas imunológicas de base celular em camundongos imunizados com linhagens recombinantes de B. subtilis e os resultados obtidos certamente contribuirão para a geração de veículos vacinais mais efetivos na profilaxia e tratamento de diferentes doenças infecciosas, como infecções virais, ou degenerativas, como o câncer. / Bacillus subtilis is a sporulated, non pathogenic, gram-positive bacterial species with a large amount of information concering its genetics and physiology, comparable only to Escherichia coli K12. Recently, genetically modified B.subtilis strains were successfully employed as vaccine vehicles but there is no information concerning the activation of antigen specific cytotoxic immune responses (T CD8+ lymphocytes) in mice vaccinated with either vegetative cells or spores. In the present report, we evaluated the activation of CD8+ T cell responses in mice immunized with B. subtilis vaccine strains genetically modified in order to express the human papillomavirus type 16 (HPV-16) E7 protein as a target antigen, isolated or genetically fused to the type I herpes simplex vírus (HSV-1) gD protein. In a second approach, we have also tested the B subunit heat labile toxin, produced by enterotoxigenic E. coli (ETEC) strains, coexpressed or genetically fused to the Mycobacterium bovis GroEL2 protein. E7 and gDE7 expression by B.subtilis vaccine strains were bellow the detection limits and did not allow the activation of antigen-specific CD8+ T cell responses in vaccinated mice. On the other hand, LTB-specific CD8+ T cell responses were detected in mice immunized with B. subtilis expressing both LTB and GroEL2. Collectively, the present study respresents an important contribution on the activation of cellular immune responses in mice immunized with genetically modified B.subtilis and should direct further analyses aiming the development of more effective vaccine vehicles employed both for preventive and therapeutic treatment of infectious and degenerative diseases, such as virus infections and cancer.

Bacillus subtilis é uma bactéria gram-positiva, não patogênica, formadora de esporos e com um grande conhecimento disponível a cerca de sua genética e fisiologia, comparável apenas à Escherichia coli K12. Recentemente, linhagens geneticamente modificadas de B. subtilis foram utilizadas como veículos vacinais mas, até o momento, não se havia avaliado a indução de respostas imunológicas citotóxicas (linfócitos T CD8+) específicas em camundongos imunizados com esporos ou células vegetativas. No presente trabalho, avaliouse a indução de linfócitos T CD8+ em animais imunizados com linhagens vacinais de B. subtilis. Inicialmente empregou-se como alvo a proteína E7 de vírus papiloma humano tipo 16 (HPV-16) fusionada ou não à proteína gD do vírus herpes simples tipo 1 (HSV-1). Em uma segunda estratégia, empregou-se como alvo a subunidade B da toxina termolábil (LTB) de E. coli enterotoxicogênica (ETEC) co-expressa ou fusionada à proteína GroEL2 de Mycobacterium bovis. As quantidades de E7 e gDE7 expressas pelas linhagens recombinantes de B.subtilis ficaram abaixo do limite de detecção e não foram suficientes para ativação de células T CD8+ específicas. Por outro lado, linhagens de B. subtilis capazes de expressar LTB e GroEL2 promoveram a ativação de linfócitos T CD8+ específicos. De um modo geral, o presente trabalho representa uma contribuição inédita sobre a ativação de respostas imunológicas de base celular em camundongos imunizados com linhagens recombinantes de B. subtilis e os resultados obtidos certamente contribuirão para a geração de veículos vacinais mais efetivos na profilaxia e tratamento de diferentes doenças infecciosas, como infecções virais, ou degenerativas, como o câncer. / Bacillus subtilis is a sporulated, non pathogenic, gram-positive bacterial species with a large amount of information concering its genetics and physiology, comparable only to Escherichia coli K12. Recently, genetically modified B.subtilis strains were successfully employed as vaccine vehicles but there is no information concerning the activation of antigen specific cytotoxic immune responses (T CD8+ lymphocytes) in mice vaccinated with either vegetative cells or spores. In the present report, we evaluated the activation of CD8+ T cell responses in mice immunized with B. subtilis vaccine strains genetically modified in order to express the human papillomavirus type 16 (HPV-16) E7 protein as a target antigen, isolated or genetically fused to the type I herpes simplex vírus (HSV-1) gD protein. In a second approach, we have also tested the B subunit heat labile toxin, produced by enterotoxigenic E. coli (ETEC) strains, coexpressed or genetically fused to the Mycobacterium bovis GroEL2 protein. E7 and gDE7 expression by B.subtilis vaccine strains were bellow the detection limits and did not allow the activation of antigen-specific CD8+ T cell responses in vaccinated mice. On the other hand, LTB-specific CD8+ T cell responses were detected in mice immunized with B. subtilis expressing both LTB and GroEL2. Collectively, the present study respresents an important contribution on the activation of cellular immune responses in mice immunized with genetically modified B.subtilis and should direct further analyses aiming the development of more effective vaccine vehicles employed both for preventive and therapeutic treatment of infectious and degenerative diseases, such as virus infections and cancer.

The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB - GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques. / <p>QC 20100726</p>

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization. / QC 20100809

SecA is a central component of the general secretion system that is essential for growth and virulence of bacteria. A series of fluorescein analogs were tested against ATPase activities of Escherichia coli SecA. Rose Bengal (RB) and Erythrosin B are potent inhibitors abolishing the activities of three forms of SecA ATPase with IC50 in µM range. Both inhibit SecA intrinsic ATPase with two mechanisms depending on ATP concentrations, indicating they influence the two non-identical nucleotide binding sites differently. RB shows different inhibitory effects against three forms of SecA ATPase activities, suggesting that the inhibition is related to the conformation of SecA. RB with IC50 at sub-µM level is the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation to date. The fluorescein analogs inhibit intrinsic ATPase of Bacillus subtilis SecA similarly, and also exhibit antibacterial effects in E. coli and B. subtilis. Our findings indicate the value of fluorescein analogs as probes for mechanistic studies of SecA and the potential development of new SecA-targeted antimicrobial agents. A series of SecA derivatives with truncated C-terminus within the first long α-helix of the helix-bundle extending the ATPase catalytic domain of N68 was analyzed. These SecA variants interact with lipids, and those containing the C-terminal portion of the long α-helix starting at residues #639 form the ring-like structure in liposomes, indicating the critical domains for forming the protein-conducting channel. The presence and length of the C-domain influence the response to RB of NBDII mutants and C-terminal truncates of SecA. Thus this region may interact with the inhibitors and is involved in the structure and regulation of SecA ATPase activity. B. subtilis SecA was analyzed for interspecies comparison. Despite sharing high homology, this SecA homolog cannot complement E. coli mutants with SecA defect. Phospholipids do not stimulate ATPase activities of B. subtilis SecA, but induce its conformational changes, leading to the lipid-specific domains and ring-like structures similar to E. coli SecA. These pore-ring structures may represent part of the protein-conducting channels. Therefore, the potential structural roles of SecA in the protein translocation machinery may be universal in both Gram-negative and Gram-positive bacteria.

In Streptococcus pyogenes, the protein SiaA (HtsA) is part of a heme uptake pathway system and involved in heme transfer from Shp to the ABC transporter. SiaA mutants, in which alanine replaces the axial histidine (H229) and methionine (M79) ligands, as well as a lysine (K61) and cysteine (C58) located near the heme propionates, are reported. Studies on a mutant of a cysteine expected to be at a distance from the propionates (C47A) are also reported. The coordination state and spin state of the selected mutants were determined via Resonance Raman studies. The pKa values of mutants ranged from 9.0 to 9.4, which were close to the pKa of the WT SiaA (9.7). The midpoint reduction potential of lysine (K61A) mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the WT protein (68 ± 3 mV). The addition of guanidinium hydrochloride resulted in protein denaturation that could show more than one process and occurred over days. The ease of protein unfolding was directly related to the extent of interaction of the residues with the heme: changes in the axial ligands resulted in far greater changes in heme protein stability than changes in the residues near the heme propionates. The causative agent of diphtheriae, Corynebacterium diphtheriae, imports heme via an ABC uptake transporter. In this research, two of the five proteins in the heme uptake pathway of C. diphtheriae were studied. These proteins were HmuT, lipoprotein component of the ABC transporter, and HtaA, the heme receptor. UV-visible spectroscopy and fluorescence spectroscopy showed that HmuT protein as isolated bound a porphyrin, rather than heme. Electrospray ionization mass spectrometry (ESI-MS) studies suggested that two tetrapyrroles were bound. To assess stability of this protein towards heme release, thermal denaturation studies were performed. For HtaA, UV-visible and fluorescence spectroscopy also showed the protein as isolated was also bound a porphyrin, rather than heme. Homology studies showed that HtaA protein is quiet distant from homologous heme uptake proteins and could be a member of novel heme binding domain family.