Invariant recognition of polychromatic images of Vibrio cholerae O1

7 pages, 5 figures.-- ©2002 Society of Photo-Optical Instrumentation Engineers.Cholera is an acute intestinal infectious disease. It has claimed many lives throughout history, and it continues to be a global health threat. Cholera is considered one of the most important emergence diseases due its relation with global climate changes. Automated methods such as optical systems represent a new trend to make more accurate measurements of the presence and quantity of this microorganism in its natural environment. Automatic systems eliminate observer bias and reduce the analysis time.We evaluate the utility of coherent optical systems with invariant correlation for the recognition of Vibrio cholerae O1. Images of scenes are recorded with a CCD camera and decomposed in three RGB channels. A numeric simulation is developed to identify the bacteria in the different samples through an invariant correlation technique. There is no variation when we repeat the correlation and the variation between images correlation is minimum. The position-, scale-, and rotation-invariant recognition is made with a scale transform through the Mellin transform. The algorithm to recognize Vibrio cholerae O1 is the presence of correlation peaks in the green channel output and their absence in red and blue channels. The discrimination criterion is the presence of correlation peaks in red, green, and blue channels.Peer reviewe

Invariant Optical Color Correlation for Recognition of Vibrio Cholerae

Abstrac

Invariant recognition of polychromatic images of Vibrio cholerae O1

Abstract. Cholera is an acute intestinal infectious disease. It has claimed many lives throughout history, and it continues to be a global health threat. Cholera is considered one of the most important emergence diseases due its relation with global climate changes. Automated methods such as optical systems represent a new trend to make more accurate measurements of the presence and quantity of this microorganism in its natural environment. Automatic systems eliminate observer bias and reduce the analysis time. We evaluate the utility of coherent optical systems with invariant correlation for the recognition of Vibrio cholerae O1. Images of scenes are recorded with a CCD camera and decomposed in three RGB channels. A numeric simulation is developed to identify the bacteria in the different samples through an invariant correlation technique. There is no variation when we repeat the correlation and the variation between images correlation is minimum. The position-, scale-, and rotation-invariant recognition is made with a scale transform through the Mellin transform. The algorithm to recognize Vibrio cholerae O1 is the presence of correlation peaks in the green channel output and their absence in red and blue channels. The discrimination criterion is the presence of correlation peaks in red, green, and blue channels

ECOLOGY OF TYPE VI SECRETION COMPETITION IN THE BENEFICIAL BACTERIAL SYMBIONT VIBRIO FISCHERI

Microbial communities perform vital ecosystem functions that impact host health and drive essential biogeochemical processes on our planet. These communities, however, do not always assemble peacefully and bacteria have evolved diverse strategies to eliminate competitors. One such mechanism is the type VI secretion system (T6SS), a contact-dependent killing mechanism found broadly distributed among both beneficial and pathogenic species. Despite the prevalence of T6SSs in host microbiomes, few studies have investigated their ecological roles due to the inherent complexity of host-associated communities. This dissertation uses the relationship between the marine bacterium Vibrio fischeri and Euprymna scolopes squid as a simplified model for studying bacterial competition during symbiosis establishment. Using novel in vitro and in vivo assays I demonstrate that V. fischeri contain a strain-specific T6SS that is used to spatially structure the microbial population within a natural host early during colonization. I developed culture conditions that mimic the physical environment within host tissue to characterize the host-specific signals that modulate T6SS competition during habitat transition, and identify a mechanism for T6SS+ cells to discriminate between potential competitor cells. This dissertation provided new tools to examine bacterial behaviors that may be relevant in a host, yet are difficult to study using traditional culturing methods and laid the foundation for the Vibrio-squid symbiosis as a powerful system to probe molecular mechanisms of interbacterial competition during natural host colonization.Doctor of Philosoph

Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

SYSTEMATIC INVESTIGATION OF QUORUM SENSING IN Escherichia coli

High throughput techniques and advanced mathematical tools have enabled systematic investigations of biological systems with unparalleled precision. Not only molecular interactions between components but mechanisms and the dynamic behaviors associated with these systems are revealed, suggesting that comprehensive systems biology can be realized in the near future. Quorum sensing, especially the auto-inducer2 (AI-2) system, has been extensively studied due to its commonality among bacteria and connections to pathogenic phenotypes. In this study, the E. coli quorum sensing AI-2 system was studied combing system-based mathematical modeling and high throughput genomic profiling. First, a Stochastic Petri Network (SPN) model was constructed based on available regulatory information. Simulations together with experimental data demonstrated that the apparent stimulation of AI-2 in the presence of glucose is not from the increased transcriptional or translational expression of AI-2 synthases luxS and pfs, nor from the increased metabolic flux associated with LuxS-related pathways but from an alternative AI-2 synthesis pathway. The conversion of adenosine with cellular extracts from both luxS and pfs mutants validated our prediction about the existence of an alternative non-LuxS related AI-2 synthesis pathway. Second, AI-2 uptake regulatory network was investigated in detail: lsrR-lacZ, lsrK-lacZ fusion reporters were constructed and the analysis found that lsrR is subject to its own repression and is induced by both lsrK and luxS. Further transcriptome analysis demonstrated that lsrR and lsrK, together with quorum signal AI-2, coregulate lsrRK regulon, which influences phenotypes (biofilm, small RNAs). Importantly, this regulation is in a distinctly different manner than that mediating the lsr operon. We hypothesize that lsrR acts together with AI-2 to mediate cellular processes and that the phosphorylation of AI-2 molecule through lsrK triggers different response pathways. These investigations demonstrated that lsrR, lsrK are indispensable for AI-2 uptake. These newly elucidated regulatory mechanisms and associations undoubtedly broaden the scope of the AI-2 quorum sensing system, and provide a solid foundation for further mathematical modeling of the dynamics and system behaviors in E. coli . Finally, a tight coupling of experimental manipulation with mathematical analysis, as demonstrated in this study, provides a good example for systematically investigating biological systems

One ring to rule them all : Identification and characterization of the type IV pili secretin associated protein TsaP and analysis of the type IV secretion system of Neisseria gonorrhoeae

Over the years, N. gonorrhoeae has evolved and acquired different mechanisms to protect itself against a variety of antibiotics and chemotherapeutic agents. One reason for the rapid spread of antibiotic resistance in gonococci is the highly effective horizontal gene transfer. The transferred DNA is either provided directly via conjugation, or via the environment via autolysis or the gonococcal type IV secretion system (T4SS), which secretes ssDNA into the extracellular milieu. DNA uptake from the environment in Neisseria involves the type IV pili (T4P) and the competence system, transporting the DNA across the outer and the inner membrane, respectively. Functional characterization of the type IV secretion system and DNA uptake system and thus the type IV pili machinery in N. gonorrhoeae could provide starting points in the exploration of new therapeutic strategies. To better understand the transcriptional regulatory network of the type IV secretion system of N. gonorrhoeae transcriptional mapping of genes essential for DNA secretion was performed. This revealed that genes essential for DNA secretion are encoded within four different operons. Additional analysis of a region, which is not essential for DNA secretion, encoding the single-stranded DNA binding protein SsbB and the topoisomerase TopB showed that these genes are significantly more highly transcribed then genes that are involved in DNA secretion, such as the coupling protein TraD and the relaxase TraI. To investigate whether the single-stranded DNA, which is secreted via the T4SS encoded within the GGI facilitates biofilm formation, biofilm formation of N. gonorrhoeae strains were analyzed in continuous flow-chamber systems by confocal laser scanning microscopy. This showed that the ssDNA secreted via the T4SS plays a role in the early stages of biofilm formation. In Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which was named TsaP, the T4P secretin-associated protein, was identified as a widely conserved component that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the outer membrane in a PilQ-dependent manner. In addition, we identified that TsaP contains apart from the LysM domain, two FlgT-like domains and a linker region, which is specific for Neisseria spp. We could show that the linker domain plays an important role in pilus biogenesis in the β-proteobacterium N. gonorrhoeae. In order to determine if TsaP directly interacts with PilQ via the B2 domain, PilQ and TsaP of N. gonorrhoeae and M. xanthus were heterologously expressed and purified. Characterization of the heterologously expressed and purified proteins showed that TsaP is able to form SDS-stable complexes, resembling a ring-like structure, and that it might interact with PilQ, forming a double ring structure. In general, we propose that TsaP anchors the secretin to the PG to enable the secretin to withstand the forces generated during pilus extension and retraction. Because T4P play an important role in the pathogenesis of many bacteria and TsaP is found in all bacteria that express T4aP and plays an important role in T4aP biogenesis, it might be an important future drug target

Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

Renibacterium salmoninarum and Aeromonas salmonicida pathogenesis and virulence in lumpfish (Cyclopterus lumpus)

Renibacterium salmoninarum, the etiological agent of Bacterial Kidney Disease (BKD), and Aeromonas salmonicida, which causes furunculosis, are economically important pathogens of marine fish. The marine teleost lumpfish (Cyclopterus lumpus) is an eco-friendly cleaner fish in Atlantic salmon (Salmo salar) farming. As the lumpfish demand in salmonid aquaculture continues to rise, understanding how lumpfish interact with well-known Gram-positive and Gram-negative fish pathogens is certainly required. Therefore, in my Ph.D. thesis, I studied the interactions between lumpfish host and Grampositive R. salmoninarum or Gram-negative A. salmonicida with a particular focus on the fundamental aspects of bacterial pathogenicity and virulence. First, I evaluated the lumpfish susceptibility and immune response to R. salmoninarum infection. Lumpfish showed typical BKD clinical signs and 35 % mortality when infected with a high dose of R. salmoninarum (1×109 cells dose-1). High bacterial loads were observed in tissues (i.e., spleen, liver, and head kidney) at 28 days post-infection (dpi), and R. salmoninarum continued to persist in tissues until 98 dpi. Further, gene expression analysis using qPCR in the fish head kidney found that R. salmoninarum causes immune suppression at 28 dpi and lumpfish induce a cell-mediated immune response at 98 dpi. Second, I profiled the lumpfish head kidney transcriptome response to R. salmoninarum at early (28 dpi) and chronic (98 dpi) infection using RNA sequencing. Compared to 98 dpi, lumpfish induced many molecular pathways and genes at 28 dpi. For instance, R. salmoninarum-induced genes at 28 dpi were linked to innate and adaptive immunity, while R. salmoninarum-suppressed genes were involved in amino acid metabolism, cellular and developmental processes. In contrast, the transcriptome response of the lumpfish head kidney to this pathogen was minimal at 98 dpi, with R. salmoninarumdependent dysregulation of genes primarily connected to cell-mediated adaptive immunity. Third, I described the riboflavin supply pathways of A. salmonicida. Using in silico tools and RT-PCR, I found that A. salmonicida has a riboflavin biosynthesis pathway (RBP) and a riboflavin transporter. Moreover, I constructed the deletion mutants of riboflavin biosynthesis genes, their duplicated copies, and the transporter (ribN) of A. salmonicida and studied their role in virulence and potential as live-attenuated vaccine candidates using the lumpfish infection model. The results showed that riboflavin biosynthesis is crucial for A. salmonicida virulence. Overall, the thesis provided fundamental insights into the pathogenicity and virulence of R. salmoninarum and A. salmonicida and lumpfish response. The findings presented here are valuable for developing immunoprophylactic measures for lumpfish against BKD and furunculosis