During colonization of the cystic fibrosis airway Pseudomonas aeruginosa converts from non-mucoid to a mucoid phenotype, characterized by the production of the exopolysaccharide alginate. Alginate production has been shown to enhance survival by promoting biofilm formation, evading complement killing, and resisting phagocytosis. The mechanism by which alginate protects P. aeruginosa from phagocytosis is unclear. To investigate the role of alginate in the inhibition of phagocytosis, a human monocytic cell line (THP-1) and a murine alveolar macrophage cell line (MH-S) were used to determine the effects of alginate on macrophage binding, signaling, and phagocytosis. Phagocytosis assays using the mucoid cystic fibrosis clinical isolate FRD1, and its non-mucoid isogenic algD mutant FRD1131, revealed that alginate inhibits opsonic and non-opsonic phagocytosis. The inhibitory effect of alginate production is intrinsic to the bacteria as exogenous alginate was unable to protect non-mucoid FRD1131 from phagocytosis. Decreased binding of FRD1 compared to FRD1131 was also demonstrated by using the actin polymerization inhibitor cytochalasin D to inhibit phagocytosis. Furthermore, studies using blocking antibodies to CD11b and CD14 found that both of these receptors were important for the phagocytosis of FRD, and it is likely that these receptors are blocked by alginate. Alginate production by P. aeruginosa may reduce lipid raft formation, however, it was not found to affect acid sphingomyelinase activity, which is important for ceramide formation within the lipid raft. Decreased binding led to decreased signaling in macrophages demonstrated by reduction in level and alteration in kinetics of phosphorylation of AKT and ERK1/2 kinases. Signaling pathway inhibitors revealed that PI3K, but not MEK, activation was critical for phagocytosis of P. aeruginosa. Despite altered intracellular signaling in murine macrophages, both mucoid and non-mucoid P. aeruginosa induced similar levels of IL-8 and MIP-2 from human and murine macrophages, respectively. By understanding the pathways involved in mediating efficient phagocytosis of clinical isolates, it may be possible to develop a treatment to promote clearance by the resident alveolar macrophages. These experiments may serve as a model to evaluate the effectiveness of such treatments. This approach also provides valuable insight into previously unknown mechanisms of phagocytosis of P. aeruginosa.

This research project presents a comprehensive functional analysis of a complex prokaryotic signal transduction system and the mechanisms underlying its evolution. The chemotaxis system regulates motility in prokaryotes and is their most complex signal transduction system. The system has been extensively characterized experimentally, but recent studies have created new questions about the function and origin of this system. Comparative genomics analyses are well-suited for studying the chemotaxis system since it is present in taxonomically diverse organisms. The first aim of this project is to understand the evolutionary history of the chemotaxis system that has resulted in the diversity of chemotaxis systems that have been experimentally. The results reveal three functional families of chemotaxis systems that regulate flagellar motility, type IV pili motility, and non-motility outputs. The flagellar family shows extensive diversity with 10 conserved classes that have variable accessory proteins, and these classes show a co-evolutionary relationship with flagella. The second aim of this project is to analyze the molecular evolution of chemotaxis system components and utilize that information to predict the contact sites involved in protein-protein interactions. The analysis supports that there is evolutionary pressure at the amino acid sequence level to maintain protein-protein interactions. From this observation, a method to predict the contact sites of protein-protein interactions from sequence information alone was developed and validated by experimental and structural information.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 155-182.

Intraflagellar transport (IFT), the bi-directional movement of particles along the length of flagella, is required for flagellar assembly. The IFT particles are moved by kinesin II from the base to the tip of the flagellum, where flagellar assembly occurs. The IFT particles are then moved in the retrograde direction by cytoplasmic dynein 1b/2 to the base of the flagellum. The IFT particles of Chlamydomonas are composed of ~16 proteins, organized into complexes A and B. Alhough IFT is believed to transport cargoes into flagella, few cargoes have been identified and little is known about how the cargos are transported. To study the mechanism of IFT and how IFT is involved in flagellar assembly, this thesis focuses on two questions. 1) In addition to a heavy chain, DHC1b, and a light chain, LC8, what other proteins are responsible for the retrograde movement of IFT particles? 2) What is the specific function of an individual IFT-particle protein? To address these two questions, I screened for Chlamydomonas mutants either defective in retrograde IFT by immunofluorescence microscopy, or defective in IFT-particle proteins and D1bLIC, a dynein light intermediate chain possibly involved in retrograde IFT, by Southern blotting. I identified several mutants defective in retrograde IFT and one of them is defective in the D1bLIC gene. I also identified several mutants defective in several IFT-particle protein genes. I then focused on the mutant defective in D1bLIC and the one defective in IFT46, which was briefly reported as an IFT complex B protein. My results show that as a subunit of the retrograde IFT motor, D1bLIC is required for the stability of DHC1b and is involved in the attachment of IFT particles to the retrograde motor. The P-loop in D1bLIC is not necessary for the function of D1bLIC in retrograde IFT. My results also show that as a complex B protein, IFT46 is necessary for complex B stability and is required for the transport of outer dynein arms into flagella. IFT46 is phosphorylated in vivo and the phosphorylation is not critical for IFT46’s function in flagellar assembly.

The first type of dynein identified, axonemel dynein (Gibbons and Rowe, 1965), slides adjacent microtubules within the axoneme, generating the force necessary for ciliary and flagellar beating. The outer dynein arm is an important component of the flagellar axoneme, providing up to 60% of the force for flagellar motility. In the absence of the outer arm, cells swim with a slow-jerky motion at about 1/3rd the speed of wild-type cells, and the flagellar beat frequency is markedly reduced. Sixteen genes (ODA1-ODA16) have been identified that are required for outer arm assembly in Chlamydomonas reinhardtii. In addition, PF13, PF22, and FLA14 are required for outer dynein arm assembly, but their phenotypes are pleiotropic, suggesting that they affect additional flagellar components. Of the uncloned genes, ODA5, ODA8, and ODA10 are of particular interest because they do not encode subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC). Mutant alleles of these genes are unable to complement in temporary dikaryons, suggesting that the gene products interact with each other (Kamiya, 1988). Since the genes encoding all of the known components of the outer dynein arm and the ODA-DC have been characterized, it is of great interest to identify the gene products of these additional, uncloned ODA alleles. The first chapter provides an introduction to the Chlamydomonasflagellum, the dyneins in general, the outer dynein arm in particular, and mutations that impinge on the assembly and regulation of this important axonemal structure. The second chapter addresses the identification and isolation of genomic DNA containing the ODA5 gene. Utilizing a NIT1-tagged oda5-insertional mutant, I identified sequences flanking the site of the inserted NIT1 gene. These sequences were used to isolate wild-type genomic clones spanning the ODA5 gene. When transformed into the oda5 mutant, the wild-type clones rescued the mutant phenotype. These results demonstrated the successful isolation of the ODA5 gene. The third chapter describes the identification of the ODA5 gene and its corresponding cDNA. The rescuing genomic fragments were sequenced. Gene modeling was used to predict intron-exon splice sites. Primers to predicted exons were designed and used to obtain the ODA5 cDNA. The gene structure of Oda5 was analyzed and its predicted amino acid sequence deduced. Secondary structure predictions indicate that Oda5p is likely to contain a series of coiled-coil domains, followed by a poly-glycine sequence and a short, highly charged region. Northern analysis demonstrated that ODA5 gene expression is upregulated by deflagellation, a hallmark of many flagellar mRNAs. Data in CHAPTER IV further characterize the Oda5 protein and its association with the axoneme. Oda5p localizes to the flagellum, consistent with the enhancement in mRNA levels in response to deflagellation. Within the flagellum, Oda5p is an axonemal component that is released from the axoneme upon high salt extraction, as are the ODA-DC and the outer dynein arm. However, Oda5p does not associate with this super-complex in the high salt extract as determined by sucrose gradient sedimentation. Oda5p assembles onto the axoneme independently of the outer dynein arm and the ODA-DC,demonstrating it does not require these complexes for localization. Furthermore, Oda5p assembles onto the axoneme in the oda8, but not the oda10 mutant, demonstrating a role for the Oda10 protein in localization of Oda5p. These data provide the first biochemical evidence for an interaction between Oda5p and Oda10p. CHAPTER V reveals the discovery of a previously unrecognized phenotype exhibited in both oda5 and oda10 mutant strains: a defect in the assembly of a previously unknown flagellar adenylate kinase (AK). The protein levels of this flagellar AK are reduced in oda5 mutant axonemes, as determined by quantitative mass spectrometry. Direct enzymatic assays confirmed a reduction in AK activity in both oda5 and oda10 mutant axonemes, providing a second line of biochemical evidence supporting a complex containing Oda5p and OdalOp. The sequence of the flagellar AK gene and its cDNA were determined. CHAPTER VI details our efforts to identify the ODA10 gene. Genomic clones were isolated, which contain sequences at, or near, the ODA10 locus. Analysis of the genomic clones yielded no insights into the identity of the ODA10 gene. The inability of these clones to rescue the Oda10-motility phenotype indicates that these clones most likely do not contain an intact ODA10 gene. And lastly, CHAPTER VII discusses future experimentation that can be done based on the data provided by the current study.

No description available.

Serratia sp. ATCC 39006 (S39006) is known for producing carbapenem and prodiginine antibiotics; 1-carbapen-2-em-3-carboxylic acid (car) and prodigiosin. It displays different motility mechanisms, such as swimming and swarming aided by flagellar rotation and biosurfactant production. In addition, S39006 produces gas vesicles to float in aqueous environments and enable colonization of air-liquid interfaces. Gas vesicles are thought to be constructed solely from proteins expressed from a gene cluster composed of two contiguous operons, gvpA1-gvpY and gvrA-gvrC. Prior to this study, three cognate regulators, GvrA, GvrB, and GvrC, encoded by the right hand operon were known to be essential for gas vesicle synthesis. Post-transcriptional regulators such as RsmA-rsmB were also known to be involved in the inverse regulation of gas vesicles and flagella based motility. Furthermore, gas vesicle formation, antibiotic production, and motility in S39006 were affected by cell population densities and de-repressed at high cellular densities through a quorum sensing (QS) system. The aim of this research study was to identify novel regulatory inputs to gas vesicle production. Mutants were generated by random transposon mutagenesis followed by extensive screening, then sequencing and bioinformatic identification of the corresponding mutant genes. After screening, 31 mutants and seven novel regulatory genes impacting on cell buoyancy were identified. Phenotypic and genetic analysis revealed that the mutations were pleiotropic and involved in cell morphology, ion transport and central metabolism. Two new pleiotropic regulators were characterized in detail. Mutations in an ion transporter gene (trkH) and a putative transcriptional regulator gene (floR) showed opposite phenotypic impacts on flotation, flagella-based motility and prodigiosin, whereas production of the carbapenem antibiotic was affected in the transcription regulator mutant. Gene expression assays with reporter fusions, phenotypic assays in single and double mutants, and proteomics suggested that these regulatory genes couple different physiological inputs to QS and RsmA-dependent and RsmA-independent pathways.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines. / text