Pseudomonas aeruginosa 1244 pilin glycosylation: substrate specificity, glycan functionality, and application for vaccine development

Pseudomonas aeruginosa expresses type IV pili, which are polymeric fibrous surface appendages extending from the poles of the cell that function in adhesion and surface motility. The monomeric pilus subunit, pilin, of P. aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Work presented here identifies the pilin structures necessary for glycosylation. Results suggested that the C-terminal Ser of pilin is the major glycosylation recognition feature, and this residue cannot be substituted at its carboxyl group. While no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur. Further work was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Data suggested that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O-repeat, and structures of the remaining sugars are irrelevant. Additional research was carried out to assess the role of the P. aeruginosa 1244 pilin glycan in pathogenesis. Competition index analysis using a mouse respiratory model comparing strain 1244 and the pilO isogenic knockout strain, 1244G7 indicated that the presence of the pilin glycan allowed for significantly greater survival in the lung environment. This suggested that the pilin glycan is a significant virulence factor and may aid in the establishment of infection. As the pilin glycan and the O-subunit are structurally identical, mice were immunized with strain 1244 glycosylated pili to test if vaccination with these fibers provided O-antigen-specific protection. Using either the mouse respiratory or the thermal injury model, protection from challenge with a pilus-deficient O-antigen-producing 1244 mutant was observed. These results provide evidence that the pilin glycan stimulates a protective response that targets the O-antigen, suggesting that this system could be utilized as a means to develop numerous protective anti-gram-negative bacterial bioconjugate vaccines

The utility of soil-phosphorus equilibrium studies as an indicator of phosphate concentration in surface and ground water.

Thesis (M.S.

Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation

Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia - the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature

A Francisella tularensis Live Vaccine Strain That Improves Stimulation of Antigen-Presenting Cells Does Not Enhance Vaccine Efficacy

Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS), does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensisinvades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks