Browsing by Subject "UTI"

Urinary tract infections (UTIs) are typically caused by bacteria that colonize different regions of the urinary tract, mainly the bladder and the kidney. Approximately 25% of women that suffer from UTIs experience a recurrent infection within 6 months of the initial bout, making UTIs a serious economic burden resulting in more than 10 million hospital visits and $3.5 billion in healthcare costs in the United States alone. Type-1 fimbriated Uropathogenic E. coli (UPEC) is the major causative agent of UTIs, accounting for almost 90 % of bacterial UTIs. The unique ability of UPEC to bind and invade the superficial bladder epithelium allows the bacteria to persist inside epithelial niches and survive antibiotic treatment. Persistent, intracellular UPEC are retained in the bladder epithelium for long periods, making them a source of recurrent UTIs. Hence, the ability of UPEC to persist in the bladder is a matter of major health and economic concern, making studies exploring the underlying mechanism of UPEC persistence highly relevant.

In my thesis, I will describe how intracellular Uropathogenic E.coli (UPEC) evade host defense mechanisms in the superficial bladder epithelium. I will also describe some of the unique traits of persistent UPEC and explore strategies to induce their clearance from the bladder. I have discovered that the UPEC virulence factor Alpha-hemolysin (HlyA) plays a key role in the survival and persistence of UPEC in the superficial bladder epithelium. In-vitro and in-vivo studies comparing intracellular survival of wild type (WT) and hemolysin deficient UPEC suggested that HlyA is vital for UPEC persistence in the superficial bladder epithelium. Further in-vitro studies revealed that hemolysin helped UPEC persist intracellularly by evading the bacterial expulsion actions of the bladder cells and remarkably, this virulence factor also helped bacteria avoid t degradation in lysosomes.

To elucidate the mechanistic basis for how hemolysin promotes UPEC persistence in the urothelium, we initially focused on how hemolysin facilitates the evasion of UPEC expulsion from bladder cells. We found that upon entry, UPEC were encased in “exocytic vesicles” but as a result of HlyA expression these bacteria escaped these vesicles and entered the cytosol. Consequently, these bacteria were able to avoid expulsion by the cellular export machinery.

Since bacteria found in the cytosol of host cells are typically recognized by the cellular autophagy pathway and transported to the lysosomes where they are degraded, we explored why this was not the case here. We observed that although cytosolic HlyA expressing UPEC were recognized and encased by the autophagy system and transported to lysosomes, the bacteria appeared to avoid degradation in these normally degradative compartments. A closer examination of the bacteria containing lysosomes revealed that they lacked V-ATPase. V-ATPase is a well-known proton pump essential for the acidification of mammalian intracellular degradative compartments, allowing for the proper functioning of degradative proteases. The absence of V-ATPase appeared to be due to hemolysin mediated alteration of the bladder cell F-actin network. From these studies, it is clear that UPEC hemolysin facilitates UPEC persistence in the superficial bladder epithelium by helping bacteria avoid expulsion by the exocytic machinery of the cell and at the same time enabling the bacteria avoid degradation when the bacteria are shuttled into the lysosomes.

Interestingly even though UPEC appear to avoid elimination from the bladder cell their ability to multiple in bladder cells seem limited.. Indeed, our in-vitro and in-vivo experiments reveal that UPEC survive in superficial bladder epithelium for extended periods of time without a significantly change in CFU numbers. Indeed, we observed these bacteria appeared quiescent in nature. This observation was supported by the observation that UPEC genetically unable to enter a quiescence phase exhibited limited ability to persist in bladder cells in vitro and in vivo, in the mouse bladder.

The studies elucidated in this thesis reveal how UPEC toxin, Alpha-hemolysin plays a significant role in promoting UPEC persistence via the modulation of the vesicular compartmentalization of UPEC at two different stages of the infection in the superficial bladder epithelium. These results highlight the importance of UPEC Alpha-hemolysin as an essential determinant of UPEC persistence in the urinary bladder.

Short peptides are poorly immunogenic when delivered sublingually – under the tongue. This challenge has prevented widespread investigation into sublingual peptide vaccines, leaving their considerable potential untapped. Sublingual immunization is logistically favorable due to its ease of administration and can raise both strong systemic immune responses and mucosal responses in tissues throughout the body. Peptide epitopes are highly specific, allowing for the generation of immune responses directed solely against precisely selected targets, without accompanying off-target antibody or T-cell production. To enable sublingual peptide immunization, we designed a strategy based on molecular self-assembly of epitopes within nanofibers. We then extract and expound several key implications from this finding, including insights into the mechanism of action, development of a translatable administration modality, and application to a currently unmet clinical need.

The design of our sublingual peptide immunization strategy is described in the first part of this thesis (Chapter 3). We sought to utilize nanomaterial delivery of peptides to enhance sublingual immunogenicity, a strategy that has proved successful in several other immunization routes. However, the salivary mucus layer is a significant barrier to nanomaterial delivery, particularly for supramolecular materials, due to its ability to entrap and clear these materials rapidly. We designed β-sheet nanofibers conjugated at a high-density to the mucus-inert polymer polyethylene glycol (PEG) to shield them from the mucus layer. Strikingly, sublingually delivered PEGylated peptide nanofibers (PEG-Q11) raised extremely durable antibody and T-cell responses against peptide epitopes when mixed with a mucosal adjuvant. We showed that PEG decreases nanofiber interactions with mucin in vitro, and extends the residence time of nanofibers at the sublingual space in vivo. Further, we showed that we could achieve similar results by adapting the use of PASylation (modification with peptide sequences rich in Pro, Ala, and Ser) to mucosal delivery.

In the second part of this thesis (Chapter 4) we designed a supramolecular strategy for enhancing sublingual nanofiber immunization. Mucosal adjuvants, such as cyclic-di-nucleotides (CDNs), can promote sublingual immune responses but must be co-delivered with the antigen to the epithelium for maximum effect. We designed peptide-polymer nanofibers displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We co-assembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. However, intermediate levels of R9 within sublingually-administered PEG-Q11 fibers were optimal for sublingual immunization, suggesting a balance between polyarginine’s ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings reveal important design considerations for the continuing development of sublingual peptide nanofiber vaccines.

We sought to enhance the translational capacity of our immunization strategy by designing a highly accessible vaccine method (described in Chapter 5). Significant barriers exist to improving vaccine coverage in lower- and middle-income countries, including the costly requirements for cold-chain distribution and trained medical personnel to administer the vaccines. To address these barriers, we built upon our sublingual nanofiber platform to design a heat-stable and highly porous tablet vaccine that can be administered via simple dissolution under the tongue. We produced SIMPL (Supramolecular Immunization with Peptides SubLingually) tablet vaccines by freeze-drying a mixture of self-assembling peptide-polymer nanofibers, sugar excipients, and adjuvant. We showed that even after heating for 1 week at 45 °C, SIMPL tablets could raise antibody responses against a peptide epitope from M. tuberculosis, in contrast to a conventional carrier vaccine (KLH) which lost sublingual efficacy after heating. Our approach directly addresses the need for a heat-stable and easily deliverable vaccine to improve equity in global vaccine coverage.

To demonstrate the clinical usefulness of our technology, we designed a sublingual vaccine against uropathogenic E. coli (UPEC), the pathogen that causes most urinary tract infections (UTIs). Sublingual peptide immunization is uniquely advantageous for immunization against UTIs, as sublingual immunization raises antibodies in the blood and urinary tract, and peptide epitopes allow for targeting UPEC without perturbing commensals. We co-assembled PEG-Q11 nanofibers containing three different B-cell epitopes from UPEC along with a helper T-cell epitope, allowing us to raise simultaneous antibody responses against three targets systemically and in the urinary tract. These antibodies were highly specific for UPEC, exhibiting no binding to a non-pathogenic strain. Further, these antibodies demonstrated clinical potential by protecting mice from an intraperitoneal UPEC challenge. Finally, we showed the ability to use SIMPL tablets to raise anti-UPEC responses in rabbits, which contain an oral cavity with key similarities to humans.

This thesis demonstrates a critical enabling technology for sublingual peptide immunization and builds upon this technology to report findings with key implications for supramolecular biomaterial design, accessible vaccination, and treatment of UPEC-mediated diseases. This interdisciplinary research synthesizes and utilizes knowledge from materials science, immunology, vaccinology, pharmaceutical sciences, and pathology to present an important original contribution to the field of immune engineering.