Browsing by Author "Kuehn, Margarethe J"

Cells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.

Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation.

Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.

The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt to changes and survive stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved a myriad of virulence factors including the production of an exopolysaccharide capsule, as well as secretion of degradative proteases and lipases that also function as defense mechanisms. Outer membrane vesicles (OMVs) acts as a secretion system to disseminate virulence factors and function as a general bacterial stress response to remove accumulated periplasmic waste. Despite the growing insights of the field into the potential functions of OMVs, the mechanism for formation remains to be fully elucidated. The three proposed mechanisms for OMV formation in P. aeruginosa are mediated by the Pseudomonas quinolone signal PQS, the AlgU envelope stress response pathway, and the periplasmic chaperone MucD. This report investigates how P. aeruginosa responds to sublethal physiological stressors with regards to OMV production levels and whether the proposed mechanisms for OMV formation are required for stress-induced OMV formation. We concluded that exposure to cell wall directed stressors increased OMV production and activity of the sigma factor that controls MucD expression, AlgU. AlgU was shown to be sufficient to induced OMV production upon overexpression; however, stress-induced OMV production was not dependent on activation of AlgU as vesiculation could be induced in strains lacking AlgU. Furthermore, MucD levels were not inversely proportional to OMV production under acute stress, and the ability to produce PQS was not required for OMV production. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that hydrogen peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. We also demonstrated that the ability for P. aeruginosa to sense oxidative stress via OxyR, was important for hydrogen peroxide-induced OMV production, by a yet to be determined method. Together these results demonstrate that current proposed mechanisms for OMV formation do not universally apply under all stress conditions, and that additional mechanisms for OMV formation are still to be identified and fully elucidated during acute stress in P. aeruginosa.

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.

Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.

Bacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (ΔnlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (ΔdegP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of ΔnlpAΔdegP correlated with reduced OMV production with respect to the hyprevesiculator ΔdegP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the ΔnlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.

Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.

Abstract

The envelope of Gram-negative bacteria is a tightly regulated, intricate system, and as an essential component of these species, it is often the target of antibacterial treatments. Bacteria monitor and protect their envelope through a multitude of distinct stress response pathways, each with its own specialized role in maintaining a functioning envelope. Although these response pathways are each specialized for a particular type of stress, they are co-regulated and act as a network of stress responses rather than isolated pathways. Studying these pathways, both individually and as a network, provides insight into bacterial survival and may aid the design of antimicrobials. In this work, envelope stress response pathways of Escherichia coli are explored to elucidate the activation mechanisms driving individual pathways and to describe the co-regulatory properties linking the pathways. The production of outer membrane vesicles is examined for an entire knock-out library using a new, high-throughput dot-blot method. Based on these data, novel biological pathways are implicated in the production of vesicles and the link between vesiculation and the SigmaE stress response pathway is investigated. Further experiments on the SigmaE pathway lead to the discovery of a second necessary activation signal. Finally, the CPX stress response pathway is categorized as a general envelope stress response that is suppressed by the SigmaE pathway, creating a potentially exploitable weakness in the bacteria's defense.

Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Although the temporal manner in which effectors are secreted is important for the proper manipulation of host cell functions, the mechanism remains a mystery. In this study, we provide several lines of evidence that T3S chaperones may impart coherence to effector secretion. In addition, we identified a new early T3S effector in Chlamydia. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. By defining proteins that associate with the three most abundant T3S chaperones, Slc1, Scc2 and Mcsc in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry, we identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form stable complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses and lack of C. trachomatis-induced morphological changes. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.