Himadri B. Thapa

Bio: Himadri B. Thapa is an academic researcher from University of Graz. The author has contributed to research in topics: Bacterial outer membrane & Enterotoxigenic Escherichia coli. The author has an hindex of 2, co-authored 4 publications receiving 5 citations.

Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake.

Abstract: Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

An Intranasal Vaccine Based on Outer Membrane Vesicles Against SARS-CoV-2.

Abstract: The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.

Outer membrane vesicles as versatile tools for therapeutic approaches

Abstract: Budding of the bacterial surface results in the formation and secretion of outer membrane vesicles, which is a conserved phenomenon observed in Gram-negative bacteria. Recent studies highlight that these sphere-shaped facsimiles of the donor bacterium's surface with enclosed periplasmic content may serve multiple purposes for their host bacterium. These include inter- and intraspecies cell–cell communication, effector delivery to target cells and bacterial adaptation strategies. This review provides a concise overview of potential medical applications to exploit outer membrane vesicles for therapeutic approaches. Due to the fact that outer membrane vesicles resemble the surface of their donor cells, they represent interesting nonliving candidates for vaccine development. Furthermore, bacterial donor species can be genetically engineered to display various proteins and glycans of interest on the outer membrane vesicle surface or in their lumen. Outer membrane vesicles also possess valuable bioreactor features as they have the natural capacity to protect, stabilize and enhance the activity of luminal enzymes. Along these features, outer membrane vesicles not only might be suitable for biotechnological applications but may also enable cell-specific delivery of designed therapeutics as they are efficiently internalized by nonprofessional phagocytes. Finally, outer membrane vesicles are potent modulators of our immune system with pro- and anti-inflammatory properties. A deeper understanding of immunoregulatory effects provoked by different outer membrane vesicles is the basis for their possible future applications ranging from inflammation and immune response modulation to anticancer therapy.

TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System

Abstract: Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

The Two Faces of Bacterial Membrane Vesicles: Pathophysiological Roles and Therapeutic Opportunities

Abstract: Bacterial membrane vesicles (MVs) are nanosized lipid particles secreted by lysis or blebbing mechanisms from Gram-negative and -positive bacteria. It is becoming increasingly evident that MVs can promote antimicrobial resistance but also provide versatile opportunities for therapeutic exploitation. As non-living facsimiles of parent bacteria, MVs can carry multiple bioactive molecules such as proteins, lipids, nucleic acids, and metabolites, which enable them to participate in intra- and interspecific communication. Although energetically costly, the release of MVs seems beneficial for bacterial fitness, especially for pathogens. In this review, we briefly discuss the current understanding of diverse MV biogenesis routes affecting MV cargo. We comprehensively highlight the physiological functions of MVs derived from human pathogens covering in vivo adaptation, colonization fitness, and effector delivery. Emphasis is given to recent findings suggesting a vicious cycle of MV biogenesis, pathophysiological function, and antibiotic therapy. We also summarize potential therapeutical applications, such as immunotherapy, vaccination, targeted delivery, and antimicrobial potency, including their experimental validation. This comparative overview identifies common and unique strategies for MV modification used along diverse applications. Thus, the review summarizes timely aspects of MV biology in a so far unprecedented combination ranging from beneficial function for bacterial pathogen survival to future medical applications.

Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles

Abstract: Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas.

Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock (INM6P.406)

Abstract: Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild type and Tlr4-deficient mice, whereas caspase 11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.

An Intranasal Vaccine Based on Outer Membrane Vesicles Against SARS-CoV-2.

Abstract: The prevailing pandemic of SARS-CoV-2 highlights the desperate need of alternative vaccine-platforms, which are safe, effective, and can be modified to carry antigens of emerging pathogens. The current SARS-CoV-2 vaccines based on mRNA and adenoviral vector technology meet some of these criteria but still face limitations regarding administration route, mass production, stability, and storage. Herein, we introduce a novel SARS-CoV-2 vaccine candidate based on bacterial outer membrane vesicles (OMVs). Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) have been genetically modified to produce increased amounts of detoxified OMVs decorated with the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein. Intranasal immunization with RBD-decorated OMVs induced not only a robust immune response against the bacterial outer membrane components but also detectable antibody titers against the Spike protein. Cell culture infection assays using a Spike-pseudotyped lentivirus confirmed the presence of SARS-CoV-2 neutralizing antibodies. Highest titers against the SARS-CoV-2 Spike protein and most potent neutralization activity were observed for an alternating immunization regimen using RBD-decorated OMVs from ETEC and V. cholerae in turn. These results highlight the versatile vaccine applications offered by OMVs via expression of heterologous antigens in the donor bacterium.