CHARACTERIZATION OF THE STRUCTURE, FUNCTION, AND PROTEIN-PROTEIN INTERACTIONS INVOLVED IN THE ASSEMBLY OF THE TYPE III SECRETION SYSTEM TIP COMPLEX AND THE TRANSLOCON OF SALMONELLA AND SHIGELLA

The type III secretion system (T3SS) is a macromolecular structure assembled by many Gram-negative bacteria in order to invade target host cells. A functional T3SS contains a syringe-like structural component known as the needle apparatus, which works in concert with an export apparatus that recognizes the cargo and an ATPase complex that energizes the transport of bacterial effector proteins. Effectors transported directly into the host cell cytoplasm modulate host cellular functions such as cytoskeletal dynamics and cellular signaling in order to enable the pathogens to invade, survive, and multiply within the host environment. Gram-negative bacteria harboring the T3SS include Salmonella, Shigella, enteropathogenic E. coli, Yersinia, Burkholderia, Pseudomonas, as well as Chlamydia. These organisms are responsible for infectious diseases in humans and pose a threat to human health worldwide. Inactivation of the T3SS, by knocking out structural or functional proteins, renders pathogens incapable of causing infection. Salmonella and Shigella are responsible for millions of cases of food-borne diarrhea annually throughout the world. In addition, large-scale food recalls due to frequent outbreaks of food poisoning has a negative impact on the food industry in the United States. No preventive vaccines are available against Salmonella and Shigella. Study of the T3SS thus has a scope for the development of strategies to combat these pathogens. The T3SS among different bacterial species share common features but also show unique structural and functional characteristics. Therefore, the T3SS provides a suitable target for the development of specific anti-infectives. The needle apparatus of the T3SS consists of a base followed by an extracellular needle. The needle is attached to a tip complex and a translocon. The tip complex serves as a platform for the assembly of the translocon that punctures a translocation pore within the host cell membrane. The tip complex is assembled from several copies of a hydrophilic tip protein and the translocon is assembled from two hydrophobic translocon proteins. This dissertation describes structural and functional studies, as well as characterization of the protein-protein interactions that are important in the assembly of the tip complex and the translocon of Salmonella and Shigella. In Salmonella, the tip complex is formed by the tip protein SipD. A translocon made up of the translocon proteins SipB and SipC is attached on the tip complex. A combination of X-ray crystallography, Nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, as well as functional assays were applied to determine the structure of the Salmonella tip protein SipD and show that the C-terminus is crucial for the function and an antiparallel β-sheet is important for the tertiary structure of SipD. The function of the T3SS can be regulated by small molecules such as bile salts. The T3SS of Salmonella is down regulated by bile salts. The tip protein SipD directly binds to the bile salt deoxycholate using an unknown mechanism. The crystal structures of SipD bound to bile salts reported herein show that the interaction between bile salts and SipD is predominantly hydrophobic. Further, deoxycholate induced the degradation of the Salmonella translocon protein SipB. These observations have led to the hypothesis that deoxycholate might interfere with the interaction between SipD and SipB leading to a down regulation of the T3SS in Salmonella. The structure of the translocon and how it is attached to the tip complex is not clear. Preliminary structural characterization of a folded, hydrophilic domain at the N-terminus of SipB was undertaken to locate two fragments of SipB within residues 82-240 and 82-226, which produced well-dispersed 2D-NMR spectra. Paramagnetic relaxation enhancement (PRE) was employed to define the interaction between SipD and the N-terminal hydrophilic domain of SipB. A region within residues Asp207-Asn283 of SipB bound to a mixed α/β region of SipD. PRE was also used to study how the Shigella tip protein IpaD binds to its needle protein MxiH in order to assemble the tip complex in Shigella. MxiH was shown to bind to the lower portion of a coiled coil region in IpaD. Secretion through the T3SS is enabled by extensive cross-talk within its components. The assembly of the needle apparatus requires polymerization of multiple copies of several different proteins. Regulation of secretion is most probably an outcome of conformational changes relayed in sequence through the needle apparatus. Work described in this dissertation shows that weak protein-protein interactions are a common theme in the assembly of the needle apparatus. Further, T3SS proteins contain discreet functional domains. For example, the coiled coil of the tip protein allows the assembly of the tip complex while the mixed α/β region attaches to the translocon. The structure of theT3SS proteins varies depending upon the role it plays. For example, the extracellular needle is assembled from a small polar protein but the translocon proteins contain both hydrophilic and hydrophobic domains. Nevertheless, further studies are required to fully appreciate certain aspects of the T3SS such as how large proteins are transported through the needle, how the T3SS switches between active and inactive states of secretion, how substrate specificity is controlled, and how effector secretion is energized. Complete understanding of theT3SS requires the determination of high resolution structures of the needle apparatus, direct binding studies analyzing how isolated components of the needle apparatus interact with each other and behave in vitro, computational modeling of larger substructures, and functional assays to test the physiological implications of these in vitro studies

Peepli [Live]: A Social Satire on Contemporary India

Films may be regarded as a powerful medium of expression that convey information on the history, civilization, philosophy, politics, education and religion of a given culture. Over decades, societal and environmental factors have been a major source of creative inspiration for filmmakers in India. Accordingly, contemporary Indian films have incorporated themes relevant to today’s times such as individual conflicts, societal hierarchy, corrupt bureaucracy, commercialization of religion, politics, educational systems, mental disorders, and terrorism among others. A powerful example of this is obtained in the film Peepli [Live] (2010) which is believed to have been produced as a response to a spate of farmer suicides that took place in India since the 1990s. It is the story of a farmer who considers committing suicide so that his family can benefit from the compensation offered by the government. The film also raises other related issues such as the general apathy of political leaders and the insensitivity of the media towards impoverished farmers. Peepli [Live] uses black humor to introduce these issues and leaves the audience with pertinent questions about current socio-political systems and the uncertainties about the nation’s future. Accordingly, the present study is devoted towards examining Peepli [Live] which remains significantly under-researched by scholars of Indian cinema. This thesis focuses on the description of three themes that have been presented in the film, namely, black humor, media and sensationalism; and gender subversion. This is achieved by drawing attention to particular elements in the film such as the background, storyline, characters,and conflicts. It is expected that this will help understand how the film was successful in highlighting rural India’s lived reality and why it has garnered tremendous national and international acclaim

Structure and Biophysics of Type III Secretion in Bacteria

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus

NMR Identification of the Binding Surfaces Involved in the Salmonella and Shigella Type III Secretion Tip-Translocon Protein-Protein Interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc

NMR Model of PrgI-SipD Interaction and its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI-SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled-coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein – the N-terminal α-helical hairpin, has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled-coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs, thus our results have wider implication in understanding other needle-tip complexes

"The fruits of independence": Satyajit Ray, Indian nationhood and the spectre of empire

Challenging the longstanding consensus that Satyajit Ray's work is largely free of ideological concerns and notable only for its humanistic richness, this article shows with reference to representations of British colonialism and Indian nationhood that Ray's films and stories are marked deeply and consistently by a distinctively Bengali variety of liberalism. Drawn from an ongoing biographical project, it commences with an overview of the nationalist milieu in which Ray grew up and emphasizes the preoccupation with colonialism and nationalism that marked his earliest unfilmed scripts. It then shows with case studies of Kanchanjangha (1962), Charulata (1964), First Class Kamra (First-Class Compartment, 1981), Pratidwandi (The Adversary, 1970), Shatranj ke Khilari (The Chess Players, 1977), Agantuk (The Stranger, 1991) and Robertsoner Ruby (Robertson's Ruby, 1992) how Ray's mature work continued to combine a strongly anti-colonial viewpoint with a shifting perspective on Indian nationhood and an unequivocal commitment to cultural cosmopolitanism. Analysing how Ray articulated his ideological positions through the quintessentially liberal device of complexly staged debates that were apparently free, but in fact closed by the scenarist/director on ideologically specific notes, this article concludes that Ray's reputation as an all-forgiving, ‘everybody-has-his-reasons’ humanist is based on simplistic or even tendentious readings of his work

Tyranny of the Beauty Myth in Joyce Carol Oates's MY SISTER, MY LOVE

Satirical in tone, Joyce Carol Oates's recent novel My Sister, My Love: The Intimate Story of Skyler Rampike presents a powerful polemic against the subjugation of contemporary women through commercialized fabrications of feminine beauty. Accordingly, the novel can be read as critique of the psychic violence suffered by women who are thrall to what Naomi Wolf described as the "beautry myth.

Promises and Perils of the Road: Female Mobility in American Women’s Road Narratives since 1970

This thesis focuses on literary road narratives published since 1970 by contemporary American woman writers to examine how the trope of female mobility, which is often viewed as a liberating and empowering experience, calls for a realistic re-evaluation such that it is interpreted as an act of tremendous courage and resilience. The trope of female road travel in American fiction, while examined by some researchers in the past as quest romances and emancipatory sagas, remains considerably unexplored with regard to the hardships and dangers that befall female travelers; and hence seeks scholarly intervention. In addition, an exploration of how fictional travelling women by crafting female alliances, adopting roles of nurturers, and resorting to emotional strength surmount the challenges posed by the road has surprisingly eluded critical attention. Accordingly, the objective of this study is to examine select American road novels which represent female mobility as acts of both subversion and identity formation and underscore how wayfaring women while exploring and expanding their consciousness also challenge and often survive the perils they face on the road. Divided into four thematic chapters, this study begins by outlining the emergence of the female wayfarer in American fiction. Having historicized the advent of the independent female quester in the road genre, this study critically examines three defining road novels published through the feminist seventies, namely, Fear of Flying, Earthly Possessions and Heading West which vocalize the emancipatory rhetoric characterizing America’s gender revolutions and advocate female mobility while also highlighting the vulnerabilities of women travelers. The discussion then proceeds to examine novels such as Housekeeping, Anywhere but Here, The Bean Trees and its sequel Pigs in Heaven that represent nurturing wayfarers and female bonding, and attest to the conservatism and maturing feminist consciousness of the eighties and nineties while showcasing the female experience of the road. Finally, this thesis examines three post-millennial road novels, namely, The Lovely Bones, Rape: A Love Story, and So Much Pretty which, while celebrating female mobility, also stridently criticize the dangers that beleaguer solo female travelers. In conclusion, this study, with the help fictional road narratives, reiterates that female journeys are not simply blissful endeavors toward deliverance but entail sacrifice, persistence and resilience

The changing politics of beauty labour in Indian cinema

This essay analyses the changing politics of beauty labour and female body image in Indian cinema. It begins by discussing how beauty, a social capital for women, is a measuring rod with which their bodies are both assessed and objectified. It claims that cinema being a visual medium demands some aesthetic capital from actors, and the beauty of leading ladies portrayed on screen often adds to the spectacle. Focusing on mainstream Bollywood films produced especially over the last two decades, this essay examines narratives upholding the beauty ideal both as a cinematic necessity and also as a plot point. Tracing developments in cinematic representations of female beauty, it then examines select postmillennial films (both mainstream Bollywood as well as regional productions) to suggest that a maturing trend in representing female bodies is emerging in Indian cinema where instead of the prettified heroine one increasingly encounters protagonists who refuse to agonize under beauty labour. Finally, it argues that owing to global debates and critical feminist interventions on female body image, a radical shift is palpable in postmillennial Indian films which showcase women who either reject or redefine the politics of beauty labour

Spatial Reconfigurations:Domesticity and Womanhood in the Works of Marilynne Robinson, Joyce Carol Oates and Anne Tyler

Home is no longer just one place. It is locations. Home is that place which enables and promotes varied and ever - changing perspectives, a place where one discovers new ways of seeing reality, frontiers of difference. One confronts and accepts dispersal and fragmentation as part of the constructi ons of a new world order that reveals more fully where we are, who we can become, an order that does not demand forgetting. — bel l hook