<p>The <italic>Chlamydiaceae</italic> are Gram-negative, obligate intracellular bacteria that are significant pathogens of humans and animals. Intracellularly, the bacteria reside in a membrane-bound vacuole, called the inclusion, from which they manipulate host processes to create a niche optimal for survival and propagation. Acquisition of host-derived lipids is essential for chlamydial growth, yet the source of lipids and mechanisms of trafficking to the inclusion are not well-established. The inclusion avoids interaction with several classical membrane and lipid transport pathways. In a functional genomic screen to identify host modulating chlamydial proteins, our lab identified cytosolic lipid droplets (LDs) as potential target organelles of <italic>Chlamydia</italic>. LDs are postulated to function in many cellular processes, such as lipid metabolism and transport, membrane trafficking, and cell signaling; therefore, we hypothesized that LDs may be important for <italic>Chlamydia</italic> pathogenesis as a source of lipids or as a platform for regulating other cellular functions. Here, we characterize the interaction between eukaryotic LDs and the chlamydial inclusion.</p><p> We find that LDs are recruited to the <italic>Chlamydia</italic> inclusion, chlamydial infection disrupts neutral lipid homeostasis, and pharmacological prevention of LD formation inhibits chlamydial replication. <italic>Chlamydia</italic> produces proteins (Ldas) that localize with LDs in yeast and mammalian cells when transiently expressed and are exported out of the inclusion to peripheral lipid-rich structures during infection. By electron microscopy and live cell imaging, we observe the translocation of intact LDs into the <italic>Chlamydia</italic> inclusion lumen. Biochemical and microscopic analysis of LDs from infected cells reveals that LD translocation may occur at specialized subregions of the inclusion membrane. The <italic>Chlamydia</italic> Lda3 protein is implicated in LD tethering to the inclusion membrane, and displacement of the protective coat protein, ADRP, from LD surfaces. This phenomenon could provide access for lipases to the LD core for utilization by the replicating bacteria. Additionally, the functional domains of Lda3 involved in binding to LD and inclusion membranes are identified. </p><p> In these studies, we identify eukaryotic lipid droplets (LDs) as a novel target organelle important for <italic>Chlamydia</italic> pathogenesis and describe a unique mechanism of whole organelle sequestration not previously observed for bacterial pathogens. These results represent a fundamental shift in our understanding of host interactions with the chlamydial inclusion, and may represent a new area for research in the field of cellular microbiology.</p> / Dissertation

<p>When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of &#946; interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.</p> / Dissertation

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 35-42).

Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 122-146).

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Irregular screening to identify infected individuals and a lack of sterilizing immunity to C. trachomatis has led to a dramatic increase in the number of reported C. trachomatis infections over the last twenty years. Repeated infections with C. trachomatis lead to serious sequelae such as pelvic inflammatory disease and ectopic pregnancy, which can result in infertility.

Infections with the bacterial pathogen Chlamydia trachomatis are a critical public health problem. Chlamydia remains the number one cause of preventable blindness worldwide and the leading cause of bacterial sexually transmitted infections in the United States. In humans, repeat and persistent infections with Chlamydia result in severe inflammation. Inflammation in the conjunctiva can result in blindness, while inflammation in the genital tract can result in pelvic inflammatory disease, ectopic pregnancy or infertility. In order to curb the increasing incidence of Chlamydia infections worldwide it will be necessary to develop a protective vaccine that affords long-term protection and prevents pathologies. To better inform vaccine development we must understand the mechanisms that drive long-term immunity in the genital tract and elucidate critical interactions between Chlamydia and host cells to uncover potential mechanisms of immune evasion.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Chlamydia trachomatis (CT) is a pestilent infection affecting upwards of 90 million people worldwide. An efficacious vaccine is needed to control the morbidities and rising healthcare cost associated with genital CT infection. We have established that protection against chlamydia infection parallels with a high frequency of T helper Type 1 cells and the associated antibodies. The current study focuses on the induction of innate immune responses involved during Chlamydia infection by a Vibrio cholera ghost-based (VCG) vaccine vector. THP-1 cells were used for dose and kinetic experiments. HeLa cells were used for infectivity assays. Based on preliminary studies, we hypothesized that the induction of immune responses by a VCG-based vaccine involves multiple innate immune signaling. Multiplex assay was used to measure T helper Type I and Type II cytokine secretion by THP-1 monocytes (Mn) or macrophages (Mϕ). Immunostimulatory cytokine secretion was significant when both cell morphologies were pulsed with VCG or VCG/murine splenocytes. We concluded that this secretion was significant enough to compliment that which would be secreted when THP-1 cells are pulsed with Chlamydia elementary bodies alone, enhancing the innate immune response during infection. Cellular supernatants (conditioned media) containing Th1-type and Th2-type cytokines were used to culture Chlamydia-infected HeLa cell monolayers. Infected HeLa monolayers cultured in the conditioned media were significantly less infected (968 IFUs) versus HeLa monolayers cultured in Earle’s minimum essential media (16,486 IFUs; p<0.001). We concluded that factors contained in conditioned media prevent and/or significantly reduce infection by Chlamydia and the development of inclusion forming units.

The overall aim was to explore the experiences of sexual risk taking among Chlamydia Trachomatis positive (CT+), HIV positive (HIV+), and HIV tested young women and men. The specific aims were to explore, from a gender perspective, the course of events, the norms, considerations and emotions involved in sexual risk taking in CT+, explore the perception of sexual risk taking in HIV+ youth, and their understanding of why they caught HIV and look at how the Law of Communicable Diseases Act impacts their sexuality. Moreover, to investigate why young adults test for HIV, how they construct the HIV risk, and what implications testing has for them. 42 informants between 17-24 years of age were recruited from a youth clinic in Umeå and from three infection clinics for HIV patients in Sweden. In depth interviews and focus group interviews were tape-recorded, transcribed verbatim and analyzed according to a Grounded Theory approach. The finding revealed that behind sexual risk taking, there was a drive to go steady, where lust and trust guided if sex would take place. In one-night stands women were expected to be less forward compared with men. We found an uneven responsibility concerning condom use where men expected women to be "condom promoters". By catching CT, women experienced guilt, while men felt content through knowing "the source of contamination". Among the HIV+ youth, socio-cultural factors such as; lack of adult supervision, naivité, love, alcohol, drugs, the macho ideal and cultures of silence blinded the informants to the risks and made them vulnerable. By grouping narratives according to degree of consensus in sexual encounters, this demonstrated that sexual risks happened in a context of gendered power relations where the informants had varied agency. The Law of Communicable Diseases Act implied both support and burden for these HIV+ youth. A lot of responsibility was put on them and to be able to handle the infromation duty they tried to switch off lust, switch off the disease, or balance lust and obedience. Among the HIV tested youth, HIV was seen a distant threat. Many had event-driven reasons for testing for HIV; multiple partners being one. Risk zones, like bars were perceived to be a milieu that often was expected to include one-night stands. Responsibility for testing was a gendered issue; "natural" for women, while men rather escaped from responsibility and had a testing resistance. Receiving a "green card" confirmed healthiness and provided relief, and made the informants felt "clean". They could restart with new ambitious, including reconsidering risk. The findings can be used in public health and in health care sectors that work with young people. We present suggestions on how to decrease the spread of STIs: To implement how men could play an equal part in sexual and reproductive health. Promote general CT screening for men. Liberal HIV testing among both young women and men. Promote safer sex behaviour from the uninfected youth, especially focusing on men??. Consider the role of gender and social background in the context of risky behaviours. Give lots of positive rewards concerning HIV disclosure to diminish the risk for HIV transmission.