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The Lyme disease bacterial pathogen,Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. FollowingB.burgdorferiinfection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.

The bacterial pathogen Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted to humans through an Ixodes tick vector. B. burgdorferi is able to survive in both mammalian and tick hosts through careful modulation of its gene expression. This allows B. burgdorferi to adapt to the environmental and nutritional changes that occur when it is transmitted between the two hosts. Distinct interactions between the spirochete and its host occur at every step of the enzootic cycle and dictate the ability of the spirochete to survive until the next stage of the cycle. Studying the interface between B. burgdorferi, the Ixodes tick vector, and the natural mammalian reservoirs has been made significantly more feasible through the complete genome sequences of the organisms and the advent of high throughput screening technologies. Ultimately, a thorough investigation of the interplay between the two domains (and two phyla within one domain) are necessary in order to completely understand how the pathogen is transmitted.

Chlamydia trachomatis is the most commonly reported sexually transmitted infection in the United States. The high prevalence of infection and lack of a vaccine indicate a critical knowledge gap surrounding the host's response to infection and how to effectively generate protective immunity. The immune response to C. trachomatis is complex, with cells of the adaptive immune system playing a crucial role in bacterial clearance. Here, we discuss the CD4+ and CD8+ T cell response to Chlamydia, the importance of antigen specificity and the role of memory T cells during the recall response. Ultimately, a deeper understanding of protective immune responses is necessary to develop a vaccine that prevents the inflammatory diseases associated with Chlamydia infection.

Summary The invasion of Chlamydia trachomatis, an obligate intracellular bacterium, into epithelial cells is driven by a complex interplay of host and bacterial factors. To comprehensively define the host genes required for pathogen invasion, we undertook a fluorescence-activated cell sorting (FACS)-based CRISPR screen in human cells. A genome-wide loss-of-function library was infected with fluorescent C. trachomatis and then sorted to enrich for invasion-deficient mutants. The screen identified heparan sulfate, a known pathogen receptor, as well as coatomer complex I (COPI). We found that COPI, through a previously unappreciated role, promotes heparan sulfate cell surface presentation, thereby facilitating C. trachomatis attachment. The heparan sulfate defect does not fully account for the resistance of COPI mutants. COPI also promotes the activity of the pathogen's type III secretion system. Together, our findings establish the requirement for COPI in C. trachomatis invasion and the utility of FACS-based CRISPR screening for the elucidation of host factors required for pathogen invasion., Graphical Abstract, Highlights • FACS-based CRISPR screen to identify host factors required for C. trachomatis invasion • Candidate genes comprise heparan sulfate biosynthesis, actin remodeling, and COPI • COPI regulates heparan sulfate cell surface presentation and C. trachomatis attachment • COPI is also required for efficient C. trachomatis T3SS translocation, Molecular Mechanism of Behavior; Medical Microbiology; Methodology in Biological Sciences; Cell Biology; Host-pathogen Interactions; Molecular Microbiology; Genetic Engineering; Genetic Screens

Borrelia burgdorferi, the causative agent of Lyme disease, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. Exhausted T cells are present in many long-term infections and mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Indeed, we have found that CD4+ T cells in the draining inguinal and popliteal lymph nodes, as well as the heart and ankle joint, significantly upregulate the immunoinhibitory molecule PD-1 following B. burgdorferi infection. While PD-1 expression on T cells is a strong indication of CD4+ T cell exhaustion, we have extended our findings by assessing expression of a ligand for PD-1, PD-L1, on antigen presenting cells in vitro and in vivo following B. burgdorferi infection. We have shown that not only is PD-L1 is significantly upregulated on both bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) following B. burgdorferi stimulation, but is also upregulated on macrophages and a subset of conventional dendritic cells (cDC2s) in the draining lymph nodes following in vivo infection. We are currently testing if mice deficient in either PD-1 or PD-L1 exhibit changes in ankle joint or heart inflammation compared to wild-type mice, which would suggest that the PD-1/PD-L1 axis is critical for regulating inflammation during B. burgdorferi infection. Together, these data will allow us to determine how this immune checkpoint regulates inflammation during B. burgdorferi infection. Supported by grants from the NIH (R01 AI150157), the Harvard Medical School Fairbairn Family Lyme Research Initiative, and the Tufts University School of Medicine Natalie Zucker Research Center for Women's Scholars Award.

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis-induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4+ and CD8+ T cells at later time points. To determine what draws these cells to C. trachomatis-infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis-induced pathology without exacerbating infection or transmission.

Chlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4+ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis., While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4+ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host’s ability to sense the cytokine gamma interferon (IFN-γ). However, it is unclear what role NR1 production or sensing of IFN-γ plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis. Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-γ−/−, and IFN-γR−/− NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis. We also determined that protection against infection requires production of IFN-γ from either NR1 T cells or endogenous cells, further highlighting the importance of IFN-γ in clearing C. trachomatis infection.

Antigen-specific CD4(+) T cells against Chlamydia are crucial for driving bacterial clearance and mediating protection against reinfection. Although the Chlamydia trachomatis protein Cta1 has been identified to be a dominant murine CD4(+) T cell antigen, its level of expression during the bacterial developmental cycle and precise localization within the host cell are unknown. Newly developed tools for Chlamydia genetic manipulation have allowed us to generate a C. trachomatis strain expressing a heterologous CD4(+) T cell epitope from ovalbumin (OVA) consisting of OVA residues 323 to 339 (OVA(323–339)). By tagging proteins expressed in C. trachomatis with OVA(323–339), we can begin to understand how protein expression, developmental regulation, and subcellular compartmentalization affect the potential of those proteins to serve as antigens. When OVA(323–339) was expressed as a fusion with green fluorescent protein, we found that we were able to elicit an OT-II T cell response in an antigen-dependent manner, but surprisingly, these T cells were unable to reduce bacterial burden in mice. These data suggest that the subcellular localization of antigen, the level of antigen expression, or the timing of expression within the developmental cycle of Chlamydia may play a crucial role in eliciting a protective CD4(+) T cell response.

Chlamydia trachomatis is the most commonly reported bacterial sexually transmitted infection in the United States. Modeling infection in animals can be challenging, as mice naturally clear C. trachomatis when it is deposited in the lower genital tract. However, C. trachomatis can productively infect mice when the lower genital tract is bypassed and bacteria are deposited directly into the upper genital tract via transcervical inoculation. Interestingly, the mouse-adapted Chlamydia species C. muridarum can infect mice both by transcervical inoculation and by natural ascension if introduced into the vaginal vault. In this study, we investigated whether the route of infection plays a role in the downstream immune responses to C. muridarum infection. We found that transcervical infection with C. muridarum results in higher bacterial burdens in the upper genital tract at earlier time points, correlating with levels of innate immune cells. When bacterial burdens were equivalent in intravaginally and transcervically infected mice at later time points, we observed substantially higher levels of adaptive immune cells in transcervically infected mice. Our data suggest that different routes of infection with the same organism can elicit different immune responses in the same tissue.

Memory antigen-specific CD4+ T cells against Chlamydia trachomatis are necessary for protection against secondary genital tract infection. While it is known that naïve antigen-specific CD4+ T cells can traffic to the genital tract in an antigen-specific manner, these T cells are not protective during primary infection. Here, we sought to compare the differences between memory and naïve antigen-specific CD4+ T cells in the same mouse following secondary infection using transgenic CD4+ T cells (NR1 T cells). Using RNA sequencing, we found that there were subtle but distinct differences between these two T cell populations. Naïve NR1 T cells significantly upregulated cell cycle genes and were more proliferative than memory NR1 T cells in the draining lymph node. In contrast, memory NR1 T cells were more activated than naïve NR1 T cells and were enriched in the genital tract. Together, our data provide insight into the differences between memory and naïve antigen-specific CD4+ T cells during C. trachomatis infection.

Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.

Immunopathology after Chlamydia trachomatis infection is the major cause of human suffering associated with this pathogen, yet the immune responses that drive pathology are not well defined. We demonstrate that a mucosal influx of neutrophils and CXCR3-driven CD4+ and CD8+ T cells is required for C. trachomatis pathology and does not contribute to bacterial clearance. Our study decouples immune-mediated pathology from immune-mediated protection for C. trachomatis, which will have important translational implications for the development of vaccines and CXCR3-mediated treatments.

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino 7-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.