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 d-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.

Infection with 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-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 -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 clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating -induced pathology without exacerbating infection or transmission.

SummaryThe 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.

Arthropod-borne diseases are becoming increasingly prevalent across the globe, and an understanding of the pathogen-arthropod interface can be an important tool in control of these diseases.Ixodes ticks carry many pathogens of human importance, including Anaplasma, Babesia, Borrelia, tick-borne encephalitis virus and Powassan virus. The most researched of these pathogens is the spirochete Borrelia burgdorferi, the causative agent of Lyme disease and the most common vector-borne disease in the United States. [1][2][3] There are three important components to maintenance of the B. burgdorferi life cycle: the spirochete, the invertebrate tick vector and the vertebrate host (Figure 1).Ixodes ticks are haematophagous and take one blood meal during the larval, nymph and adult stage of their lifecycle. B. burgdorferi is not transmitted from adult ticks to eggs, so larval ticks must acquire the spirochetes from infected animals, such as birds and mice, with the first blood meal. The spirochetes will remain within the tick after feeding and moulting into the nymphal stage. A B. burgdorferi infected nymph will feed on another reservoir host and transmit the spirochetes, continuing the enzootic cycle. After another moult into the adult stage, the adult tick will typically feed on larger animals (such as deer) that may not be competent reservoir hosts for B. burgdorferi but are critical to tick mating. Ticks in the nymphal stage are primarily responsible for transmission of B. burgdorferi to humans, but humans are not important in the enzootic cycle and are considered dead-end hosts. 1The tick salivary glands and midgut play important roles in the colonization and transmission of B. burgdorferi. During acquisition from an infected host that occurs with the tick blood meal, B. burgdorferi enters the tick with blood and interacts with tick

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. IMPORTANCE 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.