Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium Caulobacter crescentus to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series of C. crescentus mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations. IMPORTANCE Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. Caulobacter crescentus biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE1 subunits within the pilus are arranged in a zigzag architecture, containing an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, with comparatively weaker interactions at the rest of the inter-subunit interface. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which might facilitate their role in promoting cellular attachment. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili, providing a structural basis for understanding their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE pili consist of CupE1 subunits arranged in a zigzag architecture, with an N-terminal donor β-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, resulting in an overall flexible pilus arrangement. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which may facilitate their role in promoting cohesion between bacterial cells. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili and their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

Objective: The 5-year survival of patients with metastatic or recurrent osteosarcoma is less than 25%. There is an urgent need to identify new treatment options for this cancer. Osteosarcomas represent a promising indication for strategies that target the immune system (1). Here we identify interleukin 23 (IL23), an inflammatory cytokine, as a potential therapeutic target in osteosarcoma. Our interest in IL23 is reinforced by the recent genome-wide association study, where a locus at rs1906953 at 6p21.3 in the glutamate metabotropic receptor 4 (Grm4) gene was identified as having the strongest association with genetic susceptibility to osteosarcoma in humans (2). GRM4 activation has been associated with autoimmune diseases through suppression of inflammatory cytokines, including IL23 (3). Whether GRM4 plays an immune modulatory role in cancer development is unknown. Methods: We screened a panel of mouse genotypes deficient in immune-related genes or cells to identify those genotypes that would predispose or protect from the development of osteosarcoma using a radiation model of cancer. IL23 expression was examined in mouse and human osteosarcomas using in situ hybridization. We investigated targeting IL23 alone and in combination with chemotherapy in a preclinical transplant model of osteosarcoma. The role of Grm4 and its association with IL23 was investigated using Grm4 knockout mice and Grm4 agonists. Results: IL23 knockout mice were strikingly protected from the development of osteosarcomas with 80% of animals disease free at 2 years of age compared to tumors in 100% of wild-type mice (P<0.0001). This suggests that IL23 is oncogenic in cancer development. In both mouse and human osteosarcomas infiltrating dendritic cells were found to express this cytokine, contributing to an inflammatory immune environment. Antagonist blocking IL23 could suppress tumor growth and in preliminary studies combining IL23 antagonists with doxorubicin further synergized to suppress tumor growth. GRM4 knockout mice were found to have significantly enhanced IL23 expression compared to wild-type mice. In vitro agonists to Grm4 were found to down-regulate IL23 and are being explored further in preclinical models of osteosarcoma. Conclusion: We find that blocking IL23 suppresses the growth of primary osteosarcomas. Blocking IL23 may have more profound roles in suppressing metastatic disease and is currently being studied. These findings are important, as Ustekinumab a neutralizing antibody to IL23, is FDA approved for the treatment of plaque psoriasis. This antibody targets IL-12 p40, which blocks both IL12 and IL23, but more specific monoclonal antibodies to human IL23p19 are in development for autoimmune diseases and could be repurposed for the treatment of cancer. In addition, agonists targeting GRM4 are in development and we will further investigate whether these have utility in osteosarcoma. References Kansara M et al. Translational biology of osteosarcoma. Nat Rev Cancer 2014;14:722-35; doi:10.1038/nrc3838. Jiang C et al. GRM4 gene polymorphism is associated with susceptibility and prognosis of osteosarcoma in a Chinese Han population. Med Oncol 2014;31:50. Fallarino F et al. Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation. Nat Med 2010;16:897-902; doi:10.1038/nm.2183. This abstract is also being presented as Poster B30. Citation Format: Maya Kansara, Puiyi Pang, Nina Sulkowski, Michele Teng, Mark Smyth, David Thomas. Pivotal role of interleukin 23 in osteosarcoma development and its link with glutamate metabotropic 4: Identification of novel therapeutic targets for osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR13.