Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 Å from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall–degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.

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Acne is a dermatologic disease with a strong pathologic association with human commensal Cutibacterium acnes. Conspicuously, certain C. acnes phylotypes are associated with acne, whereas others are associated with healthy skin. Here we investigate if the evolution of a C. acnes enzyme contributes to health or acne. Two hyaluronidase variants exclusively expressed by C. acnes strains, HylA and HylB, demonstrate remarkable clinical correlation with acne or health. We show that HylA is strongly pro-inflammatory, and HylB is modestly anti-inflammatory in a murine (female) acne model. Structural and phylogenic studies suggest that the enzymes evolved from a common hyaluronidase that acquired distinct enzymatic activity. Health-associated HylB degrades hyaluronic acid (HA) exclusively to HA disaccharides leading to reduced inflammation, whereas HylA generates large-sized HA fragments that drive robust TLR2-dependent pathology. Replacing an amino acid, Serine to Glycine near the HylA catalytic site enhances the enzymatic activity of HylA and produces an HA degradation pattern intermediate to HylA and HylB. Selective targeting of HylA using peptide vaccine or inhibitors alleviates acne pathology. We suggest that the functional divergence of HylA and HylB is a major driving force behind C. acnes health- and acne- phenotype and propose targeting of HylA as an approach for acne therapy.

Introduction: Constitutive activation of NF-κB has been implicated as being contributive to cancer cell growth, drug resistance, and tumor recurrence in many cancers including breast cancer. Activation of NF-κB leads to nuclear translocation of RelA, a critical component of the NF-κB transcription factor complex, which subsequently binds to specific DNA sites and activates a multitude of genes involved in diverse cell functions. Studies show that triple-negative breast cancer (TNBC) cells possess constitutively active NF-κB and concomitantly have higher levels of nuclear localization of RelA than cytoplasmic RelA. This feature is considered to be associated with the response to chemotherapy. However, currently, there is no specific inhibitor to block nuclear translocation of RelA. Methods: A structure-based approach was used to develop a small-molecule inhibitor of RelA nuclear translocation. The interaction between this molecule and RelA was verified biophysically through isothermal titration calorimetry and microscale thermophoresis. TNBC cell lines MDA-MB-231 and MDA-MB-468 and a human TNBC xenograft model were used to verify in vitro and in vivo efficacy of the small molecule, respectively. Results: We found that the small molecule, CRL1101, bound specifically to RelA as indicated by the biophysical assays. Further, CRL1101 blocked RelA nuclear translocation in breast cancer cells in vitro, and markedly reduced breast tumor growth in a triple-negative breast cancer xenograft model. Conclusion: Our study demonstrates that CRL1101 may lead to new NF-κB-targeted therapeutics for TNBC. Further, blocking of nuclear translocation of shuttling transcription factors may be a useful general strategy in cancer drug development.