Compositions and method for reducing virulence of microorganisms

#Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 5,099 Byte ASCII (Text) file named “37593-203_ST25.TXT,” created on Feb. 8, 2022.

#The present disclosure relates to compositions and methods for reducing virulence of microorganisms. In particular, the present invention provides HMGB1 and variants and mimetics thereof for use in reducing virulence of microorganisms (e.g., in disease).

#Diseases caused or exacerbated by microorganisms affect hundreds of millions of patients each year. These diseases range from acute, life-threatening infections to chronic inflammatory diseases and result in staggering societal and economic losses across all age groups and ethnicities. The microorganisms involved in these diseases can be, for example, bacteria and many are initially normal body residents. Disease-causing microorganisms express virulence mechanisms that include appendages for attachment to tissues and secreted products such as toxins. Expression of these virulence mechanisms then leads to damage to body tissues and activation of the immune system. Both pathogenic and commensal microbes can produce virulence mechanisms and these are the major determinants of the clinical course and outcomes of diseases involving microorganisms.

#FIGS. 1A and 1B are immunofluorescence images showing that HMGB1 plays a physiologic role in gut antimicrobial defense. HMGB1 staining is shown in Carnoy's fixed colons from HMGB1f/f (wild type) mice (FIG. 1A) and HMGB 1f/f, Vil-CRE (deficient in HMGB1 only in intestinal epithelial cells) mice (FIG. 1B). The mucosal surface is denoted by the white arrow. FIG. 1C is a graph showing results of ELISA for HMGB1 protein in mucus isolated from the colon. FIG. 1D is a PCoA plot of 16S rRNA sequences in mucosal scrapings from mice of the indicated genotypes. R2 values reflect the influence of the genotype on the microbial community composition and indicate dysbiosis in the absence of HMGB1. FIGS. 1E and 1F are images of fluorescent in situ hybridization (FISH) using eubacterial probes on Carnoy's fixed sections of colons from HMGB1f/f (FIG. 1E) and HMGB1f/f, Vil-CRE mice (FIG. 1F). White lines indicate separation between microbes and host epithelium. In some areas in HMGB 1f/f, Vil-CRE mice there is essentially no separation. FIG. 1G is a graph showing results of q-PCR for the 16S rRNA gene in mucosal scrapings. FIGS. 1H and 1I are immunofluorescent images of Carnoy's-fixed colons from HMGB 1f/f (FIG. 1H) and HMGB1f/f, Vil-CRE mice (FIG. 1I) stained with an anti-fungal antibody that broadly recognizes commensal fungi found in the gut. HMGB1 deficient mice have increased numbers of fungi and the fungi are found in close proximity to the mucosal surface.

#FIG. 2A shows that the CAMo-1 motif is composed of conserved and variable (X) amino acids. FIG. 2B is the amino acid sequence of the mannose binding domain of E. coli FimH with CAMo-1 highlighted (SEQ ID NO: 4). FIG. 2C is an image of immunofluorescence microscopy of E. coli+/− FimH exposed to recombinant HMGB1 protein and stained with Syto9 to identify bacteria and anti-HMGB1 antibody. FIG. 2D is an image of a red blood cell (RBC) agglutination assay with FimH+E. coli (K12) and increasing amounts of recombinant HMGB1 (highest concentration 5 μM). HMGB1 blocks E. coli binding to RBC and prevents agglutination which leads to RBC sedimentation (appearance of a dot). FIGS. 2E and 2F are fluorescent images of CaCo2 cells were exposed to wild type untreated E. coli (K12) (FIG. 2E) or E. coli (K12) treated with recombinant human HMGB1 (FIG. 2F). FIG. 2G is a graph showing that exposure to HMGB1 significantly reduced the number of adherent bacteria. FIGS. 2H and 2I are graphs showing the results of flow cytometry of E. coli deficient in FimH (FimH-) or FimH- E. coli complemented with plasmids containing wild type FimH (FimH-WT) or FimH mutated in the conserved amino acids of CAMo-1 (FimH-Mut). Mutation of CAMo-1 significantly decreased HMGB1 binding to E. coli. Both the number of positive bacteria (FIG. 2H) and the amount of HMGB1 deposited on each bacterium (FIG. 2I) were decreased. FIGS. 2J and 2K are graphs showing results of flow cytometry on wild type (FimH+) or FimH knockout (FimH-) E. coli treated with Box A protein and stained with anti-HIS antibody.

#FIG. 3A is a graph showing the results of qRT-PCR for FimH gene expression in E. coli (K12) treated with 3 μM HMGB1 for 2 hours. Exposure to HMGB1 in vitro decreased expression of FimH. FIG. 3B is a graph showing results of qRT-PCR for E. coli FimH gene expression in mucosal scrapings from HMGB1f/f and HMGB1f/f Vil-CRE mice. Exposure to HMGB1 in vivo decreased expression of FimH. FIG. 3C is an image of immunoblotting for FimH in mucosal scrapings from HMGB1f/f and HMGB1f/f, Vil-CRE mice.

#To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

#As used herein, the term “subject” refers to organisms to be treated by the methods of embodiments of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the invention, the term “subject” generally refers to an individual who will receive or who has received treatment (e.g., administration of a protein or peptide of the present invention and optionally one or more other agents) for disease (e.g., inflammatory disease) or other condition requiring treatment.

#The term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, diagnostic assay (e.g., for disease) and the like.