The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping , a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island , tcpC and usp , all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

The Uropathogenic Specific Protein (USP) is an E. coli genotoxin nuclease primarily associated with urinary tract infections. Previous efforts in our laboratory found the USP gene in stool samples; more frequently found in stool samples from adenoma and CRC patients than in samples from healthy individuals. New efforts in our laboratory aim to find the USP protein in the same population of patient samples and this may require anti-USP antibodies, which at present are not commercially available. In this work, we report the first anti-USP monoclonal antibodies (mAbs) for the detection of USP in human samples. A total of 38 hybridoma supernatants containing mAbs were assayed by immunoblotting and biolayer light interferometry (BLI) for their ability to recognize purified USP(∆HNH). Results from BLI showed that the pair AB-A10 and AB-D5 presented the highest affinity with slow dissociation, moving forward for the development of a sandwich ELISA. Using AB-A10 as the capture antibody, and AB-D5 as the top antibody, results from the ELISA showed signals that were concentration-dependent above 6 ug/mL of USP. We also used the AB-D5 and AB-D10 in immunoblots for the detection of USP in proteins extracted from stool samples of patients with adenoma and CRC. The immunoblots reveal the detection of a 25 kDa protein that remains unidentified but is present only in some of the samples. This could be the first detection of USP protein directly in a biological sample. Efforts are underway to identify this protein band and confirm its identity as USP. Additional efforts are aimed at increasing the number of stool samples analyzed to establish whether USP protein is a risk factor for CRC or adenoma. Citation Format: Rachell Martínez-Ramírez, Valeria Toro-Díaz Toro-Díaz, Pearl Akamine, Kimberly Ruiz-Rosado, Ignacio Pino, Abel Baerga-Ortiz. Monoclonal antibodies for the detection of the uropathogenic specific protein (USP): A bacterial genotoxin linked to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 713.