Microarray-guided evaluation of the frequency, B-cell origins, and selectivity of human glycan-binding antibodies reveals new insights and novel antibodies.

The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG+ memory B cells, whereas none were isolated from naïve, early emigrant, or immature B cells. Therefore, our results indicate that certain B-cell subsets in our panel are enriched in antiglycan antibodies, and IgG+ memory B cells may be a promising source of such antibodies. Furthermore, some of the newly identified antibodies bound glycans for which there are no reported monoclonal antibodies available, and these may be useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future.

Enterotoxigenic Escherichia coli Degrades the Host MUC2 Mucin Barrier To Facilitate Critical Pathogen-Enterocyte Interactions in Human Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) isolates are genetically diverse pathological variants of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC strains are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease, and asymptomatic colonization with ETEC is common in low- to middle-income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains that produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery, suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL.

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family.