The Lyme disease spirochete Borrelia burgdorferi is unique among bacteria in its large number of lipoproteins that are encoded by a small, exceptionally fragmented, and predominantly linear genome. Peripherally anchored in either the inner or outer membrane and facing either the periplasm or the external environment, these lipoproteins assume varied roles. A prominent subset of lipoproteins functioning as the apparent linchpins of the enzootic tick-vertebrate infection cycle have been explored as vaccine targets. Yet, most of the B. burgdorferi lipoproteome has remained uncharacterized. Here, we comprehensively and conclusively localize the B. burgdorferi lipoproteome by applying established protein localization assays to a newly generated epitope-tagged lipoprotein expression library and by validating the obtained individual protein localization results using a sensitive global mass spectrometry approach. The derived consensus localization data indicate that 86 of the 125 analyzed lipoproteins encoded by B. burgdorferi are secreted to the bacterial surface. Thirty-one of the remaining 39 periplasmic lipoproteins are retained in the inner membrane, with only 8 lipoproteins being anchored in the periplasmic leaflet of the outer membrane. The localization of 10 lipoproteins was further defined or revised, and 52 surface and 23 periplasmic lipoproteins were newly localized. Cross-referencing prior studies revealed that the borrelial surface lipoproteome contributing to the hostpathogen interface is encoded predominantly by plasmids. Conversely, periplasmic lipoproteins are encoded mainly by chromosomal loci. These studies close a gap in our understanding of the functional lipoproteome of an important human pathogen and set the stage for more in-depth studies of thus-far-neglected spirochetal lipoproteins.IMPORTANCE The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two-thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and it encourages further functional exploration of its lipoproteome.KEYWORDS cell envelope, lipoproteins, localization, membrane biogenesis, membrane proteins, outer membrane, protein secretion, proteomics, spirochetes, surface proteins

Significance Spirochetal pathogens encode an abundance of lipoproteins that can provide a critical interface with the host environment. Borrelia burgdorferi , the model species for spirochetal biology, must survive an enzootic life cycle defined by fluctuations between vector (tick) and vertebrate host. While B. burgdorferi expresses over 80 surface lipoproteins—many of which likely contribute to host survival—the B. burgdorferi lipoproteome is poorly characterized. Here, we generated a platform to rapidly identify targets of B. burgdorferi surface lipoproteins and identified two paralogs that confer resistance to antibody-initiated complement killing that may promote survival in immunocompetent hosts. This work expands our understanding of complement evasion mechanisms and points toward a discovery approach for identifying host–pathogen interactions central to spirochete pathogenesis.

During episodes of acute inflammation, polymorphonuclear leukocytes (PMNs) are actively recruited to sites of inflammation or injury where they provide anti-microbial and wound-healing functions. One enzyme crucial for fulfilling these functions is myeloperoxidase (MPO), which generates hypochlorous acid from Cl − and hydrogen peroxide. The potential exists, however, that uncontrolled the extracellular generation of hypochlorous acid by MPO can cause bystander tissue damage and inhibit the healing response. Previous work suggests that the microbiota-derived tryptophan metabolites 1H-indole and related molecules ("indoles") are protective during intestinal inflammation, although their precise mechanism of action is unclear. In the present work, we serendipitously discovered that indoles are potent and selective inhibitors of MPO. Using both primary human PMNs and recombinant human MPO in a cell-free system, we revealed that indoles inhibit MPO at physiologic concentrations. Particularly, indoles block the chlorinating activity of MPO, a reliable marker for MPO-associated tissue damage, as measured by coulometric-coupled HPLC. Further, we observed direct interaction between indoles and MPO using the established biochemical techniques microscale thermophoresis and STD-NMR. Utilizing a murine colitis model, we demonstrate that indoles inhibit bystander tissue damage, reflected in decreased colon 3-chlorotyrosine and pro-inflammatory chemokine expression in vivo. Taken together, these results identify microbiota-derived indoles that acts as endogenous immunomodulatory compounds through their actions on MPO, suggesting a symbiotic association between the gut microbiota and host innate immune system. Such findings offer exciting new targets for future pharmacological intervention.

IL-38 is a recently discovered cytokine and member of the IL-1 Family. In the IL-1 Family, IL-38 is unique because the cytokine is primarily a B lymphocyte product and functions to suppress inflammation. Studies in humans with inflammatory bowel disease (IBD) suggest that IL-38 may be protective for ulcerative colitis or Crohn’s disease, and that IL-38 acts to maintain homeostasis in the intestinal tract. Here we investigated the role of endogenous IL-38 in experimental colitis in mice deficient in IL-38 by deletion of exons 1-4 in C57 BL/6 mice. Compared to WT mice, IL-38 deficient mice subjected to dextran sulfate sodium (DSS) showed greater severity of disease, more weight loss, increased intestinal permeability, and a worse histological phenotype including increased neutrophil influx in the colon. Mice lacking IL-38 exhibited elevated colonic Nlrp3 mRNA and protein levels, increased caspase-1 activation, and the concomitant increased processing of IL-1β precursor into active IL-1β. Expression of IL-1α, an exacerbator of IBD, was also upregulated. Colonic myleloperoxidase protein and Il17a, and Il17f mRNA levels were higher in the IL-38 deficient mice. Daily treatment of IL-38 deficient mice with an NLRP3 inhibitor attenuated diarrhea and weight loss during the recovery phase. These data implicate endogenous IL-38 as an anti-inflammatory cytokine that reduces DSS colitis severity. We propose that a relative deficiency of IL-38 contributes to IBD by disinhibition of the NLRP3 inflammasome.

Epithelial cells that line tissues such as the intestine serve as the primary barrier to the outside world. Epithelia provide selective permeability in the presence of a large constellation of microbes, termed the microbiota. Recent studies have revealed that the symbiotic relationship between the healthy host and the microbiota includes the regulation of cell–cell interactions at the level of epithelial tight junctions. The most recent findings have identified multiple microbial-derived metabolites that influence intracellular signaling pathways which elicit activities at the epithelial apical junction complex. Here, we review recent findings that place microbiota-derived metabolites as primary regulators of epithelial cell–cell interactions and ultimately mucosal permeability in health and disease.

Intestinal epithelial cells (IECs) exist in a metabolic state of low oxygen tension termed “physiologic hypoxia.” An important factor in maintaining intestinal homeostasis is the transcription factor hypoxia-inducible factor (HIF), which is stabilized under hypoxic conditions and mediates IEC homeostatic responses to low oxygen tension. To identify HIF transcriptional targets in IEC, chromatin immunoprecipitation (ChIP) was performed in Caco-2 IECs using HIF-1α or HIF-2α specific antibodies. ChIP-enriched DNA was hybridized to a custom promoter microarray (termed ChIP-chip). This unbiased approach identified autophagy as a major HIF-1 targeted pathway in IEC. Binding of HIF-1 to the ATG9A promoter, the only transmembrane component within the autophagy pathway, was particularly enriched by exposure of IEC to hypoxia. Validation of this ChIP-chip revealed prominent induction of ATG9A, and luciferase promoter assays identified a functional hypoxia response element upstream of the TSS. Hypoxia-mediated induction of ATG9A was lost in cells lacking HIF-1. Strikingly, we found that lentiviral-mediated knockdown of ATG9A in IECs prevents epithelial barrier formation by >95% and results in significant mislocalization of multiple tight junction (TJ) proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic abnormalities in the actin cytoskeleton, including mislocalization of the TJ binding protein vasodilator-stimulated phosphoprotein (VASP). These results implicate ATG9A as essential for multiple steps of epithelial TJ biogenesis and actin cytoskeletal regulation. Our findings have novel applicability for disorders that involve a compromised epithelial barrier and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

Inflammatory bowel disease (IBD) is a family of conditions characterized by chronic, relapsing inflammation of the gastrointestinal tract. IBD afflicts over 3 million adults in the United States and shows increasing prevalence in the Westernized world. Current IBD treatments center on modulation of the damaging inflammatory response and carry risks such as immunosuppression, while the development of more effective treatments is hampered by our poor understanding of the molecular mechanisms of IBD pathogenesis. Previous genome-wide association studies (GWAS) have demonstrated that gene variants linked to the cellular response to microorganisms are most strongly associated with an increased risk of IBD. These studies are supported by mechanistic work demonstrating that IBD-associated polymorphisms compromise the intestine’s anti-microbial defense. In this review, we summarize the current knowledge regarding IBD as a disease of defects in host–microbe interactions and discuss potential avenues for targeting this mechanism for future therapeutic development.

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn’s disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.