Sohini Mukherjee

Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation.

Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl–coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.

Antibacterial membrane attack by a pore-forming intestinal C-type lectin.

Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.

Multi-layered regulation of intestinal antimicrobial defense

Abstract.The gastrointestinal tract of mammals is heavily colonized with a complex and dynamic microbial community. To cope with this complex microbial challenge, multiple epithelial lineages, such as enterocytes and Paneth cells, elaborate a diverse repertoire of protein antibiotics. The gut antimicrobial arsenal encompasses multiple protein families, including defensins, cathelicidins, and C-type lectins. These antimicrobial peptides and proteins play a key role in protecting the host against pathogen challenge, and likely also function to limit invasion of indigenous microbes. It is becoming increasingly apparent that expression of mucosal antimicrobial defenses is tightly controlled. This occurs at multiple levels, including transcriptional regulation in response to bacterial cues, post-translational proteolytic processing, and bacterial regulation of Paneth cell degranulation. Impaired antimicrobial peptide expression has also been implicated in inflammatory bowel disease, underscoring the essential role of antimicrobial defenses in maintaining intestinal homeostasis.

Salmonella Secreted Factor L Deubiquitinase of Salmonella typhimurium Inhibits NF-κB, Suppresses IκBα Ubiquitination and Modulates Innate Immune Responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-κB activation downstream of IκBα kinases and impairs IκBα ubiquitination and degradation, but not IκBα phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IκBα degradation and ubiquitination, as well as NF-κB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-κB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.

Molecular basis for peptidoglycan recognition by a bactericidal lectin

RegIII proteins are secreted C-type lectins that kill Gram-positive bacteria and play a vital role in antimicrobial protection of the mammalian gut. RegIII proteins bind their bacterial targets via interactions with cell wall peptidoglycan but lack the canonical sequences that support calcium-dependent carbohydrate binding in other C-type lectins. Here, we use NMR spectroscopy to determine the molecular basis for peptidoglycan recognition by HIP/PAP, a human RegIII lectin. We show that HIP/PAP recognizes the peptidoglycan carbohydrate backbone in a calcium-independent manner via a conserved “EPN” motif that is critical for bacterial killing. While EPN sequences govern calcium-dependent carbohydrate recognition in other C-type lectins, the unusual location and calcium-independent functionality of the HIP/PAP EPN motif suggest that this sequence is a versatile functional module that can support both calcium-dependent and calcium-independent carbohydrate binding. Further, we show HIP/PAP binding affinity for carbohydrate ligands depends on carbohydrate chain length, supporting a binding model in which HIP/PAP molecules “bind and jump” along the extended polysaccharide chains of peptidoglycan, reducing dissociation rates and increasing binding affinity. We propose that dynamic recognition of highly clustered carbohydrate epitopes in native peptidoglycan is an essential mechanism governing high-affinity interactions between HIP/PAP and the bacterial cell wall.

VopA inhibits ATP binding by acetylating the catalytic loop of MAPK kinases

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors. One of these effectors, Vibrio outer protein A (VopA), inhibits MAPK signaling via a novel mechanism, distinct from those described for other bacterial toxins, that disrupts this signaling pathway. VopA is an acetyltransferase that potently inhibits MAPK signaling pathways not only by preventing the activation of MAPK kinases (MKKs) but also by inhibiting the activity of activated MKKs. VopA acetylates a conserved lysine found in the catalytic loop of all kinases and blocks the binding of ATP, but not ADP, on the MKKs, resulting in an inactive phosphorylated kinase. Acetylation of this conserved lysine inhibits kinase activity by a new mechanism of regulation that has not been observed previously. Identifying the target of VopA reveals a way that the reversible post-translational modification of lysine acetylation can be used to regulate the activity of an enzyme.

Antimicrobial Defense of the Intestine

The mammalian gastrointestinal tract is home to a dense community of resident bacteria and is also exposed to microorganisms from the external environment. The epithelial surface of the intestine plays a critical role in host protection by producing a diverse repertoire of antimicrobial proteins that directly kill or hinder the growth of microorganisms. Here we discuss the general principles that govern the mechanisms of action of epithelial antimicrobial proteins, regulation of antimicrobial protein expression and activity, and in vivo functions of intestinal antimicrobial proteins. We also consider how altered antimicrobial protein expression and function can contribute to disease and how these endogenous antibiotics might be harnessed for the benefit of human health.

HTLV-1-Tax and ICAM-1 act on T-cell signal pathways to polarize the microtubule-organizing center at the virological synapse

Human T-lymphotropic virus type 1 (HTLV-1) spreads directly between lymphocytes and other cells via a specialized cell-cell contact, termed the virological synapse. The formation of the virological synapse is accompanied by the orientation of the microtubule-organizing center (MTOC) in the infected T cell toward the cell contact region with the noninfected target cell. We previously demonstrated that the combination of intracellular Tax protein expression and the stimulation of the intercellular adhesion molecule-1 (ICAM-1) on the cell surface is sufficient to trigger MTOC polarization in the HTLV-1-infected T cell. However, the mechanism by which Tax and ICAM-1 cause the MTOC polarization is not fully understood. Here we show that the presence of Tax at the MTOC region and its ability to stimulate cyclic AMP-binding protein-dependent pathways are both required for MTOC polarization in the HTLV-1-infected T cell at the virological synapse. Furthermore, we show that the MTOC polarization induced by ICAM-1 engagement depends on activation of the Ras-MEK-ERK signaling pathway. Our findings indicate that efficient MTOC polarization at the virological synapse requires Tax-mediated stimulation of T-cell activation pathways in synergy with ICAM-1 cross-linking. The results also reveal differences in the signaling pathways used to trigger MTOC polarization between the immunologic synapse and the virological synapse.

Regulation of C-type lectin antimicrobial activity by a flexible N-terminal prosegment

Members of the RegIII family of intestinal C-type lectins are directly antibacterial proteins that play a vital role in maintaining host-bacterial homeostasis in the mammalian gut, yet little is known about the mechanisms that regulate their biological activity. Here we show that the antibacterial activities of mouse RegIIIγ and its human ortholog, HIP/PAP, are tightly controlled by an inhibitory N-terminal prosegment that is removed by trypsin in vivo. NMR spectroscopy revealed a high degree of conformational flexibility in the HIP/PAP inhibitory prosegment, and mutation of either acidic prosegment residues or basic core protein residues disrupted prosegment inhibitory activity. NMR analyses of pro-HIP/PAP variants revealed distinctive colinear backbone amide chemical shift changes that correlated with antibacterial activity, suggesting that prosegment-HIP/PAP interactions are linked to a two-state conformational switch between biologically active and inactive protein states. These findings reveal a novel regulatory mechanism governing C-type lectin biological function and yield new insight into the control of intestinal innate immunity.

Structural Analysis of Xanthomonas XopD Provides Insights into Substrate Specificity of Ubiquitin-like Protein Proteases

XopD (Xanthomonas outer protein D), a type III secreted effector from Xanthomonas campestris pv. vesicatoria, is a desumoylating enzyme with strict specificity for its plant small ubiquitin-like modifier (SUMO) substrates. Based on SUMO sequence alignments and peptidase assays with various plant, yeast, and mammalian SUMOs, we identified residues in SUMO that contribute to XopD/SUMO recognition. Further predictions regarding the enzyme/substrate specificity were made by solving the XopD crystal structure. By incorporating structural information with sequence alignments and enzyme assays, we were able to elucidate determinants of the rigid SUMO specificity exhibited by the Xanthomonas virulence factor XopD.