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Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1–5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis. This study reports the identification of a new post-translational modification, termed ADP riboxanation, which is mediated by the Shigella effector OspC3 and inactivates the cytosolic LPS sensing pathway of caspase-4 and caspase-11.

Salmonella enterica serovar Typhimurium, an intracellular Gram-negative bacterial pathogen, employs two type III secretion systems to deliver virulence effector proteins to host cells. One such effector, SseK3, is a Golgi-targeting arginine GlcNAc transferase. Here, we show that SseK3 colocalizes with cis-Golgi via lipid binding. Arg-GlcNAc-omics profiling reveals that SseK3 modifies Rab1 and some phylogenetically related Rab GTPases. These modifications are dependent on C-termini of Rabs but independent of the GTP- or GDP-bound forms. Arginine GlcNAcylation occurs in the switch II region and the third α-helix and severely disturbs the function of Rab1. The arginine GlcNAc transferase activity of SseK3 is required for the replication of Salmonella in RAW264.7 macrophages and bacterial virulence in the mouse model of Salmonella infection. Therefore, this SseK3 mechanism of action represents a new understanding of the strategy adopted by Salmonella to target host trafficking systems., Meng, Zhuang, Peng et al. study the role of a Golgi-targeting arginine GlcNAc transferase, SseK3, in the pathogenesis of Salmonella enterica. Through R-GlcNAcylated proteome analysis, they identify Rab proteins as targets for SseK3 as well as their modification sites. They demonstrate that SseK3 GlcNAc transferase activity is required for bacterial virulence in vitro and in vivo.

Chlamydia trachomatis is the cause of several diseases such as sexually transmitted urogenital disease and ocular trachoma. The pathogen contains a small genome yet, upon infection, expresses two enzymes with deubiquitinating activity, termed ChlaDUB1 and ChlaDUB2, presumed to have redundant deubiquitinase (DUB) function because of the similarity of the primary structure of their catalytic domain. Previous studies have led to structural characterization of the enzymatic properties of ChlaDUB1; however, ChlaDUB2 has yet to be investigated thoroughly. In this study, we investigated the deubiquitinase properties of ChlaDUB2 and compared them to those of ChlaDUB1. This revealed a distinct difference in hydrolytic activity with regard to di- and polyubiquitin chains while showing similar ability to cleave a monoubiquitin-based substrate, ubiquitin aminomethylcoumarin (Ub-AMC). ChlaDUB2 was unable to cleave a diubiquitin substrate efficiently, whereas ChlaDUB1 could rapidly hydrolyze this substrate like a prototypical prokaryotic DUB, SdeA. With polyubiquitinated green fluorescent protein substrate (GFP-Ubn), whereas ChlaDUB1 efficiently disassembled the polyubiquitin chains into the monoubiquitin product, the deubiquitination activity of ChlaDUB2, while showing depletion of the substrate, did not produce appreciable levels of the monoubiquitin product. We report the structures of a catalytic construct of ChlaDUB2 and its complex with ubiquitin propargyl amide. These structures revealed differences in residues involved in substrate recognition between the two Chlamydia DUBs. On the basis of the structures, we conclude that the distal ubiquitin binding is equivalent between the two DUBs, consistent with the Ub-AMC activity result. Therefore, the difference in activity with longer ubiquitinated substrates may be due to the differential recognition of these substrates involving additional ubiquitin binding sites.

Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through UbGln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination., The Legionella effector MavC catalyses the ubiquitination of the E2 enzyme UBE2N in the host cell through a transglutamination reaction, which can be inhibited by the Legionella effector Lpg2149. Here, the authors provide mechanistic insights into these processes by determining the crystal structures of the MavC/UBE2N/Ub ternary complex, the MavC/UBE2N-Ub product complex and the MavC/Lpg2149 complex.

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila.

Parenteral vaccinations are not able to elicit effective systemic and gastrointestinal immune protection simultaneously because the lymphocytes are typically restricted to primed tissues. Although all-trans retinoic acid (atRA) was reported to trigger the gut-homing of immunocytes, the bioavailability and systemic immune responses remain limited for use in robust enteric vaccinations. Here, we show that co-delivery of atRA, CpG oligodeoxynucleotides (CpG), and antigens via engineered polymer/lipid nanoparticles (PLNPs) could exploit the amplifying function of draining lymph nodes (DLNs) for potent gut tropism and immune activations. After intramuscular injection, forming an immune-potentiated environment at the injection site, the PLNPs induced the designated transfer of primed dendritic cells (DCs) to the DLNs instead of the gastrointestinal tissues. Within the DLNs, the immune-potentiated environment markedly amplified the antigen presentation and homing receptor switch among immunocytes, which simultaneously stimulated the preferential dissipation of activated lymphocytes in the peripheral and gastrointestinal tissues, that is, exerted a DLN-amplifying effect. Compared with current atRA-containing formulations, the PLNPs not only boosted potent IgG secretions and T cell activations in the peripheral tissue but also provoked robust T cell homing and antigen-specific IgA levels in the gastrointestinal tracts in both ovalbumin and EV71 vaccinations. These data indicate that exploiting DLN amplification can stimulate potent systemic and gastrointestinal responses for more efficient enteric vaccinations.

The Legionella pneumophila effector MavC is a transglutaminase that carries out atypical ubiquitination of the host ubiquitin (Ub)‐conjugation enzyme UBE2N by catalyzing the formation of an isopeptide bond between Gln40Ub and Lys92UBE2N, which leads to inhibition of signaling in the NF‐κB pathway. In the absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self‐ubiquitination. However, the mechanisms underlying these enzymatic activities of MavC are poorly understood at the molecular level. This study reports the structure of the MavC–UBE2N–Ub ternary complex representing a snapshot of MavC‐catalyzed crosslinking of UBE2N and Ub, which reveals the way by which UBE2N–Ub binds to the Insertion and Tail domains of MavC. Based on the structural and experimental data, the catalytic mechanism for the deamidase and transglutaminase activities of MavC is proposed. Finally, by comparing the structures of MavC and MvcA, the homologous protein that reverses MavC‐induced UBE2N ubiquitination, several essential regions and two key residues (Trp255MavC and Phe268MvcA) responsible for their respective enzymatic activities are identified. The results provide insights into the mechanisms for substrate recognition and ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC and MvcA in terms of regulation of UBE2N ubiquitination., The pathogen Legionella pneumophila evades immune detection by deploying the transglutaminase effector MavC to ubiquitinate the host ubiquitin‐conjugating enzyme UBE2N. This work presents the structure of MavC–UBE2N–Ub ternary complex and sheds light on the molecular basis for MavC‐induced transglutamination and its mechanistic differences with its homolog MvcA, which counteracts MavC activity by deubiquitinating modified UBE2N in later phases of infection.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme’s true physiological substrate., The Legionella pneumophila effector MavC inhibits the human ubiquitin-conjugating enzyme Ube2N. Here, the authors combine NMR, X-ray crystallography and biochemical assays and show that MavC catalyses the intramolecular transglutaminase reaction between the Ube2N and Ub subunits of the Ube2N∼Ub conjugate and present the substrate- and product-bound MavC crystal structures.

In a radical departure from the classical E1–E2–E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD(+) to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD(+) analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Essentially, all cellular functions are executed by proteins. Different physiological and pathological conditions dynamically control various properties of proteins, including expression levels, post-translational modifications (PTMs), protein-protein interactions, enzymatic activity, etc. Thus far, the vast majority of proteomic efforts have been focused on quantitative profiling of protein abundance/expression and their PTMs. In this article, we review some recent exciting progress in the development of proteomic approaches to examine protein functions from perspectives other than expression levels and PTMs. Specifically, we discuss advancements in proximity-based labeling, analysis of protein termini and newly synthesized proteins, and activity-based protein profiling.

We previously examined Salmonella proteome within infected host cells and found differential expression of many proteins with defined functional roles such as metabolism or virulence. However, the precise roles of other altered proteins in Salmonella pathogenesis are largely unknown. A putative transcriptional regulator, YdcR, was highly induced intracellularly whereas barely expressed in vitro, implicating potential relevance to bacterial infection. To unveil its physiological functions, we exploited quantitative proteomics of intracellular Salmonella and found that genetic ablation of ydcR resulted in severe repression of SrfN, a known virulence factor. Immunoblotting, qRT-PCR, and β-galactosidase assays further demonstrate YdcR-dependent transcription and expression of srfN Moreover, we found physical interaction of YdcR with the promoter region of srfN, suggesting direct activation of its transcription. Importantly, a Salmonella mutant lacking ydcR was markedly attenuated in a mouse model of infection. Our findings reveal that YdcR temporally regulates the virulence factor SrfN during infection, thus contributing to Salmonella pathogenesis. Our work also highlights the utility of combining quantitative proteomics and bacterial genetics for uncovering the functional roles of transcription factors and likely other uncharacterized proteins as well.

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.

The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin‐conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K(63)‐type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N‐Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA‐UBE2N‐Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.

Salmonella Typhimurium is one of the leading causes of foodborne bacterial infection. Nevertheless, how Salmonella adapts to distinct types of host cells during infection remains poorly understood. By contrasting intracellular Salmonella proteomes from both infected macrophages and epithelial cells, we found striking proteomic signatures specific to particular types of host cells. Notably, Salmonella proteomic remodeling exhibited quicker kinetics in macrophages than in epithelial cells with respect to bacterial virulence and flagellar and chemotaxis systems. Furthermore, we unveiled high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells, which is attributable to differing intracellular levels of this amino acid. Intriguingly, we found that a defective hisG gene renders a Salmonella strain hypersensitive to histidine shortage in macrophages. Overall, our work reveals specific Salmonella adaptation mechanisms in distinct host cells, which should aid in the development of novel anti-infection strategies., Essential to bacterial pathogenesis, Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved the capacity to quickly sense and adapt to specific intracellular environment within distinct host cells. Here we examined S. Typhimurium proteomic remodeling within macrophages, allowing direct comparison with our previous studies in epithelial cells. In addition to many shared features, our data revealed proteomic signatures highly specific to one type of host cells. Notably, intracellular S. Typhimurium differentially regulates the two type III secretion systems (T3SSs) far more quickly in macrophages than in epithelial cells; bacterial flagellar and chemotaxis systems degenerate more quickly in macrophages than in HeLa cells as well. Importantly, our comparative analysis uncovered high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells. Targeted metabolomic measurements revealed markedly lower histidine levels within macrophages. Intriguingly, further functional studies established that histidine biosynthesis that is defective (due to a hisG mutation) renders the bacterium (strain SL1344) hypersensitive to intracellular shortage of this amino acid. Indeed, another S. Typhimurium strain, namely, strain 14028s, with a fully functional biosynthetic pathway exhibited only minor induction of the his operon within infected macrophages. Our work thus provided novel insights into S. Typhimurium adaptation mechanisms within distinct host cells and also provided an elegant paradigm where proteomic profiling of intracellular pathogens is utilized to discriminate specific host environments (e.g., on the basis of nutrient availability). IMPORTANCE Salmonella Typhimurium is one of the leading causes of foodborne bacterial infection. Nevertheless, how Salmonella adapts to distinct types of host cells during infection remains poorly understood. By contrasting intracellular Salmonella proteomes from both infected macrophages and epithelial cells, we found striking proteomic signatures specific to particular types of host cells. Notably, Salmonella proteomic remodeling exhibited quicker kinetics in macrophages than in epithelial cells with respect to bacterial virulence and flagellar and chemotaxis systems. Furthermore, we unveiled high levels of induction of bacterial histidine biosynthesis in macrophages but not in epithelial cells, which is attributable to differing intracellular levels of this amino acid. Intriguingly, we found that a defective hisG gene renders a Salmonella strain hypersensitive to histidine shortage in macrophages. Overall, our work reveals specific Salmonella adaptation mechanisms in distinct host cells, which should aid in the development of novel anti-infection strategies.

BartonellaT4SS effector BepC was reported to mediate internalization of bigBartonellaaggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentationinduced byc-AMP) domain and C-terminus BID (Bartonellaintracellulardelivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.

Bartonella effector proteins (named Beps) are substrates of VirB type IV secretion system for translocation into host cells evolved in Bartonella spp. Among these, BepE has been shown to protect cells from fragmentation effects triggered by other Beps and to promote in vivo dissemination of bacteria from the dermal site of inoculation to the bloodstream. Bacterial pathogens secreted effectors to modulate the interplay with host autophagy, either to combat autophagy to escape its bactericidal effect or to exploit autophagy to benefit intracellular replication. Here, we reported a distinct phenotype that selective autophagy in host cells is activated as a countermeasure, to attack BepE via conjugation with K63 polyubiquitin chain on BepE. We found that ectopic expression of Bartonella quintana BepE specifically induced punctate structures that colocalised with an autophagy marker (LC3-II) in host cells, in addition to filopodia and membrane ruffle formation. Two tandemly arranged Bartonella Intracellular Delivery (BID) domains in the BepE C-terminus, where ubiquitination of sister pairs of lysine residues was confirmed, were essential to activate host cell autophagy. Multiple polyubiquitin chain linkages of K27, K29, K33, and K63 were found to be conjugated at sites of K222 and K365 on BepE, of which K63 polyubiquitination on BepE K365 determined the selective autophagy (p62/SQSTM1 positive autophagy) independent of the PI3K pathway. Colocalisation of BepE with LAMP1 confirmed the maturation of BepE-induced autophagosomes in which BepE were targeted for degradation. Moreover, host cells employed selective autophagy to counter-attack BepE to rescue cells from BepE-induced endocytosis deficiency.

Bacterial pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium) have to cope with fluctuating oxygen levels during infection within host gastrointestinal tracts. The global transcription factor FNR (fumarate nitrate reduction) plays a vital role in the adaptation of enteric bacteria to the low oxygen environment. Nevertheless, a comprehensive profile of the FNR regulon on the proteome level is still lacking in S. Typhimurium. Herein, we quantitatively profiled S. Typhimurium proteome of an fnr-deletion mutant during anaerobiosis in comparison to its parental strain. Notably, we found that FNR represses the expression of virulence genes of Salmonella pathogenicity island 1 (SPI-1) and negatively regulates propanediol utilization by directly binding to the promoter region of the pdu operon. Importantly, we provided evidence that S. Typhimurium lacking fnr exhibited increased antibiotics susceptibility and membrane permeability as well. Furthermore, genetic deletion of fnr leads to decreased bacterial survival in a Caenorhabditis elegans infection model, highlighting an important role of this regulator in mediating host-pathogen interactions.

Posttranslational modification of key host proteins by virulence factors is an important theme in bacterial pathogenesis. A remarkable example is the reversible modifications of the small GTPase Rab1 by multiple effectors of the bacterial pathogen Legionella pneumophila. Previous studies have shown that the effector SetA, dependent on a functional glucosyltransferase domain, interferes with host secretory pathways. However, the enzymatic substrate(s) of SetA in host cells remains unknown. Here, by using cross-linking mass spectrometry we uncovered Rab1 as the target of SetA during L. pneumophila infection. Biochemical studies establish that SetA covalently attaches a glucose moiety to Thr75 within the switch II region of Rab1, inhibiting its intrinsic GTPase activity. Moreover, we found that SetA preferentially modifies the GDP-bound form of Rab1 over its GTP-associated state and the modification of Rab1 inhibits its interaction with the GDP dissociation inhibitor GDI1, allowing for Rab1 activation. Our results thus add an extra layer of regulation on Rab1 activity and provide a mechanistic understanding of its inhibition of the host secretory pathways as well as cellular toxicity.

Shigella flexneri is an important foodborne bacterial pathogen with infectious dose as low as 10–100 cells. SlyA, a transcriptional regulator of the MarR family, has been shown to regulate virulence in a closely related bacterial pathogen, Salmonella Typhimurium. However, the regulatory role of SlyA in S. flexneri is less understood. Here we applied unbiased proteomic profiling to define the SlyA regulon in S. flexneri. We found that the genetic ablation of slyA led to the alteration of 18 bacterial proteins among over 1400 identifications. Intriguingly, most down-regulated proteins (whose expression is SlyA-dependent) were associated with bacterial acid resistance such as the glutamate decarboxylation system. We further demonstrated that SlyA directly regulates the expression of GadA, a glutamate decarboxylase, by binding to the promotor region of its coding gene. Importantly, overexpression of GadA was able to rescue the survival defect of the ΔslyA mutant under acid stress. Therefore, our study highlights a major role of SlyA in controlling S. flexneri acid resistance and provides a molecular mechanism underlying such regulation as well.

The susceptibility to autoimmune diseases is affected by genetic and environmental factors. In rheumatoid arthritis (RA), the shared epitope (SE), a five-amino acid sequence motif encoded by RA-associated HLA-DRB1 alleles, is the single most significant genetic risk factor. The risk conferred by the SE is increased in a multiplicative way by exposure to various environmental pollutants, such as cigarette smoke. The mechanism of this synergistic interaction is unknown. It is worth noting that the SE has recently been found to act as a signal transduction ligand that facilitates differentiation of Th17 cells and osteoclasts in vitro and in vivo. Intriguingly, the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the xenobiotic effects of many pollutants, including tobacco combustion products, has been found to activate similar biologic effects. Prompted by these similarities, we sought to determine whether the SE and AhR signaling pathways interact in autoimmune arthritis. Here we uncovered a nuclear factor kappa B-mediated synergistic interaction between the SE and AhR pathways that leads to markedly enhanced osteoclast differentiation and Th17 polarization in vitro. Administration of AhR pathway agonists to transgenic mice carrying human SE-coding alleles resulted in a robust increase in arthritis severity, bone destruction, overabundance of osteoclasts, and IL17-expressing cells in the inflamed joints and draining lymph nodes of arthritic mice. Thus, this study identifies a previously unrecognized mechanism of gene-environment interaction that could provide insights into the well-described but poorly understood amplification of the genetic risk for RA upon exposure to environmental pollutants.

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen, often encounters phosphate (Pi) shortage both in the environment and inside host cells. To gain a global view on its physiological responses to Pi starvation, we performed proteomic profiling of S. Typhimurium upon the shift from Pi-rich to Pi-low conditions. In addition to the Pho regulon, many metabolic processes were up-regulated, such as glycolysis, pentose phosphate pathway, pyrimidine degradation, glycogen, and trehalose metabolism, allowing us to chart an overview of S. Typhimurium carbon metabolism under Pi starvation. Furthermore, proteomic analysis of a mutant lacking phoB (that encodes a key regulator of Pi shortage response) suggested that only a small subset of the altered proteins upon Pi limitation was PhoB-dependent. Importantly, we present evidence that S. Typhimurium N-acetylglucosamine catabolism was induced under Pi-limiting conditions in a PhoB-dependent manner. Immunoblotting and β-galactosidase assays demonstrated that PhoB was required for the full activation of NagB, a key enzyme of this pathway, in response to low Pi. Thus, our study reveals that N-acetylglucosamine catabolism may represent an additional PhoB-regulated pathway to tackle bacterial Pi shortage.

Objective The mechanisms underlying bone damage in rheumatoid arthritis (RA) are incompletely understood. We recently identified the shared epitope (SE), an HLA–DRB1–coded 5–amino acid sequence motif carried by the majority of RA patients as a signal transduction ligand that interacts with cell surface calreticulin and accelerates osteoclast (OC)–mediated bone damage in collagen-induced arthritis (CIA). Given the role of the SE/calreticulin pathway in arthritis-associated bone damage, we sought to determine the therapeutic targetability of calreticulin. Methods A library of backbone-cyclized peptidomimetic compounds, all carrying an identical core DKCLA sequence, was synthesized. The ability of these compounds to inhibit SE-activated signaling and OC differentiation was tested in vitro. The effect on disease severity and OC-mediated bone damage was studied by weekly intraperitoneal administration of the compounds to DBA/1 mice with CIA. Results Two members of the peptidomimetics library were found to have SE-antagonistic effects and antiosteoclast differentiation effects at picomolar concentrations in vitro. The lead mimetic compound, designated HS(4-4)c Trp, potently ameliorated arthritis and bone damage in vivo when administered in picogram doses to mice with CIA. Another mimetic analog, designated HS(3-4)c Trp, was found to lack activity, both in vitro and in vivo. The differential activity of the 2 analogs depended on minor differences in their respective ring sizes and correlated with distinctive geometry when computationally docked to the SE binding site on calreticulin. Conclusion These findings identify calreticulin as a novel therapeutic target in erosive arthritis and provide sound rationale and early structure/activity relationships for future drug design.

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis-independent delivery of proteins to live mammalian cells are reported. By using either a "glycan" tag naturally derived from glycosylated proteins or a "traceless" tag that could reversibly label native lysines on non-glycosylated proteins, followed by bioorthogonal conjugation with cell-penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their "native" functional forms with immediate bioavailability.

Particular alleles of HLA contribute to disease susceptibility and severity in many autoimmune conditions, but the mechanisms underlying these associations are often unknown. In this study, we demonstrate that the shared epitope (SE), an HLA-DRB1–coded sequence motif that is the single most significant genetic risk factor for erosive rheumatoid arthritis, acts as a signal transduction ligand that potently activates osteoclastogenesis, both in vitro and in vivo. The SE enhanced the production of several pro-osteoclastogenic factors and facilitated osteoclast (OC) differentiation in mouse and human cells in vitro. Transgenic mice expressing a human HLA-DRB1 allele that code the SE motif demonstrated markedly higher propensity for osteoclastogenesis and enhanced bone degradation capacity ex vivo. In addition, the SE enhanced the differentiation of Th17 cells expressing the receptor activator for NF-κB ligand. When the two agents were combined, IL-17 and the SE enhanced OC differentiation synergistically. When administered in vivo to mice with collagen-induced arthritis, the SE ligand significantly increased arthritis severity, synovial tissue OC abundance, and bone erosion. Thus, the SE contributes to arthritis severity by activating an OC-mediated bone-destructive pathway. These findings suggest that besides determining the target specificity of autoimmune responses, HLA molecules may influence disease outcomes by shaping the pathogenic consequences of such responses.

Estrogen sulfotransferase (SULT1E1) catalyzes the sulfonation of estrogens, which limits estrogen mitogenicity. We recently reported that SULT1E1 expression is low in preconfluent MCF10A human breast epithelial cells but increases when the cells become confluent. Pulse-chase labeling experiments with 5-bromouridine demonstrated that the confluence-mediated increase in SULT1E1 expression was due to increased mRNA synthesis. Because aryl hydrocarbon receptor (AhR) activation has been shown to suppress SULT1E1 expression and loss of cell-cell contact has been shown to activate the AhR in other cell types, we tested whether the confluence-associated changes in SULT1E1 expression were mediated by the AhR. Relative to confluent MCF10A cells, preconfluent cells had higher levels of CYP1A1 mRNA and greater activation of an AhR-responsive luciferase reporter, demonstrating that the AhR was active in the preconfluent cells. AhR and aryl hydrocarbon receptor nuclear translocator mRNA and protein levels were also higher in preconfluent than in confluent cultures. Treatment of preconfluent cells with the AhR antagonist, 3'-methoxy-4'-nitroflavone (MNF), or AhR knockdown significantly increased SULT1E1 expression. MCF10A cells stably transfected with a luciferase reporter containing ∼7 kilobases of the SULT1E1 5'-flanking region showed both MNF- and confluence-inducible luciferase expression. Preconfluent cells transiently transfected with the reporter showed both MNF treatment- and AhR knockdown-mediated luciferase induction, but mutation of a computationally predicted dioxin response element (DRE) at nucleotide (nt) -3476 did not attenuate these effects. These results demonstrate that SULT1E1 expression in MCF10A cells is transcriptionally regulated by confluence through a suppressive action of the AhR, which is not mediated through a DRE at nt -3476.

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5'-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] -6160 and -54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4alpha bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4alpha plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4alpha activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

Systems-level analyses have the capability to offer new insight into host-pathogen interactions on the molecular level. Using Salmonella infection of host epithelial cells as a model system, we previously analyzed intracellular bacterial proteome as a window into pathogens' adaptations to their host environment [Infect. Immun. 2015; J. Proteome Res. 2017]. Herein we extended our efforts to quantitatively examine protein expression of host cells during infection. In total, we identified more than 5000 proteins with 194 differentially regulated proteins upon bacterial infection. Notably, we found marked induction of host integrin signaling and glycolytic pathways. Intriguingly, up-regulation of host glucose metabolism concurred with increased utilization of glycolysis by intracellular Salmonella during infection. In addition to immunoblotting assays, we also verified the up-regulation of PARP1 in the host nucleus by selected reaction monitoring and immunofluorescence studies. Furthermore, we provide evidence that PARP1 elevation is likely specific to Salmonella infection and independent of one of the bacterial type III secretion systems. Our work demonstrates that unbiased high-throughput proteomics can be used as a powerful approach to provide new perspectives on host-pathogen interactions.

Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways.

We have recently proposed that the shared epitope (SE) may contribute to rheumatoid arthritis pathogenesis by acting as a ligand that activates proarthritogenic signal transduction events. To examine this hypothesis, in this study we characterized a novel small SE-mimetic compound, c(HS4-4), containing the SE primary sequence motif QKRAA, which was synthesized using a backbone cyclization method. The SE-mimetic c(HS4-4) compound interacted strongly with the SE receptor calreticulin, potently activated NO and reactive oxygen species production, and markedly facilitated osteoclast differentiation and function in vitro. The pro-osteoclastogenic potency of c(HS4-4) was 100,000- to 1,000,000-fold higher than the potency of a recently described linear SE peptidic ligand. When administered in vivo at nanogram doses, c(HS4-4) enhanced Th17 expansion, and in mice with collagen-induced arthritis it facilitated disease onset, increased disease incidence and severity, enhanced osteoclast abundance in synovial tissues and osteoclastogenic propensities of bone marrow–derived cells, and augmented bone destruction. In conclusion, c(HS4-4), a highly potent small SE-mimetic compound enhances bone damage and disease severity in inflammatory arthritis. These findings support the hypothesis that the SE acts as a signal transduction ligand that activates a CRT-mediated proarthritogenic pathway.

Microarrays offer a compact solution for massively parallel screening. In recent years, microarrays have branched away from the exclusive pursuit of small molecule ‘hits’ in target centric screens, towards the sophisticated dissection of disease biology and comparative profiling of cellular states. This has led to innovative and instructive ways in which the platform may be deployed, providing new-found methods with which to harness the throughput achievable. Library design and diversity continues to drive success with peptide and small molecule microarrays. Newer synthesis and immobilization strategies extend the already wide repertoire of fabrication methods available. Herein we describe the latest advances in the small molecule and peptide microarray arena, which herald even more exciting breakthroughs in the coming decade.

A 396-member peptide-hybrid small molecule microarray was fabricated to profile small molecule ligands across 15 SH2 proteins, revealing hits against Lck and Grb2.