TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria.

Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.

The Salmonella Typhi SPI-2 injectisome enigma.

The Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system (injectisome) is assembled following uptake of bacteria into vacuoles in mammalian cells. The injectisome translocates virulence proteins (effectors) into infected cells. Numerous studies have established the requirement for a functional SPI-2 injectisome for growth of Salmonella Typhimurium in mouse macrophages, but the results of similar studies involving Salmonella Typhi and human-derived macrophages are not consistent. It is important to clarify the functions of the S. Typhi SPI-2 injectisome, not least because an inactivated SPI-2 injectisome forms the basis for live attenuated S. Typhi vaccines that have undergone extensive trials in humans. Intracellular expression of injectisome genes and effector delivery take longer in the S. Typhi/human macrophage model than for S. Typhimurium and we propose that this could explain the conflicting results. Furthermore, strains of both S. Typhimurium and S. Typhi contain intact genes for several 'core' effectors. In S. Typhimurium these cooperate to regulate the vacuole membrane and contribute to intracellular bacterial replication; similar functions are therefore likely in S. Typhi.

Speaking the host language: how Salmonella effector proteins manipulate the host.

Salmonella injects over 40 virulence factors, termed effectors, into host cells to subvert diverse host cellular processes. Of these 40 Salmonella effectors, at least 25 have been described as mediating eukaryotic-like, biochemical post-translational modifications (PTMs) of host proteins, altering the outcome of infection. The downstream changes mediated by an effector's enzymatic activity range from highly specific to multifunctional, and altogether their combined action impacts the function of an impressive array of host cellular processes, including signal transduction, membrane trafficking, and both innate and adaptive immune responses. Salmonella and related Gram-negative pathogens have been a rich resource for the discovery of unique enzymatic activities, expanding our understanding of host signalling networks, bacterial pathogenesis as well as basic biochemistry. In this review, we provide an up-to-date assessment of host manipulation mediated by the Salmonella type III secretion system injectosome, exploring the cellular effects of diverse effector activities with a particular focus on PTMs and the implications for infection outcomes. We also highlight activities and functions of numerous effectors that remain poorly characterized.

A Role for the RNA Polymerase Gene Specificity Factor σ54 in the Uniform Colony Growth of Uropathogenic Escherichia coli.

The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54's role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow. IMPORTANCE RNA synthesis is the first step of gene expression. The multisubunit RNA polymerase (RNAP) is the central enzyme responsible for RNA synthesis in bacteria. The dissociable sigma (σ) factor subunit directs the RNAP to different sets of genes to allow their expression in response to various cellular needs. Of the seven σ factors in Escherichia coli and related bacteria, σ54 exists in a class of its own. This study has uncovered that σ54 is a determinant of the uniform growth of uropathogenic E. coli on solid media. This finding suggests a role for this σ54 in gene regulation that extends beyond its known function as an RNAP gene specificity factor.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcus pneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria.

Cell-autonomous innate immune responses against bacteria attempting to colonize the cytosol of mammalian cells are incompletely understood. Polyubiquitylated proteins can accumulate on the surface of such bacteria, and bacterial growth is restricted by Tank-binding kinase (TBK1). Here we show that NDP52, not previously known to contribute to innate immunity, recognizes ubiquitin-coated Salmonella enterica in human cells and, by binding the adaptor proteins Nap1 and Sintbad, recruits TBK1. Knockdown of NDP52 and TBK1 facilitated bacterial proliferation and increased the number of cells containing ubiquitin-coated salmonella. NDP52 also recruited LC3, an autophagosomal marker, and knockdown of NDP52 impaired autophagy of salmonella. We conclude that human cells utilize the ubiquitin system and NDP52 to activate autophagy against bacteria attempting to colonize their cytosol.

Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy.

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti-bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella-associated "eat-me" signals, including host-derived glycans and K48- and K63-linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.

Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3.

The Salmonella-secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.

Methylthioadenosine Suppresses Salmonella Virulence.

In order to deploy virulence factors at appropriate times and locations, microbes must rapidly sense and respond to various metabolite signals. Previously, we showed a transient elevation of the methionine-derived metabolite methylthioadenosine (MTA) concentration in serum during systemic Salmonella enterica serovar Typhimurium infection. Here we explored the functional consequences of increased MTA concentrations on S Typhimurium virulence. We found that MTA, but not other related metabolites involved in polyamine synthesis and methionine salvage, reduced motility, host cell pyroptosis, and cellular invasion. Further, we developed a genetic model of increased bacterial endogenous MTA production by knocking out the master repressor of the methionine regulon, metJ Like MTA-treated S Typhimurium, the ΔmetJ mutant displayed reduced motility, host cell pyroptosis, and invasion. These phenotypic effects of MTA correlated with suppression of flagellar and Salmonella pathogenicity island 1 (SPI-1) networks. S Typhimurium ΔmetJ had reduced virulence in oral and intraperitoneal infection of C57BL/6J mice independently of the effects of MTA on SPI-1. Finally, ΔmetJ bacteria induced a less severe inflammatory cytokine response in a mouse sepsis model. Together, these data indicate that exposure of S Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway suppresses S Typhimurium virulence.

Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion.

Autophagy defends the mammalian cytosol against bacterial infection. Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by polyubiquitin coating. Here we show in human cells that galectin 8 (also known as LGALS8), a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin 8 monitors endosomal and lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 (also known as CALCOCO2), galectin 8 activates antibacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Because galectin 8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest that galectin 8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin 8 to combat infection by monitoring endosomal and lysosomal integrity on the basis of the specific lack of complex carbohydrates in the cytosol.

SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages.

Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of N-acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3.

Bacterial esterases reverse lipopolysaccharide ubiquitylation to block host immunity.

Aspects of how Burkholderia escape the host's intrinsic immune response to replicate in the cell cytosol remain enigmatic. Here, we show that Burkholderia has evolved two mechanisms to block the activity of Ring finger protein 213 (RNF213)-mediated non-canonical ubiquitylation of bacterial lipopolysaccharide (LPS), thereby preventing the initiation of antibacterial autophagy. First, Burkholderia's polysaccharide capsule blocks RNF213 association with bacteria and second, the Burkholderia deubiquitylase (DUB), TssM, directly reverses the activity of RNF213 through a previously unrecognized esterase activity. Structural analysis provides insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from its isopeptidase function. Furthermore, a putative TssM homolog also displays esterase activity and removes ubiquitin from LPS, establishing this as a virulence mechanism. Of note, we also find that additional immune-evasion mechanisms exist, revealing that overcoming this arm of the host's immune response is critical to the pathogen.

Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression.

Non-typhoidal Salmonella enterica cause an estimated 1 million cases of gastroenteritis annually in the United States. These serovars use secreted protein effectors to mimic and reprogram host cellular functions. We previously discovered that the secreted effector SarA (Salmonella anti-inflammatory response activator; also known as SteE) was required for increased intracellular replication of S. Typhimurium and production of the anti-inflammatory cytokine interleukin-10 (IL-10). SarA facilitates phosphorylation of STAT3 through a region of homology with the host cytokine receptor gp130. Here, we demonstrate that a single amino acid difference between SarA and gp130 is critical for the anti-inflammatory bias of SarA-STAT3 signaling. An isoleucine at the pY+1 position of the YxxQ motif in SarA (which binds the SH2 domain in STAT3) causes increased STAT3 phosphorylation and expression of anti-inflammatory target genes. This isoleucine, completely conserved in ~4000 Salmonella isolates, renders SarA a better substrate for tyrosine phosphorylation by GSK-3. GSK-3 is canonically a serine/threonine kinase that nonetheless undergoes tyrosine autophosphorylation at a motif that has an invariant isoleucine at the pY+1 position. Our results provide a molecular basis for how a Salmonella secreted effector achieves supraphysiological levels of STAT3 activation to control host genes during infection.

Ready, STAT3, Go! Bacteria in the race for M2 macrophage polarisation.

Despite macrophages representing professional immune cells that are integral to the host defences against microbial threats, several intracellular bacteria not only infect, but survive, replicate and often persist in these cells. This is perhaps possible because not all macrophages are the same. Instead, macrophages are loosely divided into two classes: the M1 'classically activated' pro-inflammatory subset and the M2 'alternatively activated' cells that are generally anti-inflammatory and infection-permissive. In this review, we summarise recent findings explaining how several intracellular pathogens, often using secreted effectors, rewire host circuitry in favour of an anti-inflammatory niche. A common theme is the phosphorylation and activation of the signal transducer and activator of transcription-3 (STAT3) transcription factor. We describe and compare the diverse mechanisms employed and reflect how such non-canonical processes may have evolved to circumvent regulation by the host, providing a potent means by which different pathogens manipulate the cells they infect.

Membrane properties modulation by SanA: implications for xenobiotic resistance in Salmonella Typhimurium.

Introduction: Multidrug resistance in bacteria is a pressing concern, particularly among clinical isolates. Gram-negative bacteria like Salmonella employ various strategies, such as altering membrane properties, to resist treatment. Their two-membrane structure affects susceptibility to antibiotics, whereas specific proteins and the peptidoglycan layer maintain envelope integrity. Disruptions can compromise stability and resistance profile toward xenobiotics. In this study, we investigated the unexplored protein SanA's role in modifying bacterial membranes, impacting antibiotic resistance, and intracellular replication within host cells. Methods: We generated a sanA deletion mutant and complemented it in trans to assess its biological function. High-throughput phenotypic profiling with Biolog Phenotype microarrays was conducted using 240 xenobiotics. Membrane properties and permeability were analyzed via cytochrome c binding, hexadecane adhesion, nile red, and ethidium bromide uptake assays, respectively. For intracellular replication analysis, primary bone marrow macrophages served as a host cells model. Results: Our findings demonstrated that the absence of sanA increased membrane permeability, hydrophilicity, and positive charge, resulting in enhanced resistance to certain antibiotics that target peptidoglycan synthesis. Furthermore, the sanA deletion mutant demonstrated enhanced replication rates within primary macrophages, highlighting its ability to evade the bactericidal effects of the immune system. Taking together, we provide valuable insights into a poorly known SanA protein, highlighting the complex interplay among bacterial genetics, membrane physiology, and antibiotic resistance, underscoring its significance in understanding Salmonella pathogenicity.

Dedicated bacterial esterases reverse lipopolysaccharide ubiquitylation to block immune sensing.

Pathogenic bacteria have evolved diverse mechanisms to counteract cell-autonomous immunity, which otherwise guards both immune and non-immune cells from the onset of an infection1,2. The versatile immunity protein Ring finger protein 213 (RNF213)3-6 mediates the non-canonical ester-linked ubiquitylation of lipopolysaccharide (LPS), marking bacteria that sporadically enter the cytosol for destruction by antibacterial autophagy4. However, whether cytosol-adapted pathogens are ubiquitylated on their LPS and whether they escape RNF213-mediated immunity, remains unknown. Here we show that Burkholderia deubiquitylase (DUB), TssM7-9, is a potent esterase that directly reverses the ubiquitylation of LPS. Without TssM, cytosolic Burkholderia became coated in polyubiquitin and autophagy receptors in an RNF213-dependent fashion. Whereas the expression of TssM was sufficient to enable the replication of the non-cytosol adapted pathogen Salmonella, we demonstrate that Burkholderia has evolved a multi-layered defence system to proliferate in the host cell cytosol, including a block in antibacterial autophagy10-12. Structural analysis provided insight into the molecular basis of TssM esterase activity, allowing it to be uncoupled from isopeptidase function. TssM homologs conserved in another Gram-negative pathogen also reversed non-canonical LPS ubiquitylation, establishing esterase activity as a bacterial virulence mechanism to subvert host cell-autonomous immunity.

Dorzolamide suppresses PKCδ -TIRAP-p38 MAPK signaling axis to dampen the inflammatory response.

Background: Sepsis is a syndrome due to microbial infection causing impaired multiorgan function. Its underlying cause is immune dysfunction and macrophages play an essential role. Methods: TIRAP interaction with PKCδ in macrophage was studied, revealing downstream signaling by Western blot and quantitative reverse transcriptase PCR. Dorzolamide (DZD) disrupting TIRAP-PKCδ interaction was identified by virtual screening and validated in vitro and in septic mice. Results: The study highlights the indispensable role of TIRAP-PKCδ in p38 MAPK-activation, NF-κB- and AP-1-mediated proinflammatory cytokines expression, whereas DZD significantly attenuated the signaling. Conclusion: Targeting TIRAP-PKCδ interaction by DZD is a novel therapeutic approach for treating sepsis.