Figure - available from: International Journal of Molecular Sciences (IJMS)
This content is subject to copyright.
TBCB interacts with SseK1 in bacteria and host cells. (A) Bacterial lysates from strain SV7071 (14028 sseK1::3xFLAG) expressing glutathione S-transferase (GST) or GST-TBCB were prepared in NP40 buffer. Then, purified GST fusion proteins were blotted on nitrocellulose membranes, stained with Ponceau S red (left panel), and developed with monoclonal anti-FLAG (right panel). Aliquots of the original lysates were also included. The molecular weights of the marker bands (M) are indicated. (B) HeLa cells were transiently transfected with a derivative of pBABEpuro expressing SseK1-3xFLAG, a derivative of pCS2 expressing 3xHA-TBCB or cotransfected with the same plasmid and a derivative of pBABEpuro expressing SseK1-3xFLAG. NP40 lysates from 6 × 10⁶ transfected cells were subjected to immunoprecipitation (IP) with anti-FLAG monoclonal antibodies and, after washing, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and developed with monoclonal anti-HA. Total lysates from 5 × 10⁵ transfected cells are included in the blot. M, molecular weight marker. Molecular weights in kDa are indicated on the left.
Source publication
Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding part...
Similar publications
The type III secretion system (T3SS) is an important virulence factor of Gram-negative bacteria, including the genus Aeromonas , a group of aquatic bacteria capable of both mutualistic and pathogenic interactions. Aeromonas species are increasingly recognized as opportunistic human pathogens. The type strain A. schubertii ATCC 43700 encodes two dis...
The assembly of the bacterial flagellum is orchestrated by secretion of distinct early and late secretion substrates via the flagellar‐specific type‐III secretion system (fT3SS). However, how the fT3SS is able to distinguish between the different (early and late) substrate classes during flagellar assembly remains poorly understood. Here, we invest...
The iron-sulfur cluster coordinating transcription factor IscR is important for the virulence of Yersinia pseudotuberculosis and a number of other bacterial pathogens. However, the IscR regulon has not yet been defined in any organism. To determine the Yersinia IscR regulon and identify IscR-dependent functions important for virulence, we employed...
Outer membrane vesicles (OMVs) are proteoliposomes shed by Gram-negative bacteria. Their secretion is enhanced by the transition into the intra-host milieu, and OMVs play critical roles during pathogenesis. Enterohemorrhagic Escherichia coli O157 (EHEC) can cause diarrheal disease in humans, and soluble toxins including Shiga-like toxins that contr...
Ralstonia solanacearum gram-negative phytopathogenic bacterium exerts its virulence through a type III secretion system (T3SS) that translocates type III effectors (T3Es) directly into the host cells. T3E secretion is finely controlled at the posttranslational level by helper proteins, T3SS control proteins, and type III chaperones. The HpaP protei...
Citations
... Reviews submitted summarize our current knowledge on important pathogens such as Corynebacterium diphtheriae [7], Staphylococcus aureus [8,9], Legionella species [10] and Neisseria meningitidis [11], while research articles highlight specific aspects of host-pathogen interaction. Different mycobacterial pathogens [12,13], Corynebacterium species [14,15], Helicobacter pylori [16], Mycoplasma fermentans [17], rickettsiae [18] and S. enterica [19] were studied in respect to their interaction with human cell lines. In addition to other human pathogens [20][21][22][23][24], animal- [25,26] and plant-pathogenic bacteria [27][28][29][30] were investigated. ...
Microorganisms can interact with plants, animals and humans in many different ways, e [...]
... These enzymes are important to bacterial virulence because they disrupt host innate immune signaling pathways by Arg-glycosylating multiple proteins, including the FAS-associated death domain-containing protein (FADD) and the tumor necrosis factor receptor (TNFR) type 1-associated death domain protein (TRADD) 6,7 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the transcriptional regulator of cellular O 2 homeostasis Hif1α, and the tubulin-binding cofactor B (TBCB), are also glycosylation targets of some of the NleB/SseK orthologs [11][12][13][14] . Subcellular fractionation experiments facilitated the identification of the Rab GTPases Rab1, Rab5, and Rab11 as targets of SseK3 15 . ...
... Flag-SseK1 pFLAG-CTC-sseK1 10 Flag-SseK1 (HEN) pFLAG-CTC-sseK1 H244A E255A N256A 10 GST-SseK1 pET42a-sseK1 11 GST-SseK1 (HEN) pET42a-sseK1 H244A E255A N256A 10 His GFP reporter assay. A low-copy number plasmid (pHG165) carrying nagB, glmU, or glmR promoter transcriptional fusions to gfp was electroporated into Salmonella. ...
The Salmonella enterica SseK1 protein is a type three secretion system effector that glycosylates host proteins during infection on specific arginine residues with N-acetyl glucosamine (GlcNAc). SseK1 also Arg-glycosylates endogenous bacterial proteins and we thus hypothesized that SseK1 activities might be integrated with regulating the intrabacterial abundance of UPD-GlcNAc, the sugar-nucleotide donor used by this effector. After searching for new SseK1 substrates, we found that SseK1 glycosylates arginine residues in the dual repressor-activator protein NagC, leading to increased DNA-binding affinity and enhanced expression of the NagC-regulated genes glmU and glmS. SseK1 also glycosylates arginine residues in GlmR, a protein that enhances GlmS activity. This Arg-glycosylation improves the ability of GlmR to enhance GlmS activity. We also discovered that NagC is a direct activator of glmR expression. Salmonella lacking SseK1 produce significantly reduced amounts of UDP-GlcNAc as compared with Salmonella expressing SseK1. Overall, we conclude that SseK1 up-regulates UDP-GlcNAc synthesis both by enhancing the DNA-binding activity of NagC and by increasing GlmS activity through GlmR glycosylation. Such regulatory activities may have evolved to maintain sufficient levels of UDP-GlcNAc for both bacterial cell wall precursors and for SseK1 to modify other bacterial and host targets in response to environmental changes and during infection.
Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.
Pathogenic bacteria encode virulent glycosyltransferases that conjugate various glycans onto host crucial proteins, which allows adhesion to mammalian cells and modulates host cellular processes for pathogenesis. Escherichia coli NleB1, Citrobacter rodentium NleB, and Salmonella enterica SseK1/3 type III effectors fatally glycosyltransfer N-acetyl glucosamine (GlcNAc) from UDP-GlcNAc to arginine residues of death domain-containing proteins that regulate host inflammation, intra-bacterial proteins, and themselves, whose post-translational modification disrupts host immune functions and prolongs bacterial viability inside host cells. However, unlike the similar NleB1/SseK1/SseK3, E. coli NleB2 and S. enterica SseK2 show deficient GlcNAcylation and neither intra-bacterial glycosylation nor auto-glycosylation. Here, as the major factor in SseK2/NleB2 deficiency, we focused on the catalytic Asp-x-Asp (DxD) motif conserved throughout all O-/N-glycosyltransferases to coordinate Mn²⁺. All DxD motifs in apo-glycosyltransferases form Type-I-turns for binding Mn²⁺, similar to the ligand-bound DxD motif, whereas TcnA/SseK2/NleB2 DxD motifs form Asx-turns, which are unable to bind Mn²⁺. Interestingly, methionine of the NleB2 DMD motif forms triple Met–aromatic interactions, as found in age-associated diseases and tumor necrosis factor (TNF) ligand-receptor complexes. The NleB1 A222M mutation induces triple Met–aromatic interactions to steeply attenuate glycosylation activity to 3% of that in the wild type. Thus, the characteristic conformation of the DxD motif is essential for binding Mn²⁺, donors, and glycosylate targets. This explains why SseK2/NleB2 effectors with the DxD motif caged in the Asp-/Asn-turn (Asx-turn) and triple Met–aromatic interactions have lower glycosyltransferase activity than that of other fatal NleB1/SseK1/SseK3 toxins.
Intracellular bacteria have developed a multitude of mechanisms to influence the post-translational modifications (PTMs) of host proteins to pathogen advantages. The recent explosion of insights into the diversity and sophistication of host PTMs and their manipulation by infectious agents challenges us to formulate a comprehensive vision of this complex and dynamic facet of the host-pathogen interaction landscape. As new discoveries continue to shed light on the central roles of PTMs in infectious diseases, technological advances foster our capacity to detect old and new PTMs and investigate their control and impact during pathogenesis, opening new possibilities for chemical intervention and infection treatment. Here, we present a comprehensive overview of these pathogenic mechanisms and offer perspectives on how these insights may contribute to the development of a new class of therapeutics that are urgently needed to face rising antibiotic resistances.
The bacterial genus Salmonella includes a large group of food-borne pathogens that cause a variety of gastrointestinal or systemic diseases in hosts. Salmonella use several secretion devices to inject various effectors targeting eukaryotic hosts, or bacteria. In the past few years, considerable progress has been made towards understanding the structural features and molecular mechanisms of the secretion systems of Salmonella, particularly regarding their roles in host-pathogen interactions. In this review, we summarize the current advances about the main characteristics of the Salmonella secretion systems. Clarifying the roles of the secretion systems in the process of infecting various hosts will broaden our understanding of the importance of microbial interactions in maintaining human health and will provide information for developing novel therapeutic approaches.
