scholarly journals Global Lysine Crotonylation Alterations of Host Cell Proteins Caused by Brucella Effector BspF

2021 ◽  
Vol 10 ◽  
Author(s):  
Jinying Zhu ◽  
Qiao Dong ◽  
Changpeng Dong ◽  
Xi Zhang ◽  
Huan Zhang ◽  
...  
Keyword(s):  
Type Iv Secretion ◽  
Proteomics Data ◽  
Host Proteins ◽  
Activity Data ◽  
Acetyltransferase Activity ◽  
Type Iv ◽  
Host Cell Proteins ◽  
Host Innate Immune Response ◽  
Acetyltransferase Domain

In Brucella spp., the type IV secretion system (T4SS) is essential for bacterial intracellular survival and inhibition of the host innate immune response. The Brucella T4SS secretes 15 different effectors to escape host immunity and promote intracellular replication. Among them, BspF has a GNAT-family acetyltransferase domain, implying its acetyltransferase activity. We confirmed that BspF has acetyltransferase activity (data not shown) and de-crotonyltransferase activity. However, BspF overexpressed in HEK-293T cells can also enhance octamer crotonylation in vitro. Then we enriched crotonylated proteins and conducted LC-MS to study the crotonylation changes of proteins in HEK-293T cells caused by BspF overexpression. A total of 5,559 crotonylation sites were identified on 1,525 different proteins, of which 331 sites on 265 proteins were significantly changed. We found that Rab9A and RAP1B in proteomics data have a great impact on Brucella survival, so we speculate that BspF may influence the function of host proteins by altering crotonylation, thereby promoting the intracellular propagation of Brucella.

scholarly journals Cytotoxicity in Macrophages Infected with Rough Brucella Mutants Is Type IV Secretion System Dependent

2007 ◽  
Vol 76 (1) ◽  
pp. 30-37 ◽  
Author(s):  
Jianwu Pei ◽  
Qingmin Wu ◽  
Melissa Kahl-McDonagh ◽  
Thomas A. Ficht
Keyword(s):  
Cell Death ◽  
Brucella Melitensis ◽  
Regulatory Element ◽  
Random Mutagenesis ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Mutant Bank

ABSTRACT Smooth Brucella spp. inhibit macrophage apoptosis, whereas rough Brucella mutants induce macrophage oncotic and necrotic cell death. However, the mechanisms and genes responsible for Brucella cytotoxicity have not been identified. In the current study, a random mutagenesis approach was used to create a mutant bank consisting of 11,354 mutants by mariner transposon mutagenesis using Brucella melitensis rough mutant 16MΔmanBA as the parental strain. Subsequent screening identified 56 mutants (0.49% of the mutant bank) that failed to cause macrophage cell death (release of 10% or less of the lactate dehydrogenase). The absence of cytotoxicity during infection with these mutants was independent of demonstrable defects in in vitro bacterial growth or uptake and survival in macrophages. Interrupted genes in 51 mutants were identified by DNA sequence analysis, and the mutations included interruptions in virB encoding the type IV secretion system (T4SS) (n = 36) and in vjbR encoding a LuxR-like regulatory element previously shown to be required for virB expression (n = 3), as well as additional mutations (n = 12), one of which also has predicted roles in virB expression. These results suggest that the T4SS is associated with Brucella cytotoxicity in macrophages. To verify this, deletion mutants were constructed in B. melitensis 16M by removing genes encoding phosphomannomutase/phosphomannoisomerase (ΔmanBA) and the T4SS (ΔvirB). As predicted, deletion of virB from 16MΔmanBA and 16M resulted in a complete loss of cytotoxicity in rough strains, as well as the low level cytotoxicity observed with smooth strains at extreme multiplicities of infection (>1,000). Taken together, these results demonstrate that Brucella cytotoxicity in macrophages is T4SS dependent.

scholarly journals Requirement for TAFII250 Acetyltransferase Activity in Cell Cycle Progression

2000 ◽  
Vol 20 (4) ◽  
pp. 1134-1139 ◽  
Author(s):  
Elizabeth L. Dunphy ◽  
Theron Johnson ◽  
Scott S. Auerbach ◽  
Edith H. Wang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Acetyltransferase Activity ◽  
Cycle Arrest ◽  
Protein Encoding ◽  
Acetyltransferase Domain ◽  
Mutant Cells

ABSTRACT The TATA-binding protein (TBP)-associated factor TAFII250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAFII250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G1. At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G1 cyclins, is dramatically reduced in the mutant cells. Here we demonstrate that the ability of the ts13 allele of TAFII250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5°C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAFII250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAFII150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or transcriptional defect observed at 39.5°C. These data suggest that TAFII250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes.

scholarly journals Analysis of Cell Type-Specific Responses Mediated by the Type IV Secretion System of Helicobacter pylori

2005 ◽  
Vol 73 (8) ◽  
pp. 4643-4652 ◽  
Author(s):  
Bianca Bauer ◽  
Stefan Moese ◽  
Sina Bartfeld ◽  
Thomas F. Meyer ◽  
Matthias Selbach
Keyword(s):  
Helicobacter Pylori ◽  
Host Cell ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Type Iv ◽  
Host Cell Proteins ◽  
Mammalian Cell Lines ◽  
Host Cell Factors ◽  
H Pylori

ABSTRACT Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.

scholarly journals T4SS-dependent TLR5 activation by Helicobacter pylori infection

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Suneesh Kumar Pachathundikandi ◽  
Nicole Tegtmeyer ◽  
Isabelle Catherine Arnold ◽  
Judith Lind ◽  
Matthias Neddermann ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Pathogenic Bacteria ◽  
Type Iv Secretion ◽  
Downstream Signaling ◽  
Type Iv ◽  
Efficient Control ◽  
H Pylori ◽  
Highly Virulent

AbstractToll-like receptor TLR5 recognizes a conserved domain, termed D1, that is present in flagellins of several pathogenic bacteria but not in Helicobacter pylori. Highly virulent H. pylori strains possess a type IV secretion system (T4SS) for delivery of virulence factors into gastric epithelial cells. Here, we show that one of the H. pylori T4SS components, protein CagL, can act as a flagellin-independent TLR5 activator. CagL contains a D1-like motif that mediates adherence to TLR5+ epithelial cells, TLR5 activation, and downstream signaling in vitro. TLR5 expression is associated with H. pylori infection and gastric lesions in human biopsies. Using Tlr5-knockout and wild-type mice, we show that TLR5 is important for efficient control of H. pylori infection. Our results indicate that CagL, by activating TLR5, may modulate immune responses to H. pylori.

scholarly journals Type IV Secretion System Is Not Involved in Infection Process in Citrus

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Tiago Rinaldi Jacob ◽  
Marcelo Luiz de Laia ◽  
Leandro Marcio Moreira ◽  
Janaína Fernandes Gonçalves ◽  
Flavia Maria de Souza Carvalho ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Positive Control ◽  
Secretion System ◽  
Infection Process ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Type Iv Secretion System ◽  
In Planta ◽  
Type Iv

The type IV secretion system (T4SS) is used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells.Xanthomonas citrisubsp.citricontains two copies of the T4SS, one in the chromosome and the other is plasmid-encoded. To understand the conditions that induce expression of the T4SS inXcc, we analyzed,in vitroandin planta, the expression of 18 ORFs from the T4SS and 7 hypothetical flanking genes by RT-qPCR. As a positive control, we also evaluated the expression of 29 ORFs from the type III secretion system (T3SS), since these genes are known to be expressed during plant infection condition, but not necessarily in standard culture medium. From the 29 T3SS genes analyzed by qPCR, onlyhrpAwas downregulated at 72 h after inoculation. All genes associated with the T4SS were downregulated onCitrusleaves 72 h after inoculation. Our results showed that unlike the T3SS, the T4SS is not induced during the infection process.

scholarly journals Enterococcus faecalis PcfC, a Spatially Localized Substrate Receptor for Type IV Secretion of the pCF10 Transfer Intermediate

2008 ◽  
Vol 190 (10) ◽  
pp. 3632-3645 ◽  
Author(s):  
Yuqing Chen ◽  
Xiaolin Zhang ◽  
Dawn Manias ◽  
Hye-Jeong Yeo ◽  
Gary M. Dunny ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Transmembrane Domain ◽  
Type Iv Secretion ◽  
Nucleoside Triphosphate ◽  
Mutant Proteins ◽  
Type Iv ◽  
Accessory Factor ◽  
Processed Form

ABSTRACT Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCΔN103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCΔN103 bound pCF10 but not pcfG or ΔoriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCΔN103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

scholarly journals Haemophilusducreyi Requires an Intact flp Gene Cluster for VirulenceinHumans

2003 ◽  
Vol 71 (12) ◽  
pp. 7178-7182 ◽  
Author(s):  
Stanley M. Spinola ◽  
Kate R. Fortney ◽  
Barry P. Katz ◽  
Jo L. Latimer ◽  
Jason R. Mock ◽  
...  
Keyword(s):  
Animal Model ◽  
Gene Cluster ◽  
Rabbit Model ◽  
Type Iv Secretion ◽  
Haemophilus Ducreyi ◽  
Secretion Systems ◽  
Temperature Dependent ◽  
Type Iv ◽  
Human Volunteers

ABSTRACT An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.

scholarly journals Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

2012 ◽  
Vol 80 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Ana I. Martín-Martín ◽  
Pilar Sancho ◽  
María Jesús de Miguel ◽  
Luis Fernández-Lago ◽  
Nieves Vizcaíno
Keyword(s):  
Quorum Sensing ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Multiplication Rate ◽  
Content Type ◽  
Murine Macrophages ◽  
Brucella Ovis ◽  
Type Iv

ABSTRACTBrucella ovisis a rough bacterium—lacking O-polysaccharide chains in the lipopolysaccharide—that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguishB. ovisfrom smoothBrucella, which require O-polysaccharide chains for virulence, we have analyzed the significance inB. ovisof the main virulence factors described for smoothBrucella. Attempts to obtain strains of virulentB. ovisstrain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system forin vitrosurvival, while the inactivation ofbacA—in contrast to the results seen with smoothBrucella—did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake ofB. ovisPA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by thevirBoperon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described forB. melitensis, VjbR regulates the transcription of thevirBoperon positively, and theN-dodecanoyl-dl-homoserine lactone (C12-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of thefliFflagellar gene was observed inB. ovis, probably due to the two deletions detected upstream offliF. These results, together with others reported in the text, point to similarities between rough virulentB. ovisand smoothBrucellaspecies as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics ofB. ovis.

scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center

2021 ◽  
Author(s):  
Pratick Khara ◽  
Liqiang Song ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Electron Microscopy ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

ABSTRACTBacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1-VirB11 and VirD4. We visualized the pKM101-encoded T4SS in the native cellular context by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but differs strikingly from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers-of-dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially-dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.SIGNIFICANCEBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate early-stage machine biogenesis and substrate entry passage through the T4SS. The VirB4 ATPases are therefore excellent targets for development of intervention strategies aimed at suppressing the action of T4SS nanomachines.

scholarly journals Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

2021 ◽  
Author(s):  
Sofya Kuzmich ◽  
Dorota Skotnicka ◽  
Dobromir Szadkowski ◽  
Philipp Klos ◽  
María Pérez‐Burgos ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Intact Protein ◽  
Dependent Manner ◽  
Chemosensory System ◽  
Type Iv ◽  
Acetyltransferase Domain

In bacteria, the nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ-domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system, and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA and PixB act in independent pathways and have distinct functions in regulation of motility. Importance c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts acts in Frz-independent mannerto regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

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