Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Expression of cytokine and Apoptosis-Associated genes in mice bone Marrow-Derived Macrophages stimulated with Brucella recombinant type IV secretion effectors.

BACKGROUND: Brucellosis is an economically important infectious caused by most commonly by Brucella. Detection of infected animals at the early stage is important for controlling the disease. The diagnostic antigens, usually protein antigens, have attracted much interest. However, the accurate mechanism of immune response is still unknown. The secretory effectors (BPE005, BPE275, and BPE123) of the type IV secretion system (T4SS) were involved in the intracellular circulation process of Brucella and the immune responses of the host. METHODS: Genes encoding three B. abortus effector proteins (BPE005, BPE275, and BPE123) of T4SS were cloned and the recombinant proteins were expressed and purified. The purified recombinant proteins were named rBPE005, rBPE275 and rBPE123. Then, the expressions of Th1- and Th2-related cytokine genes were analyzed in mice bone marrow-derived macrophages (BMDMs) after stimulation with rBPE005, rBPE275, and rBPE123. Furthermore, four apoptosis-associated genes (Caspase-3, Caspase-8, Bax, and Bcl-2) were also detected to explore the damage of the proteins to the cells. RESULTS: Expressions of all Th1- and Th2-related cytokine genes were induced with three proteins, and different cytokine expression patterns induced by each protein depend on the stimulation time and dose of protein. However, expressions of apoptosis-related genes did not change. CONCLUSION: These results showed that the secreted antigens of Brucella induced an immune reaction via the production of Th1- and Th2-type cytokines in BMDMs without exerting any damage on the cells.

Structural insights into the assembly pathway of the Helicobacter pylori CagT4SS outer membrane core complex.

Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of Helicobacter pylori. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and ß strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.

Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.

Cryo-EM structure of the R388 plasmid conjugative pilus reveals a helical polymer characterized by an unusual pilin/phospholipid binary complex.

Bacterial conjugation is a process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It is the main means by which antibiotic resistance genes spread among bacterial populations. It is crucially dependent upon the elaboration of an extracellular appendage, termed "pilus," by a large double-membrane-spanning secretion system termed conjugative "type IV secretion system." Here we present the structure of the conjugative pilus encoded by the R388 plasmid. We demonstrate that, as opposed to all conjugative pili produced so far for cryoelectron microscopy (cryo-EM) structure determination, the conjugative pilus encoded by the R388 plasmid is greatly stimulated by the presence of recipient cells. Comparison of its cryo-EM structure with existing conjugative pilus structures highlights a number of important differences between the R388 pilus structure and that of its homologs, the most prominent being the highly distinctive conformation of its bound lipid.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli, indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCEStenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa. We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

Cryo-EM structure of a conjugative type IV secretion system suggests a molecular switch regulating pilus biogenesis.

Conjugative type IV secretion systems (T4SS) mediate bacterial conjugation, a process that enables the unidirectional exchange of genetic materials between a donor and a recipient bacterial cell. Bacterial conjugation is the primary means by which antibiotic resistance genes spread among bacterial populations (Barlow 2009; Virolle et al, 2020). Conjugative T4SSs form pili: long extracellular filaments that connect with recipient cells. Previously, we solved the cryo-electron microscopy (cryo-EM) structure of a conjugative T4SS. In this article, based on additional data, we present a more complete T4SS cryo-EM structure than that published earlier. Novel structural features include details of the mismatch symmetry within the OMCC, the presence of a fourth VirB8 subunit in the asymmetric unit of both the arches and the inner membrane complex (IMC), and a hydrophobic VirB5 tip in the distal end of the stalk. Additionally, we provide previously undescribed structural insights into the protein VirB10 and identify a novel regulation mechanism of T4SS-mediated pilus biogenesis by this protein, that we believe is a key checkpoint for this process.

Structure and dispersion of the conjugative mobilome in surface ocean bacterioplankton.

Mobile genetic elements (MGEs), collectively referred to as the "mobilome", can have a significant impact on the fitness of microbial communities and therefore on ecological processes. Marine MGEs have mainly been associated with wide geographical and phylogenetic dispersal of adaptative traits. However, whether the structure of this mobilome exhibits deterministic patterns in the natural community is still an open question. The aim of this study was to characterize the structure of the conjugative mobilome in the ocean surface bacterioplankton by searching the publicly available marine metagenomes from the TARA Oceans survey, together with molecular markers, such as relaxases and type IV coupling proteins of the type IV secretion system (T4SS). The T4SS machinery was retrieved in more abundance than relaxases in the surface marine bacterioplankton. Moreover, among the identified MGEs, mobilizable elements were the most abundant, outnumbering self-conjugative sequences. Detection of a high number of incomplete T4SSs provides insight into possible strategies related to trans-acting activity between MGEs, and accessory functions of the T4SS (e.g. protein secretion), allowing the host to maintain a lower metabolic burden in the highly dynamic marine system. Additionally, the results demonstrate a wide geographical dispersion of MGEs throughout oceanic regions, while the Southern Ocean appears segregated from other regions. The marine mobilome also showed a high similarity of functions present in known plasmid databases. Moreover, cargo genes were mostly related to DNA processing, but scarcely associated with antibiotic resistance. Finally, within the MGEs, integrative and conjugative elements showed wider marine geographic dispersion than plasmids.

T6SS and T4SS Redundantly Secrete Effectors to Govern the Virulence and Bacterial Competition in Pectobacterium PccS1.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria.

Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection.

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Cancer-associated SNPs in bacteria: lessons from Helicobacter pylori.

Several single-nucleotide polymorphisms (SNPs) in human chromosomes are known to predispose to cancer. However, cancer-associated SNPs in bacterial pathogens were unknown until discovered in the stomach pathogen Helicobacter pylori. Those include an alanine-threonine polymorphism in the EPIYA-B phosphorylation motif of the injected effector protein CagA that affects cancer risk by modifying inflammatory responses and loss of host cell polarity. A serine-to-leucine change in serine protease HtrA is associated with boosted proteolytic cleavage of epithelial junction proteins and introduction of DNA double-strand breaks (DSBs) in host chromosomes, which co-operatively elicit malignant alterations. In addition, H. pylori genome-wide association studies (GWAS) identified several other SNPs potentially associated with increased gastric cancer (GC) risk. Here we discuss the clinical importance, evolutionary origin, and functional advantage of the H. pylori SNPs. These exciting new data highlight cancer-associated SNPs in bacteria, which should be explored in more detail in future studies.

T4SEpp: A pipeline integrating protein language models to predict bacterial type IV secreted effectors.

Many pathogenic bacteria use type IV secretion systems (T4SSs) to deliver effectors (T4SEs) into the cytoplasm of eukaryotic cells, causing diseases. The identification of effectors is a crucial step in understanding the mechanisms of bacterial pathogenicity, but this remains a major challenge. In this study, we used the full-length embedding features generated by six pre-trained protein language models to train classifiers predicting T4SEs and compared their performance. We integrated three modules into a model called T4SEpp. The first module searched for full-length homologs of known T4SEs, signal sequences, and effector domains; the second module fine-tuned a machine learning model using data for a signal sequence feature; and the third module used the three best-performing pre-trained protein language models. T4SEpp outperformed other state-of-the-art (SOTA) software tools, achieving ∼0.98 accuracy at a high specificity of ∼0.99, based on the assessment of an independent validation dataset. T4SEpp predicted 13 T4SEs from Helicobacter pylori, including the well-known CagA and 12 other potential ones, among which eleven could potentially interact with human proteins. This suggests that these potential T4SEs may be associated with the pathogenicity of H. pylori. Overall, T4SEpp provides a better solution to assist in the identification of bacterial T4SEs and facilitates studies of bacterial pathogenicity. T4SEpp is freely accessible at https://bis.zju.edu.cn/T4SEpp.

Identification of atypical T4SS effector proteins mediating bacterial defense.

To remain competitive, proteobacteria use various contact-dependent weapon systems to defend against microbial competitors. The bacterial-killing type IV secretion system (T4SS) is one such powerful weapon. It commonly controls the killing/competition between species by secreting the lethal T4SS effector (T4E) proteins carrying conserved XVIPCD domains into competing cells. In this study, we sought knowledge to understand whether the bacterial-killing T4SS-producing bacteria encode T4E-like proteins and further explore their biological functions. To achieve this, we designed a T4E-guided approach to discover T4E-like proteins that are designated as atypical T4Es. Initially, this approach required scientists to perform simple BlastP search to identify T4E homologs that lack the XVIPCD domain in the genomes of T4SS-producing bacteria. These homologous genes were then screened in Escherichia coli to identify antibacterial candidates (atypical T4Es) and their neighboring detoxification proteins, followed by testing their gene cotranscription and validating their physical interactions. Using this approach, we did discover two atypical T4E proteins from the plant-beneficial Lysobacter enzymogenes and the phytopathogen Xanthomonas citri. We also provided substantial evidence to show that the atypical T4E protein Le1637-mediated bacterial defense in interspecies interactions between L. enzymogenes and its competitors. Therefore, the newly designed T4E-guided approach holds promise for detecting functional atypical T4E proteins in bacterial cells.

Caracterização de dois pares efetor/inibidor associados ao sistema de secreção tipo IV de Xanthomonas citri / Characterization of the two effector/inhibitor pair associated with the type IV secretion system of Xanthomonas citri

O sistema de secreção tipo IV (T4SS) da família de bactérias Xanthomonadaceae transfere efetores (X-Tfes) com a capacidade de matar outras bactérias, conferindo uma vantagem em comunidades bacterianas mistas para colonizar diferentes nichos como o solo ou as superfícies das plantas. Os X-Tfes possuem diferentes domínios putativos com atividades hidrolíticas contra componentes do envelope celular bacteriano do tipo: glicohidrolases, transglicosilases, amidases e lipases. Os X-Tfes por sua atividade biológica inata podem ocasionar dano intracelular para a bactéria que os produz. Para se proteger contra estas atividades, também são produzidas lipoproteínas com função inibitoria (X-Tfis) localizadas no periplasma. Os genes que codificam os X-Tfes e os X-Tfis estão organizados em operons, o que permite gerar os pares efetor/inibidor simultaneamente. Entre os potenciais X-Tfes do fitopatógeno Xanthomonas citri estão Xac1918 e Xac0574. Xac1918 é uma proteína com um domínio da superfamília da lisozima e um domínio conhecido como RTX (Repeats in Toxin) de ligação ao cálcio, enquanto Xac0574 tem um domínio da superfamília da lipase 3. Os seus possíveis inibidores, Xac1917 e Xac0573 respectivamente, apresentam um peptídeo sinal no N-terminal contendo o lipobox representativo das lipoproteínas. As proteínas Xac0574 e Xac0573 são monômeros em solução que formam um complexo estável 1:1, favorecido termodinamicamente (ΔG°= -12 Kcal/mol) com uma constante de dissociação de 2,45 nM, garantindo que a bactéria fique protegida contra os efeitos nocivos de Xac0574 quando é produzida intracelularmente. Xac0574 é uma fosfolipase A1, sem atividade lisofosfolipase, com a capacidade de hidrolisar os três fosfolipídios majoritários que compõem a membrana celular bacteriana, fosfatidilglicerol (PG), cardiolipina e fosfatidiletanolamina (PE), mostrando uma aparente preferência pelo último. A atividade enzimática de Xac0574 explica a forte inibição do crescimento celular em E. coli após da sua indução heteróloga, já que gera uma diminuição de quase 10 vezes da população celular comparada com a cultura não induzida com a mesma construção. Poroutro lado, Xac0573 inibe efetivamente a atividade enzimática de Xac0574 ao formar o complexo, além de não ter atividade fosfolipase nem lisofosfolipase. Foram produzidos cristais da Xac1918 e Xac0573 que difrataram com uma resolução de 3,0 e 2,5 Å, respectivamente. Porém, só foi gerado um modelo de Xac0573. Xac0573 está composta por duas folhas ß antiparalelas com uma topologia característica de ß sanduíche Com uma pequena hélice e duas voltas. Um alinhamento de homólogos de Xac0573 identificou nas extremidades da proteína as regiões conservadas, constituindo duas possíveis interfaces de interação que podem ser as responsáveis por bloquear o acesso dos fosfolipídios ao sítio catalítico ou impedir os rearranjos estruturais de Xac0574 que são necessários para a sua atividade enzimática. Adicionalmente, a topologia da Xac0573 é semelhante do domínio C2, conhecido em eucariotos como domínio de ligação ao lipídio e ao cálcio, e está envolvido em processos de sinalização de segundos mensageiros lipídicos, proteínas de trafego de membranas e mecanismos de fusão de membranas. Nossos resultados apontam para uma nova função biológica do domínio C2 como um inibidor enzimático intracelular em bactérias

The type IV secretion system (T4SS) of the bacteria family Xanthomonadaceae transfers effectors (X-Tfes) with that can kill other bacterial cells, conferring an advantage to the bacterial community during colonization of different niches in the soil or on the plant surface. The X-Tfes possess different putative domains with hydrolytic activity against components of the bacterial cellular envelope, including glycohydrolase, transglycolase, amidase and lipase domain. The innate biological activity of X-Tfes can cause intracellular damage. Therefore, the bacteria that produce them also produce lipoproteins with inhibitor function (X-Tfis) located in the periplasm for their protection. The genes that code for X-Tfes and X-Tfis are organized in operons that allow for their simultaneous expression. Among the X-Tfes of the phytopathogen Xanthomonas citri are Xac1918 and Xac0574. Xac1918 is carries a lysozyme superfamily domain, as well as a domain known as RTX (Repeats in Toxic) predict to bind calcium, while, Xac0574 has a domain belonging to the lipase 3 superfamily. Their possible inhibitors, Xac1917 e Xac0573 respectively, carry an N-terminal signal peptide containing a lipobox found in bacterial lipoproteins. The Xac0574 and Xac0573 proteins are both monomers in solution, They can form a stable 1:1 complex, that is thermodynamically favored (ΔG°= -12 Kcal/mol) with a dissociation constant of 2,45 nM. This affinity ensure that the bacterium is protected against the harmful effects of Xac0574 when it is produced intracellularly. We show that Xac0574 is a phospholipase A1, without lisophospholipase activity, and is able to hydrolyze the three most common phospholipids found in the membranes of Gram negative bacteria, namely phosphatidylglycerol (PG), cardiolipin and phosphatidylethanolamine (PE), presenting an apparent preference for PE. The enzymatic activity of Xac0574 explains the strong inhibition of growth of E. coli cells after its heterologous induction: a nearly 10-fold decrease in the cell population is observed when compared to the non-induced culture with the same construct. On the other hand, Xac0573 effectively inhibits the enzymatic activity of Xac0574. Furthermore, Xac0573 does not possess when forming the complex, besides not having phospholipase nor lysophospholipase activity.Crystals of Xac1918 and Xac0573 were produced which diffracted with to resolution of 3.0 and 2.5 Å, respectively. However, we were able to resolve the structure of only Xac0573. Xac0573 is composed of two anti-parallel sheet that form a ß-sandwich with three small helices. An alignment to Xac0573 homologs identified conserved regions at the ends of the protein that constitute two possible interfaces of interaction that may be responsible for blocking the access of the phospholipids to the catalytic site or impede the structural rearrangements of Xac0574 that are necessary for its enzymatic activity. Additionally, the topology of Xac0573 is similar to that to C2 domains, known in eukaryotes to bind lipids and calcium and to be involved in signaling processes mediated by lipid second messengers, membrane trafficking and membrane fusion mechanisms. Our results point to a new biological function of the C2 domain as an intracellular enzyme inhibitor in bacteria

Estudos funcionais e bioquímicos sobre o reconhecimento e inibição de efetores de um sistema de secreção tipo IV de Xanthomonas citri subsp. citri. / Functional and biochemical son the recognition and Inhibition of effectors of a Type IV secretion System of Xanthomonas citri subsp. citri

Sistemas de Secreção Tipo IV (T4SSs), normalmente compostos por 12 proteínas (VirB1-VirB11 e VirD4) são tipicamente associados às funções de conjugação bacteriana e transferência de fatores de patogenicidade para células hospedeiras. Mas também, muitas espécies da ordem Xanthomonadales possuem um T4SS associado a matar bactérias. O modelo atual de morte de uma célula-alvo mediada pelo T4SS é baseado na secreção de toxinas denominadas XVIPs ("Xanthomonas VirD4 interacting proteins") ou X-Tfe (Xanthomonadaceae-T4SS effector) no qual cada XVIP/X-Tfe apresenta uma proteína de imunidade cognata denominada X-Tfi (Xanthomonadaceae-T4SS immunity protein). Demonstramos que um XVIP, XAC2609, é secretado através do T4SS de modo que depende de contato célula-célula e do seu domínio XVIPCD ("XVIP conserved domains"). A porção N-terminal de XAC2609 codifica um domínio GH19 que cliva a peptideoglicana de E. coli, mas perde a sua atividade na presença do seu inibidor cognato, o X-Tfi XAC2610. Portanto, XAC2609/XAC2610 formam um par de proteínas efetora/imunidade associado ao T4SS de X. citri. Através de diferentes técnicas de microscopias utilizando a cepa Δxac2610, foi observado que XAC2610 protege o envelope celular de X. citri contra efeitos de autólise celular promovidos pela atividade de XAC2609. Ensaios funcionais baseados nas observações de fenótipos de colônias e de formação de biofilme mostraram que XAC2610 confere imunidade para X. citri contra uma atividade 7 intrínseca de XAC2609. A proteína com o papel de reconhecer os substratos através da interação com os sinais de secreção do T4SS é VirD4. No T4SS de X. citri, existe a hipótese de que o domínio XVIPCD seja o sinal de secreção presente nas XVIPs. Logo, os aspectos bioquímicos e biofísicos da interação VirD4-XVIPCD foram investigados através de experimentos de co-purificação por cromatografia de afinidade e exclusão molecular, RMN e SAXS. Demonstramos que o domínio AAD de VirD4 (VirD4AAD) está associado a interagir especificamente com o domínio XVIPCD de XAC2609 (XAC2609XVIPCD), formando um heterodímero em solução. VirD4AAD é um domínio globular e monomérico e XAC2609XVIPCD é desenovelado mas se enovela concomitante à interação com VirD4AAD. Construções de XAC2609 contendo mutações pontuais no domínio XVIPCD foram utilizadas em ensaios in vivo de secreção pela X. citri e ensaios in vitro de interação com VirD4AAD por titulação monitorada por calorimetria isotérmica (ITC). Através desses experimentos, observamos que uma forte interação entre VirD4AAD-XAC2609XVIPCD é essencial para secreção de XAC2609 via o T4SS. Esses resultados permitem concluir que o domínio XVIPCD é o sinal de secreção dos substratos do T4SS de X. citri e que o AAD confere especificidade à VirD4 por interagir com o XVIPCD. Finalmente, através de ensaios de competições bacterianas entre E. coli e X. citri, foram observados diferentes fenótipos associados à função do T4SS: i) nocautes gênicos das subunidades estruturais VirB5, VirB11 abolem a função do T4SS em X. citri.; ii) nocautes de xac2611, apresentaram uma maior vantagem adaptativa do que a cepa selvagem de X. citri em competições e a expressão epissomal de XAC2611 inibe fortemente a função do T4SS e iii) a atividade ATPásica de VirD4 é essencial para a função do sistema e a expressão de mutantes 8 de VirD4 exerce um fenótipo de dominância negativa sobre a função do T4SS em X. citri

The Type IV secretion System (T4SS) is typically associated with the function of bacterial conjugation and as a pathogenicity factor. T4SSs are normally composed of 12 proteins, VirB1-VirB11 and VirD4. Many species of the order Xanthomonadales possess a T4SS associated with killing bacteria. The current model of the T4SS killing is based on the secretion of toxins denominated XVIPs/X-Tfes (Xanthomonas VirD4 interacting proteins) /(Xanthomonadaceae-T4SS effector) in which each XVIP/X-Tfe has a cognate immunity protein denominated X-Tfi (Xanthomonadaceae-T4SS immunity protein). We demonstrate that an XVIP, XAC2609, is secreted through the T4SS so that it depends on cell-cell contact and its XVIPCD domain ("XVIP conserved domains"). The N-terminal portion of XAC2609 encodes a GH19 domain which cleaves the E. coli peptidoglycan but loses its activity in the presence of its cognate inhibitor, X-Tfi XAC2610. Therefore, XAC2609 /XAC2610 form a pair of effector/immunity proteins associated with X. citri T4SS. By using the X. citri Δxac2610 strain, has been shown through different microscopic techniques that XAC2610 protects the cell envelope of X. citri against the effects of cellular autolysis promoted by XAC2609 activity. Functional assays based on observations of colony phenotypes and biofilm formation has shown that XAC2610 confers immunity to X. citri against an intrinsic activity of XAC2609. VirD4 is the protein that recognizes the substrates through the interaction with the T4SS secretion signals. In the T4SS of X. citri, is hypothesized that the XVIPCD domain is the secretion signal present in the XVIPs. Here, the biochemical and biophysical aspects of the VirD4-XVIPCD interaction were investigated through Pull- Down, Molecular Exclusion Chromatography, NMR and SAXS assays. It has been shown the AAD domain of VirD4 (VirD4AAD) is associated with specifically interacting with the XAC2609XVIPCD domain (XAC2609XVIPCD), forming a heterodimer in solution. VirD4AAD is a globular and monomeric domain while XAC2609XVIPCD is elongated, but upon interaction with VirD4AAD goes through structural compaction process. Constructs of XAC2609 containing point mutations in the XVIPCD domain were used to perform secretion experiments in X. citri and Isothermal titration calorimetry against VirD4AAD. Through these assays, it has been characterized that a strong interaction between VirD4AAD-XAC2609XVIPCD is essential for secretion of XAC2609 via T4SS. Consequently, these results allow concluding that the XVIPCD domain is the secretion signal of X. citri T4SS substrate and the AAD confer specificity to VirD4 by interact with the XVIPCD domains. Finally, bacterial competitions between E. coli and X. citri showed different phenotypes associated with T4SS function: i) virB5, virB11 knockouts abolish the function of T4SS in X. citri.; ii) knockouts of xac2611 exhibited a higher adaptive efficiency than the wild-type X. citri strain in competitions, but the expression of XAC2611 abolishes the function of T4SS in the wild strain of X. citri; iii) The ATPase activity of VirD4 is essential and exerts a negative dominance over the T4SS function in X.citri