Metabolism of leukotrienes

Summary The in vitro metabolism of leukotriene B₄ is initiated by -hydroxylation. This reaction is followed by oxidation of the -hydroxyl group to a carboxyl group. In vivo extensive -oxidation occurs and the main excreted products after administration of leukotriene B₄ are water and carbon dioxide.Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal y-glutamyl residue, catalyzed by the enzyme -glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E₄, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C₄. In rats, the two major fecal metabolities of leukotriene C₄4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E₄. These compounds are formed in reactions between leukotriene E₄ or 11-trans leukotriene E₄ and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.     

Ras Promotes Transforming Growth Factor-β (TGF-β)-induced Epithelial-Mesenchymal Transition via a Leukotriene B4 Receptor-2-linked Cascade in Mammary Epithelial Cells

Inflammation and inflammatory mediators are inextricably linked with epithelial-mesenchymal transition (EMT) through complex pathways in the tumor microenvironment. However, the mechanism by which inflammatory mediators, such as the lipid inflammatory mediators, eicosanoids, contribute to EMT is largely unknown. In the present study we observed that BLT2, leukotriene B₄ receptor-2, is markedly up-regulated by oncogenic Ras and promotes EMT in response to transforming growth factor-β (TGF-β) in mammary epithelial cells. Blockade of BLT2 by the BLT2 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY255283","term_id":"1257961172","term_text":"LY255283"}}LY255283 or by siRNA reduced EMT induced by Ras in the presence of TGF-β. In addition, stimulation of BLT2 by the addition of a BLT2 ligand, such as leukotriene B₄, restored EMT in the presence of TGF-β in human immortalized mammary epithelial MCF-10A cells. We further searched BLT2 downstream components and identified reactive oxygen species and nuclear factor κB as critical components that contribute to EMT. Taken together, these results demonstrate for the first time that a BLT2-linked inflammatory pathway contributes to EMT. This provides valuable insight into the mechanism of EMT in mammary epithelial cells. In addition, considering the implications of EMT with the stemness of cancer cells, our finding may contribute to a better understanding of tumor progression.     

Antibiotic treatment modulates protein components of cytotoxic outer membrane vesicles of multidrug-resistant clinical strain, <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> DU202

Background

Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs following antibiotic treatment by proteogenomic analysis.

Methods

Purified OMVs from A. baumannii DU202 grown in different antibiotic culture conditions were screened for pathogenic and immunogenic effects, and subjected to quantitative proteomic analysis by one-dimensional electrophoresis and liquid chromatography combined with tandem mass spectrometry (1DE-LC-MS/MS). Protein components modulated by imipenem were identified and discussed.

Results

OMV secretion was increased >?twofold following imipenem treatment, and cytotoxicity toward A549 human lung carcinoma cells was elevated. A total of 277 proteins were identified as components of OMVs by imipenem treatment, among which β-lactamase OXA-23, various proteases, outer membrane proteins, β-barrel assembly machine proteins, peptidyl-prolyl cis–trans isomerases and inherent prophage head subunit proteins were significantly upregulated.

Conclusion

In vitro stress such as antibiotic treatment can modulate proteome components in A. baumannii OMVs and thereby influence pathogenicity.

     

Evolution of the facial musculature in basal ray-finned fishes

Background

The facial musculature is a remarkable anatomical complex involved in vital activities of fishes, such as food capture and gill ventilation. The evolution of the facial muscles is largely unknown in most major fish lineages, such as the Actinopterygii. This megadiverse group includes all ray-finned fishes and comprises approximately half of the living vertebrate species. The Polypteriformes, Acipenseriformes, Lepisosteiformes, Amiiformes, Elopiformes, and Hiodontiformes occupy basal positions in the actinopterygian phylogeny and a comparative study of their facial musculature is crucial for understanding the cranial evolution of bony fishes (Osteichthyes) as a whole.

Results

The facial musculature of basal actinopterygians is revised, redescribed, and analyzed under an evolutionary perspective. We identified twenty main muscle components ontogenetically and evolutionarily derived from three primordial muscles. Homologies of these components are clarified and serve as basis for the proposition of a standardized and unifying myological terminology for all ray-finned fishes. The evolutionary changes in the facial musculature are optimized on the osteichthyan tree and several new synapomorphies are identified for its largest clades, including the Actinopterygii, Neopterygii, and Teleostei. Myological data alone ambiguously support the monophyly of the Holostei. A newly identified specialization constitutes the first unequivocal morphological synapomorphy for the Elopiformes. The myological survey additionally allowed a reinterpretation of the homologies of ossifications in the upper jaw of acipenseriforms.

Conclusions

The facial musculature proved to be extremely informative for the higher-level phylogeny of bony fishes. These muscles have undergone remarkable changes during the early radiation of ray-finned fishes, with significant implications for the knowledge of the musculoskeletal evolution of both derived actinopterygians and lobe-finned fishes (Sarcopterygii).

     

Coupling solid phase microextraction to complementary separation platforms for metabotyping of <Emphasis Type="Italic">E. coli</Emphasis> metabolome in response to natural antibacterial agents

Introduction

Essential oils are known to possess antimicrobial activity; thus, their use has played an important role over the years in medicine and for food preservation purposes.

Objective

The effect of clove oil and its major constituents as bactericidal agents on the global metabolic profiling of E. coli bacteria was assessed by means of metabolic alterations, using solid phase microextraction (SPME) as a sample preparation method coupled to complementary analytical platforms.

Method

E. Coli cultures treated with clove oil and its major individual components were sampled by HS-SPME-GCxGC-ToF/MS and SPME-UPLC–MS. Full factorial design was applied in order to estimate the most effective antibacterial agent towards E. coli. Central composite design and factorial design were applied to investigate parameters influencing metabolite coverage and efficiency by SPME.

Results

The metabolic profile, including 500 metabolites identified by LC–MS and 789 components detected by GCxGC-ToF/MS, 125 of which were identified as dysregulated metabolites, revealed changes in the metabolome provoked by the antibacterial activity of clove oil, and in particular its major constituent eugenol. Analyses of individual components selected using orthogonal projections to latent structures discriminant analysis showed a neat differentiation between control samples in comparison to treated samples in various sets of metabolic pathways.

Conclusions

The combination of a sample preparation method capable of providing cleaner extracts coupled to different analytical platforms was successful in uncovering changes in metabolic pathways associated with lipids biodegradation, changes in the TCA cycle, amino acids, and enzyme inhibitors in response to antibacterial treatment.

     

COPD phenotype description using principal components analysis

Background

Airway inflammation in COPD can be measured using biomarkers such as induced sputum and Fe_NO. This study set out to explore the heterogeneity of COPD using biomarkers of airway and systemic inflammation and pulmonary function by principal components analysis (PCA).

Subjects and Methods

In 127 COPD patients (mean FEV₁ 61%), pulmonary function, Fe_NO, plasma CRP and TNF-α, sputum differential cell counts and sputum IL8 (pg/ml) were measured. Principal components analysis as well as multivariate analysis was performed.

Results

PCA identified four main components (% variance): (1) sputum neutrophil cell count and supernatant IL8 and plasma TNF-α (20.2%), (2) Sputum eosinophils % and Fe_NO (18.2%), (3) Bronchodilator reversibility, FEV₁ and IC (15.1%) and (4) CRP (11.4%). These results were confirmed by linear regression multivariate analyses which showed strong associations between the variables within components 1 and 2.

Conclusion

COPD is a multi dimensional disease. Unrelated components of disease were identified, including neutrophilic airway inflammation which was associated with systemic inflammation, and sputum eosinophils which were related to increased Fe_NO. We confirm dissociation between airway inflammation and lung function in this cohort of patients.     

In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.     

Female Responses to Synthetic Pheromone and Plant Compounds in <Emphasis Type="Italic">Agriotes brevis</Emphasis> Candeze (Coleoptera: Elateridae)

Traps baited with synthetic pheromone components of Agriotes brevis [geranyl butanoate + (E,E)-farnesyl butanoate] captured significantly higher numbers of not only male, but also female beetles, compared to unbaited controls. Catches of both sexes showed a clear positive relationship with increasing doses. In electroantennogram tests, antennal responses of females and males to a number of known Agriotes pheromone components, identified from pheromone glands, showed a similar trend, with geranyl butanoate eliciting the strongest responses. This suggests that the female and male A. brevis antennae are similar with respect to the perception of pheromone compounds, and female beetles have the sensory capabilities to perceive the pheromone components which they produce. Addition of the plant-derived compounds (Z)-3-hexenyl acetate, methyl benzoate, (Z)-3-hexenol and methyl salicylate (identified earlier from foliage as attractive for A. brevis) to the synthetic pheromone significantly increased catches. All the above results suggest that geranyl butanoate and (E,E)-farnesyl butanoate are constituents of an aggregation pheromone of A. brevis, in contrast to the general view of click beetle pheromones being “classical” sex pheromones. These findings could be useful for more precise monitoring and forecasting of damage, based on female catches.     

Linear and nonlinear stiffness and friction in biological rhythmic movements

Biological rhythmic movements can be viewed as instances of self-sustained oscillators. Auto-oscillatory phenomena must involve a nonlinear friction function, and usually involve a nonlinear elastic function. With respect to rhythmic movements, the question is: What kinds of nonlinear friction and elastic functions are involved? The nonlinear friction functions of the kind identified by Rayleigh (involving terms such as $\dot \theta ^3 $ ) and van der Pol (involving terms such as $\theta ^2 \dot \theta $ ), and the nonlinear elastic functions identified by Duffing (involving terms such as $\theta ^3 $ ), constitute elementary nonlinear components for the assembling of self-sustained oscillators. Recently, additional elementary nonlinear friction and stiffness functions expressed, respectively, through terms such as $\theta ^2 \dot \theta ^3 $ and $\theta \dot \theta ^2 $ , and a methodology for evaluating the contribution of the elementary components to any given cyclic activity have been identified. The methodology uses a quantification of the continuous deviation of oscillatory motion from ideal (harmonic) motion. Multiple regression of this quantity on the elementary linear and nonlinear terms reveals the individual contribution of each term to the oscillator's non-harmonic behavior. In the present article the methodology was applied to the data from three experiments in which human subjects produced pendular rhythmic movements under manipulations of rotational inertia (experiment 1), rotational inertia and frequency (experiment 2), and rotational inertia and amplitude (experiment 3). The analysis revealed that the pendular oscillators assembled in the three experiments were compositionally rich, braiding linear and nonlinear friction and elastic functions in a manner that depended on the nature of the task.     

Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis

Introduction

Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)–producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.

Methods

We utilized whole blood–derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.

Results

Our data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release–activated channel–mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP⁺ cells capable of F-actin ring formation. These effects were dependent on Ca²⁺ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.

Conclusions

In conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca²⁺ signaling and the LTB4 signaling cascade.     

QTLRel: an R Package for Genome-wide Association Studies in which Relatedness is a Concern

Background

Existing software for quantitative trait mapping is either not able to model polygenic variation or does not allow incorporation of more than one genetic variance component. Improperly modeling the genetic relatedness among subjects can result in excessive false positives. We have developed an R package, QTLRel, to enable more flexible modeling of genetic relatedness as well as covariates and non-genetic variance components.

Results

We have successfully used the package to analyze many datasets, including F₃₄ body weight data that contains 688 individuals genotyped at 3105 SNP markers and identified 11 QTL. It took 295 seconds to estimate variance components and 70 seconds to perform the genome scan on an Linux machine equipped with a 2.40GHz Intel(R) Core(TM)2 Quad CPU.

Conclusions

QTLRel provides a toolkit for genome-wide association studies that is capable of calculating genetic incidence matrices from pedigrees, estimating variance components, performing genome scans, incorporating interactive covariates and genetic and non-genetic variance components, as well as other functionalities such as multiple-QTL mapping and genome-wide epistasis.     

A novel rice protein family of OsHIGDs may be involved in early signalling of hypoxia-promoted stem growth in deepwater rice

Key message

OsHIGDs was identified as a novel hypoxia-responsive protein family. Among them, OsHIGD2 is characterized as a mitochondrial protein and is related to hypoxia signalling through interacting with mitochondrial proteins of critical functions in reducing cell damages caused by hypoxia.

Abstract

Recent evidence supports ethylene as a key factor in modulating plant responses to submergence stress. Meanwhile, there has been general consent that ethylene is not the only signal for the submergence-induced stem growth. In this study, we confirmed that hypoxia also promotes stem elongation in deepwater rice even in the absence of ethylene. As components of ethylene-independent hypoxia signalling, five HIGD (hypoxia-induced gene domain) protein genes were identified. Among the genes, OsHIGD2 showed the fastest and strongest induction by hypoxia as well as submergence. Co-expression analysis indicated that OsHIGD2 had a simultaneous expression pattern with fermentation-related genes, such as ADH1 (alcohol dehydrogenase 1) and PDC2 (pyruvate decarboxylase 2). Transient expression of OsHIGD2 in leaf epidermal cells of Nicotiana benthamiana provided evidence that the protein is localized to mitochondria. We further identified OsHIGD2-interacting proteins through the yeast two-hybrid assay using OsHIGD2 as bait. As a result, three mitochondrial proteins were discovered that function in the regulation of redox potential or reduction of protein damages caused by reactive oxygen species. In this report, we propose that OsHIGD2 is a mitochondrial protein which takes part in the early stage of hypoxia signalling by interacting with proteins that are related to oxygen utilization.

     

Subunits of tetrameric α-amylase inhibitors of Hordeum chilense are encoded by genes located in chromosomes 4Hch and 7Hch

Summary Three proteins (components 1, 2, and 4) of the non-prolamin, 70% ethanol soluble fraction from the endosperm of Hordeum chilense have been identified as putative subunits of the tetrameric inhibitors active against insect -amylases. In experiments carried out with the synthetic alloploid Tritordeum (H. chilense x Triticum turgidum conv. durum), previously described proteins from T. turgidum, designated CM2, CM3 and CM 16, have been also identified as subunits of -amylase inhibitors. Genes for components 1 and 4 of H. chilense have been located in chromosomes 4H^ch and 7H^ch, based on the analysis of H. chilense-T.turgidum addition lines. Subunits of the inhibitors from wheat and from cultivated barley had been previously assigned to chromosomes of the same homoeology groups.     

Terpenoids and phenethyl glucosides from Hyssopus cuspidatus (Labiatae)

Monoterpenoids (3 and 4), sesquiterpenoid (2), diterpenoid (1) and four phenethyl glucosides (5-8), together with fourteen known compounds, were isolated from the whole herb of Hyssopus cuspidatus. Their structures were determined by spectroscopic means. The abietane-type diterpenoids (1, 9, 10), rosmarinic acid (15) and salvigenin (17) inhibited leukotriene (LT) C₄ secretion from primary alveolar cells of Wistar rats.     

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein.Helicobacter pylori infects 50% of the world population. Stomach infection with this bacterium is associated with the development of several gastric diseases, including chronic active gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. Factors influencing disease outcomes are not completely understood, but bacterial, host, and environmental factors have been identified that affect the dynamics of this bacterium-host interaction (30). A hallmark of H. pylori infection is the induction of mucosal inflammation, which is a risk factor for developing more severe pathology (27).Epidemiological studies have established that infection with strains harboring the cag pathogenicity island (PAI) leads to a higher risk for development of severe disease (27). The cag PAI size varies between 35 and 40 kb and encodes 27 putative proteins (1, 13). Several of the encoded proteins share sequence similarities with components of the prototypical type IV secretion (T4S) system VirB/D4 of Agrobacterium tumefaciens (15, 16). Based on research done in A. tumefaciens, the components of the molecular machinery have been divided into channel or core complex components (VirB6, VirB7, VirB8, VirB9, and VirB10), energetic components (VirB11, VirB4, and VirD4), and extracellular appendage components (VirB2 and VirB5). VirB6, VirB8, and VirB10 are components anchored at the inner membrane with domains spanning the periplasm, while VirB7 and VirB9 are located at the outer membrane. Energetic components are located at the inner membrane, and pilus components include the main subunit VirB2 and accessory components, such as VirB5, which functions as an adhesin (15, 16). The VirB/D4 T4S is thought to be energized by the inner membrane ATPases, and this energy is transduced to VirB10 and the outer membrane complex for protein translocation (11). The lipoprotein VirB7 is critical for the stability of HpVirB9 at the outer membrane (19).While the extent of homology of the H. pylori cag T4S components is often limited, sequence analysis has allowed the identification of the VirB11 (HP0525 and HpVirB11), VirB10 (HP0527 and HpVirB10), VirB9 (HP0528 and HpVirB9), and VirD4 (HP0524 and HpVirD4) homologues as summarized in Table S1 of the supplemental material (1, 13, 28). HpVirB9 and HpVirB10 homologies are not distributed along the entire length of the protein. For example, HpVirB10 is a very large protein with only a short domain similar to VirB10. HpVirB10 is also reported to localize on the external surface of the pilus (31), while VirB10 is tethered in the inner membrane. HP0529 (HpVirB6) and HP0530 (HpVirB8) have been assigned as homologs of VirB6 and VirB8, respectively (28). HP0523 (HpVirB1) has lytic transglycosylase activity, supporting its designation as a VirB1 homolog (38). HP0532 (HpVirB7) has a lipoprotein attachment site, suggesting a role as a VirB7 homolog (1, 28), and has been suggested to stabilize a Cag T4S outer membrane subcomplex containing CagM, HpVirB9, and HpVirB10 (28).The activity of the cag PAI-encoded T4S system is responsible for the translocation of the effector protein CagA and induction of proinflammatory chemokine and cytokine secretion, including the chemokine interleukin-8 (IL-8) (7). CagA T4S-mediated translocation into host cells is followed by tyrosine phosphorylation on specific tyrosine phosphorylation motifs (EPIYA motifs) at the C-terminal region of the protein and both phosphorylation-dependent and -independent interference with host cellular pathways. The induction of proinflammatory chemokine production is mediated by a still-uncharacterized Cag T4S-mediated delivery of peptidoglycan into host cells and subsequent activation of Nod receptors (37), and it has also been reported that CagA itself has proinflammatory properties (9). The molecular mechanisms responsible for Cag T4S system assembly and activity remain unclear.Null alleles of the genes with homology to T4S components (HpVirB11, HpVirB4, HpVirB6, HpVirB7, HpVirB8, HpVirB9, and HpVirB10) abolish both CagA translocation and IL-8 induction, with the exception of HpVirD4, which affects CagA translocation but not IL-8 induction (20). Other genes of the island also essential for Cag T4S function do not share sequence or structural homology with known T4S components. More detailed analysis of these Cag T4S essential genes allowed the recent assignment of several proteins as functional homologs of additional VirB components. HP0546 was suggested as a VirB2 homolog, the main subunit of other T4S system pili (3). Ultrastructural work suggested that HpVirB10 is also a major subunit of the Cag T4S system pilus (31, 35), but clear evidence that either HpVirB2 or HpVirB10 is the main pilus subunit is still lacking. CagL (HP0539) has been identified (29) as an adhesin (functionally similar to VirB5) whose binding to host cell receptors is required for activation of the secretion process, and CagF (HP0543) has been characterized as a CagA chaperone (17). CagD (HP0545) has been recently reported as a multifunctional Cag T4S component essential for CagA translocation and full IL-8 secretion induction (12).We have characterized the biochemical role of an additional essential H. pylori-specific gene, HP0522/cag3, in Cag T4S. A previous yeast two-hybrid screen that investigated interactions among cag PAI proteins suggested Cag3 could interact with HpVirB8, HpVirB7, CagM (HP0537), and CagG (HP0542) (10). To begin to understand the molecular basis of Cag3 function in T4S we investigated the subcellular localization of the Cag3 protein and the protein-protein interactions this protein establishes in H. pylori cells. We found evidence suggesting that Cag3 is an integral part of the Cag T4S outer membrane subcomplex required to maintain HpVirB7 levels.