RESUMEN
Many viral, protozoal, and fungal pathogens represent major human and animal health problems due to their great potential of causing infectious diseases. Research on these pathogens has contributed substantially to our current understanding of both microbial virulence determinants and host key factors during infection. Countless studies have also shed light on the molecular mechanisms of host-pathogen interactions that are employed by these microbes. For example, actin cytoskeletal dynamics play critical roles in effective adhesion, host cell entry, and intracellular movements of intruding pathogens. Cortactin is an eminent host cell protein that stimulates actin polymerization and signal transduction, and recently emerged as fundamental player during host-pathogen crosstalk. Here we review the important role of cortactin as major target for various prominent viral, protozoal and fungal pathogens in humans, and its role in human disease development and cancer progression. Most if not all of these important classes of pathogens have been reported to hijack cortactin during infection through mediating up- or downregulation of cortactin mRNA and protein expression as well as signaling. In particular, pathogen-induced changes in tyrosine and serine phosphorylation status of cortactin at its major phospho-sites (Y-421, Y-470, Y-486, S-113, S-298, S-405, and S-418) are addressed. As has been reported for various Gram-negative and Gram-positive bacteria, many pathogenic viruses, protozoa, and fungi also control these regulatory phospho-sites, for example, by activating kinases such as Src, PAK, ERK1/2, and PKD, which are known to phosphorylate cortactin. In addition, the recruitment of cortactin and its interaction partners, like the Arp2/3 complex and F-actin, to the contact sites between pathogens and host cells is highlighted, as this plays an important role in the infection process and internalization of several pathogens. However, there are also other ways in which the pathogens can exploit the function of cortactin for their needs, as the cortactin-mediated regulation of cellular processes is complex and involves numerous different interaction partners. Here, the current state of knowledge is summarized.
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Cortactina , Hongos , Interacciones Huésped-Patógeno , Cortactina/metabolismo , Humanos , Animales , Hongos/metabolismo , Hongos/patogenicidad , Virus/metabolismo , Virus/patogenicidad , Transducción de Señal , Fosforilación , Virosis/metabolismoRESUMEN
Gastric cancer is a very serious and deadly disease worldwide with about one million new cases every year. Most gastric cancer subtypes are associated with genetic and epigenetic aberrations caused by chromosome instability, microsatellite instability or Epstein-Barr virus infection. Another risk factor is an infection with Helicobacter pylori, which also triggers severe alterations in the host genome. This pathogen expresses an extraordinary repertoire of virulence determinants that take over control of important host cell signaling functions. In fact, H. pylori is a paradigm of persistent infection, chronic inflammation and cellular destruction. In particular, H. pylori profoundly induces chromosomal DNA damage by introducing double-strand breaks (DSBs) followed by genomic instability. DSBs appear in response to oxidative stress and pro-inflammatory transcription during the S-phase of the epithelial cell cycle, which mainly depends on the presence of the bacterial cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). This scenario is closely connected with the T4SS-mediated injection of ADP-glycero-ß-D-manno-heptose (ADP-heptose) and oncoprotein CagA. While ADP-heptose links transcription factor NF-κB-induced innate immune signaling with RNA-loop-mediated DNA replication stress and introduction of DSBs, intracellular CagA targets the tumor suppressor BRCA1. The latter scenario promotes BRCAness, a disease characterized by the deficiency of effective DSB repair. In addition, genetic studies of patients demonstrated the presence of gastric cancer-associated single nucleotide polymorphisms (SNPs) in immune-regulatory and other genes as well as specific pathogenic germline variants in several crucial genes involved in homologous recombination and DNA repair, all of which are connected to H. pylori infection. Here we review the molecular mechanisms leading to chromosomal DNA damage and specific genetic aberrations in the presence or absence of H. pylori infection, and discuss their importance in gastric carcinogenesis.
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Infecciones por Virus de Epstein-Barr , Helicobacter pylori , Neoplasias Gástricas , Humanos , ADN , Daño del ADN , Helicobacter pylori/genética , Heptosas , Herpesvirus Humano 4 , Neoplasias Gástricas/genéticaRESUMEN
Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions.
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Adhesinas Bacterianas , Helicobacter pylori , Leucina , Serina , Helicobacter pylori/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Humanos , Serina/genética , Serina/metabolismo , Leucina/genética , Leucina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/genética , AnimalesRESUMEN
Pathogenic bacteria possess a great potential of causing infectious diseases and represent a serious threat to human and animal health. Understanding the molecular basis of infection development can provide new valuable strategies for disease prevention and better control. In host-pathogen interactions, actin-cytoskeletal dynamics play a crucial role in the successful adherence, invasion, and intracellular motility of many intruding microbial pathogens. Cortactin, a major cellular factor that promotes actin polymerization and other functions, appears as a central regulator of host-pathogen interactions and different human diseases including cancer development. Various important microbes have been reported to hijack cortactin signaling during infection. The primary regulation of cortactin appears to proceed via serine and/or tyrosine phosphorylation events by upstream kinases, acetylation, and interaction with various other host proteins, including the Arp2/3 complex, filamentous actin, the actin nucleation promoting factor N-WASP, focal adhesion kinase FAK, the large GTPase dynamin-2, the guanine nucleotide exchange factor Vav2, and the actin-stabilizing protein CD2AP. Given that many signaling factors can affect cortactin activities, several microbes target certain unique pathways, while also sharing some common features. Here we review our current knowledge of the hallmarks of cortactin as a major target for eminent Gram-negative and Gram-positive bacterial pathogens in humans.
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Actinas , Cortactina , Bacterias Gramnegativas , Bacterias Grampositivas , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cortactina/metabolismo , Citoesqueleto/metabolismo , FosforilaciónRESUMEN
The human pathogen Helicobacter pylori is a major risk factor for gastric disease development. Serine protease HtrA is an important bacterial virulence factor that cleaves the cell junction proteins occludin, claudin-8 and E-cadherin, which causes gastric tissue damage. Using casein zymography, we discovered that HtrA trimer stability varies in clinical H. pylori strains. Subsequent sequence analyses revealed that HtrA trimer stability correlated with the presence of leucine or serine residue at position 171. The importance of these amino acids in determining trimer stability was confirmed by leucine-to-serine swapping experiments using isogenic H. pylori mutant strains as well as recombinant HtrA proteins. In addition, this sequence position displays a high sequence variability among various bacterial species, but generally exhibits a preference for hydrophilic amino acids. This natural L/S171 polymorphism in H. pylori may affect the protease activity of HtrA during infection, which could be of clinical importance and may determine gastric disease development.
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Helicobacter pylori , Humanos , Proteínas Bacterianas/metabolismo , Leucina/genética , Leucina/metabolismo , Serina Proteasas/genética , Serina Proteasas/metabolismo , Proteínas Recombinantes/genética , Mutación , Serina/genética , Serina/metabolismoRESUMEN
The human gastric pathogen Helicobacter pylori is a major causative agent of gastritis, peptic ulcer disease, and gastric cancer. As part of its adhesive lifestyle, the bacterium targets members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family by the conserved outer membrane adhesin HopQ. The HopQ-CEACAM1 interaction is associated with inflammatory responses and enables the intracellular delivery and phosphorylation of the CagA oncoprotein via a yet unknown mechanism. Here, we generated crystal structures of HopQ isotypes I and II bound to the N-terminal domain of human CEACAM1 (C1ND) and elucidated the structural basis of H. pylori specificity toward human CEACAM receptors. Both HopQ alleles target the ß-strands G, F, and C of C1ND, which form the trans dimerization interface in homo- and heterophilic CEACAM interactions. Using SAXS, we show that the HopQ ectodomain is sufficient to induce C1ND monomerization and thus providing H. pylori a route to influence CEACAM-mediated cell adherence and signaling events.
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Antígenos CD/fisiología , Proteínas Bacterianas/fisiología , Moléculas de Adhesión Celular/fisiología , Helicobacter pylori/fisiología , Animales , Antígenos CD/química , Proteínas Bacterianas/química , Células CHO , Moléculas de Adhesión Celular/química , Línea Celular Tumoral , Cricetulus , Humanos , Multimerización de ProteínaRESUMEN
Campylobacter jejuni and Campylobacter coli can be frequently isolated from poultry and poultry-derived products, and in combination these two species cause a large portion of human bacterial gastroenteritis cases. While birds are typically colonized by these Campylobacter species without clinical symptoms, in humans they cause (foodborne) infections at high frequencies, estimated to cost billions of dollars worldwide every year. The clinical outcome of Campylobacter infections comprises malaise, diarrhea, abdominal pain and fever. Symptoms may continue for up to two weeks and are generally self-limiting, though occasionally the disease can be more severe or result in post-infection sequelae. The virulence properties of these pathogens have been best-characterized for C. jejuni, and their actions are reviewed here. Various virulence-associated bacterial determinants include the flagellum, numerous flagellar secreted factors, protein adhesins, cytolethal distending toxin (CDT), lipooligosaccharide (LOS), serine protease HtrA and others. These factors are involved in several pathogenicity-linked properties that can be divided into bacterial chemotaxis, motility, attachment, invasion, survival, cellular transmigration and spread to deeper tissue. All of these steps require intimate interactions between bacteria and host cells (including immune cells), enabled by the collection of bacterial and host factors that have already been identified. The assortment of pathogenicity-associated factors now recognized for C. jejuni, their function and the proposed host cell factors that are involved in crucial steps leading to disease are discussed in detail.
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Campylobacter coli , Campylobacter jejuni , Campylobacter , Campylobacter jejuni/genética , Interacciones Huésped-Patógeno , Humanos , Factores de Virulencia/genéticaRESUMEN
Cortactin represents an important actin-binding factor, which controls actin-cytoskeletal remodelling in host cells. In this way, cortactin has been shown to exhibit crucial functions both for cell movement and tumour cell invasion. In addition, the cortactin gene cttn is amplified in various cancer types of humans. Helicobacter pylori is the causative agent of multiple gastric diseases and represents a significant risk factor for the development of gastric adenocarcinoma. It has been repeatedly shown that H. pylori manipulates cancer-related signal transduction events in infected gastric epithelial cells such as the phosphorylation status of cortactin. In fact, H. pylori modifies the activity of cortactin's binding partners to stimulate changes in the actin-cytoskeleton, cell adhesion and motility. Here we show that H. pylori infection of cultured AGS and Caco-2 cells for 24-48 hr leads to the overexpression of cortactin by 2-3 fold at the protein level. We demonstrate that this activity requires the integrity of the type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI) as well as the translocated effector protein CagA. We further show that ectopic expression of CagA is sufficient to stimulate cortactin overexpression. Furthermore, phosphorylation of CagA at the EPIYA-repeat region is not required, suggesting that this CagA activity proceeds in a phosphorylation-independent fashion. Inhibitor studies further demonstrate that the involved signalling pathway comprises the mitogen-activated protein kinase JNK (c-Jun N-terminal kinase), but not ERK1/2 or p38. Taken together, using H. pylori as a model system, this study discovered a previously unrecognised cortactin activation cascade by a microbial pathogen. We suggest that H. pylori targets cortactin to manipulate the cellular architecture and epithelial barrier functions that can impact gastric cancer development. TAKE AWAYS: Helicobacter pylori infection induces overexpression of cortactin at the protein level Cortactin upregulation requires the T4SS and effector protein CagA Ectopic expression of CagA is sufficient to stimulate cortactin overexpression Overexpression of cortactin proceeds CagA phosphorylation-independent The involved host cell signalling pathway comprises the MAP kinase JNK.
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Cortactina/metabolismo , Infecciones por Helicobacter , Helicobacter pylori , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células CACO-2 , Cortactina/genética , Células Epiteliales/metabolismo , Helicobacter pylori/metabolismo , Humanos , Fosforilación , Sistemas de Secreción Tipo IVRESUMEN
The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.
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Infecciones por Helicobacter , Helicobacter pylori , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citotoxinas/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Cortactin is a well-known regulatory protein of the host actin cytoskeleton and represents an attractive target of microbial pathogens like Helicobacter pylori. H. pylori manipulates cortactin's phosphorylation status by type-IV secretion-dependent injection of its virulence protein CagA. Multiple host tyrosine kinases, like FAK, Src, and Abl, are activated during infection, but the pathway(s) involved is (are) not yet fully established. Among them, Src and Abl target CagA and stimulate tyrosine phosphorylation of the latter at its EPIYA-motifs. To investigate the role of cortactin in more detail, we generated a CRISPR/Cas9 knockout of cortactin in AGS gastric epithelial cells. Surprisingly, we found that FAK, Src, and Abl kinase activities were dramatically downregulated associated with widely diminished CagA phosphorylation in cortactin knockout cells compared to the parental control. Together, we report here a yet unrecognized cortactin-dependent signaling pathway involving FAK, Src, and Abl activation, and controlling efficient phosphorylation of injected CagA during infection. Thus, the cortactin status could serve as a potential new biomarker of gastric cancer development.
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Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Quinasa 1 de Adhesión Focal/genética , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Proteínas Oncogénicas v-abl/genética , Regulación Bacteriana de la Expresión Génica/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Fosforilación/genética , Familia-src Quinasas/genéticaRESUMEN
Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c-Src and c-Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ-521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA-dependent fashion. In contrast, we discovered that AZ-521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin-ß1 or carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ-521 cells readily express integrin-ß1 , but overexpression of integrin-ß1 constructs did not restore the T4SS defect. We further show that AZ-521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ-521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ-521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori-infected AGS cells. Our results suggest the existence of an integrin-ß1 - and CEACAM1- or CEACAM5-dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.
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Antígenos Bacterianos/metabolismo , Antígenos CD/metabolismo , Proteínas Bacterianas/metabolismo , Antígeno Carcinoembrionario/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Helicobacter pylori/metabolismo , Interacciones Huésped-Patógeno , Línea Celular , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Transporte de Proteínas , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Helicobacter pylori is a very successful Gram-negative pathogen colonizing the stomach of humans worldwide. Infections with this bacterium can generate pathologies ranging from chronic gastritis and peptic ulceration to gastric cancer. The best characterized H. pylori virulence factors that cause direct cell damage include an effector protein encoded by the cytotoxin-associated gene A (CagA), a type IV secretion system (T4SS) encoded in the cag-pathogenicity island (cag PAI), vacuolating cytotoxin A (VacA), γ-glutamyl transpeptidase (GGT), high temperature requirement A (HtrA, a serine protease) and cholesterol glycosyl-transferase (CGT). Since these H. pylori factors are either surface-exposed, secreted or translocated, they can directly interact with host cell molecules and are able to hijack cellular functions. Studies on these bacterial factors have progressed substantially in recent years. Here, we review the current status in the characterization of signaling cascades by these factors in vivo and in vitro, which comprise the disruption of cell-to-cell junctions, induction of membrane rearrangements, cytoskeletal dynamics, proliferative, pro-inflammatory, as well as, pro-apoptotic and anti-apoptotic responses or immune evasion. The impact of these signal transduction modules in the pathogenesis of H. pylori infections is discussed.
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Infecciones por Helicobacter , Helicobacter pylori , Factores de Virulencia , Proteínas Bacterianas/metabolismo , Islas Genómicas , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , HumanosRESUMEN
Helicobacter pylori is a paradigm of persistent pathogens and major risk factor for developing severe diseases including adenocarcinoma in the human stomach. An important bacterial factor linked to gastric disease progression is the cag pathogenicity island-encoded type-IV secretion system (T4SS) effector protein CagA. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-motifs and then activates or inactivates multiple host signaling proteins in a phosphorylation-dependent and phosphorylation-independent fashion. In this way, intracellular CagA acts as a 'masterkey' or 'picklock', which evolved during evolution to hijack key host cell signal transduction functions. Crucial targets of CagA represent a variety of serine/threonine and tyrosine kinases, which control major checkpoints of eukaryotic signaling. Here we review the signal transmission by translocated CagA on multiple receptor kinases (c-Met and EGFR) and non-receptor kinases (Src, Abl, Csk, aPKC, Par1, PI3K, Akt, FAK, GSK-3, JAK, PAK1, PAK2 and MAP kinases), manipulating a selection of fundamental processes in the human gastric epithelium such as cell adhesion, polarity, proliferation, motility, receptor endocytosis, cytoskeletal rearrangements, apoptosis, inflammation and cell cycle progression. This enormous complexity generates a highly remarkable and puzzling scenario during H. pylori infection. The contribution of these signaling pathways to bacterial survival, persistence and gastric pathogenesis is discussed.
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Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Helicobacter pylori/genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mucosa Gástrica/metabolismo , Islas Genómicas , Glucógeno Sintasa Quinasa 3/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Interacciones Huésped-Patógeno , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Fosfotransferasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Sistemas de Secreción Tipo IV/metabolismoRESUMEN
Culture-independent studies have identified DNA of bacterial pathogens in the gallbladder under pathological conditions, yet reports on the isolation of corresponding live bacteria are rare. Thus, it is unclear which pathogens, or pathogen communities, can colonize the gallbladder and cause disease. Using light microscopy, scanning electron microscopy, culture techniques, phylogenetic analysis, urease assays and Western blotting, we investigated the presence of live bacterial communities in the gallbladder of a cholecystitis patient after cholecystectomy. 16S rRNA gene sequencing of isolated bacterial colonies revealed the presence of pathogens most closely resembling Corynebacterium urinapleomorphum nov. sp., Staphylococcus saprophyticus and Helicobacter pylori. The latter colonies were confirmed as H. pylori by immunohistochemistry and biochemical methods. H. pylori cultured from the gallbladder exhibited both the same DNA fingerprinting and Western cagA gene sequence with ABC-type EPIYA (Glu-Pro-Ile-Tyr-Ala) phosphorylation motifs as isolates recovered from the gastric mucus of the same patient, suggesting that gastric H. pylori can also colonize other organs in the human body. Taken together, here we report, for the first time, the identification and characterization of a community consisting of live S. saprophyticus; C. urinapleomorphum, and H. pylori in the gallbladder of a patient with acute cholecystitis. Their potential infection routes and roles in pathogenesis are discussed.
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Infecciones Bacterianas/microbiología , Colecistitis Aguda/microbiología , Corynebacterium/patogenicidad , Vesícula Biliar/microbiología , Helicobacter pylori/patogenicidad , Staphylococcus saprophyticus/patogenicidad , Antígenos Bacterianos/genética , Infecciones Bacterianas/patología , Infecciones Bacterianas/cirugía , Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Colecistitis Aguda/patología , Colecistitis Aguda/cirugía , Corynebacterium/clasificación , Corynebacterium/genética , Corynebacterium/aislamiento & purificación , Vesícula Biliar/patología , Vesícula Biliar/cirugía , Expresión Génica , Helicobacter pylori/clasificación , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Filogenia , ARN Ribosómico 16S/genética , Staphylococcus saprophyticus/clasificación , Staphylococcus saprophyticus/genética , Staphylococcus saprophyticus/aislamiento & purificación , Estómago/microbiología , Estómago/patologíaRESUMEN
HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour-suppressor E-cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA-sequencing (dRNA-seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacterâ htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti-bacterial therapy.
Asunto(s)
Helicobacter pylori/enzimología , Helicobacter pylori/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cadherinas/genética , Cadherinas/metabolismo , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Bacterianos , Genes Esenciales , Variación Genética , Humanos , Datos de Secuencia Molecular , Operón , Periplasma/genética , Periplasma/metabolismo , Análisis de Secuencia de ARNAsunto(s)
Helicobacter pylori , Integrina alfaV , Integrina beta1 , Integrinas , Elevación , FenotipoRESUMEN
Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Células Epiteliales/microbiología , Helicobacter pylori/genética , Polimorfismo de Nucleótido Simple , Secuencias de Aminoácidos , Línea Celular , Técnicas de Cocultivo , Interacciones Huésped-Patógeno/genética , Humanos , Immunoblotting , Inmunoprecipitación , FosforilaciónRESUMEN
BACKGROUND: Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming. RESULTS: This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells. CONCLUSIONS: The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/inmunología , Fosfotirosina/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/aislamiento & purificación , Fosfotirosina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Alineación de Secuencia , Transducción de Señal , Estómago/microbiología , Estómago/patología , Neoplasias Gástricas/microbiología , Sistemas de Secreción Tipo IVRESUMEN
The Helicobacter pylori gene JHP0940 has been shown to encode a serine/threonine kinase which can induce cytokines in gastric epithelial cells relevant to chronic gastric inflammation. Here we demonstrate that JHP0940 can be secreted by the bacteria, triggers apoptosis in cultured mouse macrophages and acts as an auto-phosphorylating tyrosine kinase. Recombinant JHP0940 protein was found to decrease the viability of RAW264.7 cells (a mouse macrophage cell line) up to 55% within 24h of co-incubation. The decreased cellular viability was due to apoptosis, which was confirmed by TUNEL assay and Fas expression analysis by flow-cytometry. Further, we found that caspase-1 and IL-1beta were activated upon treatment with JHP0940. These results point towards possible action through the host inflammasome. Our in vitro studies using tyrosine kinase assays further demonstrated that JHP0940 acts as auto-phosphorylating tyrosine kinase and induces pro-inflammatory cytokines in RAW264.7 cells. Upon exposure with JHP0940, these cells secreted IL-1beta, TNF-alpha and IL-6, in a dose- and time-dependent manner, as detected by ELISA and transcript profiling by q-RT-PCR. The pro-inflammatory, pro-apoptotic and other regulatory responses triggered by JHP0940 lead to the assumption of its possible role in inducing chronic inflammation for enhanced bacterial persistence and escape from host innate immune responses by apoptosis of macrophages.
Asunto(s)
Apoptosis , Proteínas Bacterianas/metabolismo , Helicobacter pylori/enzimología , Interacciones Huésped-Patógeno , Macrófagos/microbiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Virulencia/metabolismo , Animales , Línea Celular , Supervivencia Celular , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Macrófagos/fisiología , Ratones , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
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.