RESUMO
We assembled a collection of 73 Capnocytophaga canimorsus isolates obtained from blood cultures taken from patients treated at Helsinki University Hospital (Helsinki, Finland) during 2000-2017. We serotyped these isolates by PCR and Western blot and attempted to correlate pathogen serovar with patient characteristics. Our analyses showed, in agreement with previous research, that 3 C. canimorsus serovars (A-C) caused most (91.8%) human infections, despite constituting only 7.6% of isolates found in dogs. The 3 fatalities that occurred in our cohort were equally represented by these serovars. We found 2 untypeable isolates, which we designated serovars J and K. We did not detect an association between serovar and disease severity, immune status, alcohol abuse, or smoking status, but dog bites occurred more frequently among patients infected with non-A-C serovars. Future research is needed to confirm serovar virulence and develop strategies to reduce risk for these infections in humans.
Assuntos
Capnocytophaga/classificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Doenças dos Animais/epidemiologia , Doenças dos Animais/microbiologia , Animais , Capnocytophaga/genética , Capnocytophaga/imunologia , Capnocytophaga/isolamento & purificação , Gatos , Cães , Finlândia/epidemiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/história , História do Século XXI , Humanos , RNA Ribossômico 16S/genética , Sorogrupo , Índice de Gravidade de Doença , VirulênciaRESUMO
Capnocytophaga canimorsus is a dog oral commensal that causes rare but severe infections in humans. C. canimorsus was recently shown to be endowed with a capsular polysaccharide implicated in resistance to the innate immune system of the host. Here, we developed the first C. canimorsus capsular serotyping scheme. We describe nine different serovars (A to I), and this serotyping scheme allowed typing of 25/25 isolates from human infections but only 18/52 isolates from dog mouths, indicating that the repertoire of capsules in the species is vast. However, while only three serovars (A, B, and C) covered 88% of the human isolates tested (22/25), they covered only 7.7% of the dog isolates (4/52). Serovars A, B, and C were found 22.9-, 14.6-, and 4.2-fold more often, respectively, among human isolates than among dog isolates, with no geographical bias, implying that isolates endowed with these three capsular types are more virulent for humans than other isolates. Capsular serotyping would thus allow identification of virulent isolates in dogs, which could contribute to the prevention of these infections. To this end, we developed a PCR typing method based on the amplification of specific capsular genes.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Capnocytophaga/classificação , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Polissacarídeos Bacterianos/análise , Sorotipagem/métodos , Fatores de Virulência/análise , Animais , Capnocytophaga/isolamento & purificação , Cães , HumanosRESUMO
Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus.
Assuntos
Cápsulas Bacterianas/química , Capnocytophaga/química , Lipopolissacarídeos/química , Polissacarídeos Bacterianos/química , Animais , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Capnocytophaga/imunologia , Capnocytophaga/patogenicidade , Gatos , Cães , Infecções por Bactérias Gram-Negativas/imunologia , Humanos , Lipopolissacarídeos/imunologia , Polissacarídeos Bacterianos/imunologiaRESUMO
Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.
Assuntos
Capnocytophaga/genética , Capnocytophaga/metabolismo , Lipídeo A/biossíntese , Fosfatos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Capnocytophaga/efeitos dos fármacos , Capnocytophaga/enzimologia , Cães , Genes Bacterianos/genética , Teste de Complementação Genética , Genoma Bacteriano , Humanos , Lipídeo A/química , Lipídeo A/genética , Espectrometria de Massas , Óperon , Deleção de SequênciaRESUMO
Capnocytophaga canimorsus and Capnocytophaga cynodegmi can be transmitted from dogs or cats and cause serious human infections. We aimed to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify these two Capnocytophaga species. Ninety-four C. canimorsus and 10 C. cynodegmi isolates identified by 16S rRNA gene sequencing were analyzed. Using the MALDI BioTyper database, correct identification was achieved for only 16 of 94 (17%) C. canimorsus and all 10 C. cynodegmi strains, according to the manufacturer's log score specifications. Following the establishment of a complementary homemade reference database by addition of 51 C. canimorsus and 8 C. cynodegmi mass spectra, MALDI-TOF MS provided reliable identification to the species level for 100% of the 45 blind-coded Capnocytophaga isolates tested. MALDI-TOF MS can accurately identify C. canimorsus and C. cynodegmi using an enriched database and thus constitutes a valuable diagnostic tool in the clinical laboratory.
Assuntos
Técnicas Bacteriológicas/métodos , Capnocytophaga/isolamento & purificação , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/veterinária , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Zoonoses/diagnóstico , Animais , Capnocytophaga/química , Gatos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Cães , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Zoonoses/microbiologiaRESUMO
Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.
Assuntos
Sistemas de Secreção Tipo III/farmacologia , Células 3T3 , Animais , Apoptose , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Peixe-ZebraRESUMO
Capnocytophaga canimorsus is a bacterium from the normal oral flora of dogs and cats that causes rare generalized infections in humans. In an attempt to determine whether infections could be caused by a subset of strains and to identify pathogenicity factors, we sequenced the genomes of three strains isolated from human infections.
RESUMO
Here, we present the draft genome sequences of three strains of Capnocytophaga canimorsus, each isolated from a different dog's mouth. Genome analysis provided evidence that these organisms may belong to a different nonpathogenic subtype of C. canimorsus.
RESUMO
Here, we present the draft genome sequences of three strains of Capnocytophaga cynodegmi. In contrast to the very close relationship among them, C. cynodegmi and Capnocytophaga canimorsus differ dramatically in terms of virulence in humans. Comparative genomics provided some understanding on how Capnocytophaga species may switch from being dog commensals to human pathogens.
RESUMO
Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca(2+) ), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca(2+) ), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100 nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Transporte Biológico , Tomografia com Microscopia Eletrônica , Microscopia de Fluorescência , Regulação para Cima , Yersinia enterocolitica/ultraestruturaRESUMO
Capnocytophaga canimorsus, a dog mouth commensal and a member of the Bacteroidetes phylum, causes rare but often fatal septicemia in humans that have been in contact with a dog. Here, we show that C. canimorsus strains isolated from human infections grow readily in heat-inactivated human serum and that this property depends on a typical polysaccharide utilization locus (PUL), namely, PUL3 in strain Cc5. PUL are a hallmark of Bacteroidetes, and they encode various products, including surface protein complexes that capture and process polysaccharides or glycoproteins. The archetype system is the Bacteroides thetaiotaomicron Sus system, devoted to starch utilization. Unexpectedly, PUL3 conferred the capacity to acquire iron from serotransferrin (STF), and this capacity required each of the seven encoded proteins, indicating that a whole Sus-like machinery is acting as an iron capture system (ICS), a new and unexpected function for Sus-like machinery. No siderophore could be detected in the culture supernatant of C. canimorsus, suggesting that the Sus-like machinery captures iron directly from transferrin, but this could not be formally demonstrated. The seven genes of the ICS were found in the genomes of several opportunistic pathogens from the Capnocytophaga and Prevotella genera, in different isolates of the severe poultry pathogen Riemerella anatipestifer, and in strains of Bacteroides fragilis and Odoribacter splanchnicus isolated from human infections. Thus, this study describes a new type of ICS that evolved in Bacteroidetes from a polysaccharide utilization system and most likely represents an important virulence factor in this group.
Assuntos
Bacteroidetes/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Bacteroidetes/genética , Bacteroidetes/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Soro/microbiologiaRESUMO
We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.
Assuntos
Capnocytophaga/química , Lipopolissacarídeos/química , Espectroscopia de Ressonância Magnética/métodos , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Lipopolissacarídeos/isolamento & purificação , Dados de Sequência MolecularRESUMO
The type III secretion system machinery, also known as the injectisome, delivers bacterial effector proteins into eukaryotic cells during infection. The outer membrane YscC secretin is a major part of Yersinia enterocolitica's injectisome and is among the first components to assemble, solely assisted by its pilotin, YscW. We have determined the three-dimensional structures of the native complex and its protease-resistant core to 12 Å resolution by cryo-electron microscopy (cryo-EM) and show that YscC forms a dodecameric complex. Cryo-EM of YscC reconstituted into proteoliposomes defines the secretin's membrane-spanning region. Native YscC consists of an outer membrane ring connected via a thin cylindrical wall to a conical, periplasmic region that exposes N-terminal petals connected by flexible linkers. These petals harbor the binding site of YscD, a component of the inner membrane ring. A change in their orientation adapts the length of the YscC secretin and facilitates its interaction with YscD.
Assuntos
Proteínas de Bactérias/química , Secretina/química , Tripsina/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Pressão Osmótica , Conformação Proteica , Multimerização Proteica , Secretina/genética , Secretina/metabolismoRESUMO
Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30-40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance. DOI:http://dx.doi.org/10.7554/eLife.00792.001.
Assuntos
Proteínas de Membrana/metabolismo , Yersinia enterocolitica/metabolismo , Microscopia Crioeletrônica , Proteínas de Membrana/química , Pressão Osmótica , Conformação ProteicaRESUMO
The assembly of the type III secretion injectisome culminates in the formation of the needle. In Yersinia, this step requires not only the needle subunit (YscF), but also the small components YscI, YscO, YscX and YscY. We found that these elements act after the completion of the transmembrane export apparatus. YscX and YscY co-purified with the export apparatus protein YscV, even in the absence of any other protein. YscY-EGFP formed fluorescent spots, suggesting its presence in multiple copies. YscO and YscX were required for export of the early substrates YscF, YscI and YscP, but were only exported themselves after the substrate specificity switch had occurred. Unlike its flagellar homologue FliJ, YscO was not required for the assembly of the ATPase YscN. Finally, we investigated the role of the small proteins in export across the inner membrane. No export of the reporter substrate YscP(1-137) -PhoA into the periplasm was observed in absence of YscI, YscO or YscX, confirming that these proteins are required for export of the first substrates. In contrast, YscP(1-137) -PhoA accumulated in the periplasm in the absence of YscF, suggesting that YscF is not required for the function of the export apparatus, but that its polymerization opens the secretin YscC.
Assuntos
Proteínas de Bactérias/metabolismo , Yersinia/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Immunoblotting , Imunoprecipitação , Lipoproteínas/genética , Lipoproteínas/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Microscopia de Fluorescência , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Yersinia/genéticaRESUMO
The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.
Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/metabolismo , Fibrose Cística/microbiologia , Proteínas de Membrana/metabolismo , Infecções por Pseudomonas/genética , Pseudomonas aeruginosa , Transdução de Sinais/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fibrose Cística/complicações , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Conformação Proteica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Infecções Respiratórias/genética , Infecções Respiratórias/microbiologiaRESUMO
Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a "hybrid backbone" lacking the 4' phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 - lipid A complex in case the 4' phosphate is not present.
Assuntos
Capnocytophaga/patogenicidade , Endotoxinas/química , Endotoxinas/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Proteínas de Fase Aguda/metabolismo , Animais , Antígenos CD/metabolismo , Capnocytophaga/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Cães , Células HEK293 , Humanos , Interleucina-6/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Açúcares Ácidos/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in Yersinia enterocolitica) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.