RESUMO
Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.
Assuntos
Chlamydia trachomatis/fisiologia , Proteínas de Bactérias , Biotinilação , Chlamydia trachomatis/genética , Chlamydia trachomatis/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes , EstreptavidinaRESUMO
The obligate intracellular bacterial pathogen, Chlamydia trachomatis, develops within a membrane-bound vacuole termed the inclusion. Affinity purification-mass spectrometry (AP-MS) experiments to study the interactions that occur at the chlamydial inclusion membrane have been performed and, more recently, combined with advances in C. trachomatis genetics. However, each of the four AP-MS published reports used either different experimental approaches or statistical tools to identify proteins that localize at the inclusion. We critically analyzed each experimental approach and performed a meta-analysis of the reported statistically significant proteins for each study, finding that only a few eukaryotic proteins were commonly identified between all four experimental approaches. The two similarly conducted in vivo labeling studies were compared using the same statistical analysis tool, Significance Analysis of INTeractome (SAINT), which revealed a disparity in the number of significant proteins identified by the original analysis. We further examined methods to identify potential background contaminant proteins that remain after statistical analysis. Overall, this meta-analysis highlights the importance of carefully controlling and analyzing the AP-MS data so that pertinent information can be obtained from these various AP-MS experimental approaches. This study provides important guidelines and considerations for using this methodology to study intracellular pathogens residing within a membrane-bound compartment. SIGNIFICANCE: Chlamydia trachomatis, an obligate intracellular pathogen, grows within a membrane-bound vacuole termed the inclusion. The inclusion is studded with bacterial membrane proteins that likely orchestrate numerous interactions with the host cell. Although maintenance of the intracellular niche is vital, an understanding of the host-pathogen interactions that occur at the inclusion membrane is limited by the difficulty in purifying membrane protein fractions from infected host cells. The experimental procedures necessary to solubilize hydrophobic proteins fail to maintain transient protein-protein interactions. Advances in C. trachomatis genetics has allowed us and others to use various experimental approaches in combination with affinity purification mass spectrometry (AP-MS) to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. For the first time, two groups have published AP-MS studies using the same tool, the ascorbate peroxidase proximity labeling system (APEX2), which overcomes past experimental limitations because membrane protein interactions are labeled in vivo in the context of infection. The utility of this system is highlighted by its ability to study chlamydial type III secreted inclusion membrane protein (Inc) interactions. Incs act as the mediators of host-pathogen interactions at the inclusion during C. trachomatis infection. When carefully controlled and analyzed, the data obtained can yield copious amounts of useful information. Here, we critically analyzed four previously published studies, including statistical analysis of AP-MS datasets related to Chlamydia-host interactions, to contextualize the data and to identify the best practices in interpreting these types of complex outputs.
Assuntos
Proteínas de Bactérias/análise , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Eucariotos/metabolismo , Corpos de Inclusão/metabolismo , Proteínas de Membrana/análise , Proteômica/métodos , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/isolamento & purificação , Cromatografia de Afinidade/métodos , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Corpos de Inclusão/microbiologia , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Vacúolos/química , Vacúolos/metabolismo , Vacúolos/microbiologiaRESUMO
In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host-pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein-protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein-protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3'-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.
Assuntos
Infecções por Chlamydia/patologia , Chlamydia trachomatis/fisiologia , Interações Hospedeiro-Patógeno , Corpos de Inclusão/microbiologia , Ascorbato Peroxidases/análise , Proteínas de Bactérias/análise , Biotinilação , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/isolamento & purificação , Células HeLa , Humanos , Corpos de Inclusão/patologia , Coloração e Rotulagem/métodosRESUMO
The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Adenilil Ciclases/metabolismo , Clonagem Molecular/métodos , Escherichia coli/enzimologia , Vetores Genéticos , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido , Toxina Adenilato Ciclase/genética , Adenilil Ciclases/genética , AMP Cíclico/metabolismo , Óperon , Ligação ProteicaRESUMO
Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncA TM -APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.