Recombinant Shigella sonnei Uncharacterized protein ytcA (ytcA)

What is known about the basic properties of the ytcA protein in Shigella sonnei?

The ytcA protein from Shigella sonnei is currently classified as an uncharacterized protein, indicating limited knowledge about its structure, function, and biological role. Commercially available recombinant versions of this protein, such as that from MyBioSource, enable researchers to study its properties in vitro . Similar to other uncharacterized proteins in Shigella species, initial characterization typically involves sequence analysis, molecular weight determination, and comparison with homologous proteins in related bacterial species. While not specifically addressing ytcA, research on other uncharacterized Shigella proteins, like YfiH in S. flexneri, has progressed to crystal structure determination, offering a methodological blueprint for ytcA characterization .

How does the genomic context of the ytcA gene inform potential functional characterization approaches?

When studying an uncharacterized protein like ytcA, examining the genomic neighborhood can provide valuable insights into potential function. Researchers should analyze:

• Adjacent genes that may form an operon with ytcA

• Regulatory elements upstream of the coding sequence

• Conservation of the genetic locus across Shigella strains and related Enterobacteriaceae

• Presence of known functional domains or motifs

This genomic context analysis can suggest whether ytcA might be involved in metabolic pathways, stress response, virulence, or other cellular processes. Similar approaches have been employed in the study of other Shigella proteins such as YnfA, where genomic analysis helped establish its role in antimicrobial resistance .

What expression systems are most effective for producing recombinant ytcA protein for in vitro studies?

Based on methodologies employed for other Shigella proteins, researchers should consider multiple expression systems when working with recombinant ytcA:

For functional characterization studies, consider using expression vectors with cleavable affinity tags (His6, GST, etc.) to facilitate purification while allowing tag removal for downstream functional assays. Expression protocols similar to those used for S. flexneri proteins like YnfA could be adapted, which typically employ bacterial expression systems optimized for microbial protein production .

What are the most effective methods for determining the cellular localization of ytcA protein in Shigella sonnei?

Determining the cellular localization of ytcA is crucial for understanding its function. Researchers should employ a multi-faceted approach:

• Computational prediction tools:

  • SignalP for signal peptide prediction

  • TMHMM or HMMTOP for transmembrane domain prediction

  • PSORTb for general bacterial protein localization prediction

• Experimental approaches:

  • Subcellular fractionation followed by Western blotting

  • Immunofluorescence microscopy using anti-ytcA antibodies

  • Fusion protein approaches (ytcA-GFP) with appropriate controls

  • Protease accessibility assays if membrane association is predicted

The methodology should be similar to those employed in studies of other Shigella proteins. For instance, in the characterization of YnfA in S. flexneri, researchers employed computational predictions combined with experimental validation to establish its localization as a membrane-embedded efflux transporter .

What genetic approaches are most suitable for investigating the function of ytcA in Shigella sonnei?

Given the uncharacterized nature of ytcA, genetic manipulation approaches offer powerful tools for functional investigation:

• Gene knockout strategies:

  • CRISPR-Cas9 mediated deletion

  • Lambda Red recombination system for precise deletions

  • Transposon mutagenesis for initial screening

• Complementation and overexpression studies:

  • Plasmid-based complementation of knockout strains

  • Controlled overexpression using inducible promoters

  • Heterologous expression in other bacterial systems

• Phenotypic assessment:

  • Growth curve analysis under various conditions

  • Virulence assays in cellular and animal models

  • Stress response profiling (oxidative, acid, temperature stress)

  • Antimicrobial susceptibility testing

How can researchers resolve contradictory data when studying the function of ytcA?

When studying uncharacterized proteins like ytcA, contradictory results often emerge. A systematic approach to resolving such contradictions should include:

• Methodological validation:

  • Confirm antibody specificity with appropriate controls

  • Verify knockout strains by PCR and sequencing

  • Ensure recombinant protein folding through circular dichroism or limited proteolysis

• Multiple complementary approaches:

  • Combine in silico, in vitro, and in vivo methods

  • Use both gain-of-function and loss-of-function approaches

  • Employ different bacterial strains and growth conditions

• Rigorous controls:

  • Include wild-type strains in all experiments

  • Use empty vector controls for complementation studies

  • Perform rescue experiments with the native protein

• Contextual analysis:

  • Consider the impact of experimental conditions on protein function

  • Evaluate strain-specific differences in ytcA expression or function

  • Compare results with closely related proteins in other Shigella species

This approach mirrors successful strategies used in characterizing other initially confounding bacterial proteins, such as the efforts to understand the functional role of YnfA in S. flexneri, which required multiple complementary techniques to establish its role in antimicrobial resistance .

What computational approaches can predict the structure and potential function of ytcA?

For uncharacterized proteins like ytcA, computational approaches provide valuable initial insights:

• Sequence-based predictions:

  • Homology detection using PSI-BLAST and HMM-based methods

  • Motif identification using InterProScan and PROSITE

  • Secondary structure prediction using PSIPRED

• Structure prediction tools:

  • AlphaFold for accurate 3D structure prediction

  • I-TASSER for threading-based structure modeling

  • SWISS-MODEL for homology modeling if templates exist

• Functional inference:

  • Conserved domain analysis

  • Structural similarity to characterized proteins

  • Binding site prediction using COACH or 3DLigandSite

Such computational approaches have proven valuable in similar studies, such as the structural characterization of YnfA from S. flexneri, where I-TASSER was employed to predict its functional 3D structure, which was then validated using the AlphaFold protein structure database .

What experimental methods are most appropriate for determining the crystal structure of ytcA?

Based on approaches used for other Shigella proteins like YfiH , researchers should consider the following workflow for ytcA structural determination:

This comprehensive approach has been successfully applied to determine the crystal structure of the hypothetical protein YfiH from S. flexneri , providing a methodological framework for structural studies of ytcA.

How can researchers identify potential protein-protein interactions involving ytcA?

Understanding protein-protein interactions is crucial for deciphering the function of uncharacterized proteins like ytcA. Researchers should employ complementary approaches:

• In vivo methods:

  • Bacterial two-hybrid assays

  • Co-immunoprecipitation followed by mass spectrometry

  • Protein fragment complementation assays

• In vitro methods:

  • Pull-down assays with purified recombinant ytcA

  • Surface plasmon resonance for interaction kinetics

  • Isothermal titration calorimetry for binding thermodynamics

• Cross-linking approaches:

  • Chemical cross-linking coupled with mass spectrometry

  • Photo-cross-linking with modified amino acids

  • Proximity labeling approaches (BioID, APEX)

• Computational prediction:

  • Interolog mapping based on homologs in related species

  • Interface prediction using structural models

  • Co-evolution analysis of potentially interacting partners

When interpreting protein interaction data, researchers should be mindful that transient interactions may be missed, and false positives can occur. Validation through multiple methods is essential for establishing physiologically relevant interactions. Similar methodologies have been applied to study protein-protein interactions in Shigella research, including investigations of efflux transporters like YnfA .

How can researchers determine if ytcA plays a role in Shigella sonnei virulence?

To investigate the potential involvement of ytcA in S. sonnei virulence, researchers should consider a systematic approach combining genetic manipulation and infection models:

• Genetic manipulation:

  • Generate ytcA knockout strains

  • Create complemented strains expressing wild-type ytcA

  • Develop conditional expression systems if ytcA is essential

• In vitro infection models:

  • Epithelial cell invasion assays (e.g., HeLa, Caco-2)

  • Macrophage survival assays

  • Intracellular replication assessment

  • Cell-to-cell spread quantification

• Ex vivo approaches:

  • Intestinal tissue explant infection models

  • Organoid infection models representing human intestinal epithelium

• In vivo models:

  • Mouse pulmonary infection model

  • Guinea pig keratoconjunctivitis model

  • Human challenge model as used for S. sonnei strain 53G

The human challenge model described in search result provides a particularly relevant framework for evaluating virulence factors in S. sonnei. In this model, healthy adult volunteers were challenged with defined doses of S. sonnei strain 53G, with clinical disease endpoints carefully monitored . While such models are primarily used for vaccine development, they could potentially be adapted to study isogenic strains differing in ytcA expression, provided appropriate ethical approvals.

What approaches can determine if ytcA contributes to antimicrobial resistance in Shigella sonnei?

Given that some uncharacterized proteins in Shigella have been found to contribute to antimicrobial resistance, such as YnfA in S. flexneri , investigating ytcA's potential role in this area is warranted:

• Susceptibility testing:

  • Minimum inhibitory concentration (MIC) determination for wild-type versus ytcA knockout strains

  • Growth inhibition zone assays with various antimicrobials

  • Time-kill kinetics to assess bactericidal effects

• Efflux activity assessment:

  • Accumulation assays using fluorescent substrates (e.g., ethidium bromide, acriflavine)

  • Real-time efflux monitoring with fluorescence-based assays

  • Competitive transport assays with known efflux pump substrates

• Expression analysis:

  • qRT-PCR to measure ytcA expression in response to antibiotic exposure

  • Western blotting to quantify protein levels under different conditions

  • Transcriptome analysis to identify co-regulated genes

• Structural and computational approaches:

  • Molecular docking of antibiotics to predicted ytcA structure

  • Simulation of substrate transport if ytcA resembles known transporters

  • Mutational analysis of predicted binding sites

These approaches mirror those used in the study of YnfA in S. flexneri, where genetic, computational, and biochemical techniques demonstrated that disrupting the YnfA transporter rendered the mutant strain more susceptible to certain antimicrobial compounds and affected transport activity against ethidium bromide and acriflavine .

How does ytcA expression change during different stages of Shigella infection?

Understanding the temporal expression pattern of ytcA during infection can provide insights into its potential role. Researchers should consider:

• In vitro infection time course:

  • qRT-PCR analysis of ytcA expression at different infection stages

  • Western blot analysis of protein levels during infection

  • Reporter gene fusions (ytcA promoter-GFP) to monitor expression in real-time

• Transcriptional regulation analysis:

  • Identification of transcription factors binding to the ytcA promoter

  • Characterization of environmental signals influencing expression

  • ChIP-seq to identify DNA-protein interactions at the ytcA locus

• In vivo expression profiling:

  • RNA-seq from bacteria recovered from infection models

  • In vivo expression technology (IVET) to identify in vivo-induced genes

  • Recombination-based in vivo expression technology (RIVET) for temporal analysis

• Host response correlation:

  • Correlation of ytcA expression with host inflammatory markers

  • Analysis of expression in response to host defense mechanisms

  • Dual RNA-seq to simultaneously profile bacterial and host responses

This approach is informed by methodology used in studies of Shigella pathogenesis, including the human challenge model established for S. sonnei, which could potentially be leveraged to study gene expression during defined stages of infection .

How conserved is ytcA among different Shigella species and other Enterobacteriaceae?

Comparative genomic analysis of ytcA can provide evolutionary insights and functional clues:

• Sequence conservation analysis:

  • Multiple sequence alignment of ytcA homologs

  • Phylogenetic tree construction

  • Calculation of selection pressure (dN/dS ratios)

  • Identification of highly conserved regions

• Genomic context comparison:

  • Synteny analysis across species

  • Operon structure conservation

  • Regulatory element comparison

  • Mobile genetic element association

• Domain architecture analysis:

  • Identification of domain shuffling events

  • Insertion/deletion patterns in homologs

  • Comparison with functionally characterized homologs in other species

• Expression pattern comparison:

  • Transcriptomic data mining across species

  • Condition-specific expression comparison

  • Regulatory network conservation

A comprehensive table comparing ytcA conservation could be structured as:

This comparative approach has been effectively employed in studies of other Shigella proteins, providing valuable insights into their evolutionary history and potential functional significance .

What functional insights can be gained by comparing ytcA to structurally characterized homologs in other species?

Structural comparison with characterized homologs can accelerate functional understanding:

• Structural alignment approaches:

  • Superimposition of predicted ytcA structure with solved homolog structures

  • Binding site conservation analysis

  • Identification of structurally conserved but sequence-divergent regions

  • Electrostatic surface potential comparison

• Functional inference methods:

  • Identification of conserved catalytic residues

  • Substrate binding pocket comparison

  • Structural motif recognition

  • Conformational dynamics analysis through molecular modeling

• Experimental validation approaches:

  • Site-directed mutagenesis of conserved residues

  • Complementation studies with homologs from other species

  • Chimeric protein construction and functional testing

  • Substrate specificity comparison across homologs

This approach could follow the methodology used for the structural characterization of YnfA from S. flexneri, where the I-TASSER tool was employed to predict its structure based on the already resolved crystal structure of the EmrE transporter . Similar approaches could be applied to ytcA, particularly if it shares structural features with characterized proteins.

How can researchers evaluate ytcA as a potential vaccine antigen against Shigella sonnei?

Assessment of ytcA as a vaccine candidate should follow a systematic approach:

• Antigenicity and immunogenicity assessment:

  • Epitope prediction using computational tools

  • Antibody response characterization in animal models

  • T-cell epitope mapping

  • Cross-reactivity testing with other Shigella species

• Protection studies:

  • Active immunization followed by challenge in animal models

  • Passive immunization with anti-ytcA antibodies

  • Correlates of protection analysis

  • Longevity of immune response evaluation

• Vaccine formulation optimization:

  • Adjuvant screening for enhanced immunogenicity

  • Delivery system evaluation (e.g., liposomes, virus-like particles)

  • Combination with other Shigella antigens

  • Stability and storage assessment

• Human immune response prediction:

  • HLA binding prediction for population coverage

  • Ex vivo stimulation of human immune cells

  • Analysis of natural antibody responses in convalescent patients

The human challenge model established for S. sonnei provides a valuable framework for evaluating vaccine candidates . The model identified that a dose of 1680 CFU of S. sonnei 53G was required to elicit clinical disease in 75% of healthy Thai adults . Such challenge models could potentially be utilized to evaluate the protective efficacy of ytcA-based vaccine candidates in conjunction with appropriate immunization protocols.

What methodological approaches are most effective for developing diagnostic assays based on ytcA detection?

If ytcA proves to be a suitable diagnostic target, researchers should consider:

• Antibody-based detection methods:

  • Monoclonal antibody development against ytcA

  • ELISA development for protein detection

  • Lateral flow assay design for point-of-care testing

  • Flow cytometry for bacterial cell surface detection if applicable

• Nucleic acid-based detection:

  • PCR primer design specific to ytcA gene

  • LAMP assay development for field-deployable diagnostics

  • Microarray probe design for multiplex detection

  • CRISPR-based detection methods

• Aptamer and biosensor approaches:

  • Selection of ytcA-specific aptamers

  • Electrochemical biosensor development

  • Surface plasmon resonance-based detection

  • Piezoelectric biosensor applications

• Validation strategies:

  • Analytical sensitivity and specificity determination

  • Clinical sample validation

  • Comparison with gold standard diagnostic methods

  • Field testing in endemic regions

Diagnostic assay development could benefit from knowledge of the human challenge model for S. sonnei, which demonstrated that all subjects who excreted S. sonnei showed positive immune responses, regardless of clinical symptoms . This suggests that detecting bacterial shedding, potentially through ytcA-targeted assays, could be a sensitive approach for identifying infections.

What are the major technical challenges in studying uncharacterized proteins like ytcA in Shigella sonnei?

Researchers face several challenges when investigating uncharacterized proteins:

• Expression and purification obstacles:

  • Protein solubility issues

  • Inclusion body formation

  • Improper folding in heterologous systems

  • Post-translational modification requirements

• Functional assessment limitations:

  • Lack of known interaction partners

  • Absence of predicted functional domains

  • Potential redundancy with other proteins

  • Context-dependent functionality

• Methodological constraints:

  • Limited availability of specific antibodies

  • Challenges in generating viable knockout strains if essential

  • Difficulty in establishing relevant phenotypic assays

  • Limited in vivo models for Shigella infection

• Data interpretation complexities:

  • Distinguishing direct from indirect effects

  • Separating physiological from artifacts

  • Reconciling contradictory results across methods

  • Translating in vitro findings to in vivo relevance

Similar challenges have been addressed in studies of other initially uncharacterized Shigella proteins, such as YnfA in S. flexneri, where multiple complementary approaches were required to establish its functional role .

How can high-throughput approaches accelerate the functional characterization of ytcA?

Modern high-throughput methods offer powerful tools for investigating uncharacterized proteins:

• Omics-based approaches:

  • Transcriptomics to identify co-expressed genes

  • Proteomics to map interaction networks

  • Metabolomics to detect metabolic perturbations in knockout strains

  • Phenomics for comprehensive phenotypic profiling

• High-throughput screening methods:

  • Chemical genetic screening to identify compounds affecting ytcA function

  • Synthetic genetic array analysis for genetic interaction mapping

  • Arrayed CRISPR screens for functional genomics

  • Small molecule microarray screening for ligand identification

• Next-generation structural biology:

  • Cryo-EM for rapid structure determination

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

  • Fragment-based screening for ligand discovery

  • Integrative structural biology combining multiple data sources

• Advanced computational methods:

  • Machine learning for function prediction

  • Molecular dynamics simulations to study conformational changes

  • Network analysis for contextual function prediction

  • Literature mining for hypothesis generation

These approaches could significantly accelerate the characterization of ytcA, similar to how advanced methodologies have been applied to study other Shigella proteins like YnfA and YfiH .

What interdisciplinary collaborations would most benefit ytcA research?

Advancing understanding of ytcA would benefit from collaborative approaches:

• Structural biologists and computational scientists:

  • Combining experimental structure determination with computational modeling

  • Integrating dynamics simulations with functional assays

  • Developing structure-based functional predictions

• Microbiologists and immunologists:

  • Linking bacterial physiology to host-pathogen interactions

  • Evaluating immune recognition and evasion mechanisms

  • Developing infection models relevant to human disease

• Biochemists and systems biologists:

  • Characterizing enzymatic activities and metabolic impacts

  • Mapping protein interaction networks

  • Integrating multi-omics data for system-level understanding

• Clinicians and epidemiologists:

  • Correlating ytcA variants with clinical outcomes

  • Evaluating geographical distribution and evolution

  • Assessing relevance to human disease burden

Such interdisciplinary approaches have proven valuable in Shigella research, as exemplified by the establishment of the human challenge model for S. sonnei, which required collaboration between clinical researchers, microbiologists, and immunologists .

What is the current state of knowledge regarding ytcA, and what are the most promising research directions?

While ytcA in Shigella sonnei remains largely uncharacterized, methodologies successfully applied to other hypothetical proteins in Shigella species provide a roadmap for its investigation. The most promising research directions include:

• Structural characterization using computational prediction followed by experimental validation, similar to approaches used for YnfA and YfiH in S. flexneri .

• Functional genomics approaches including gene knockout studies paired with comprehensive phenotypic profiling, particularly focusing on potential roles in antimicrobial resistance based on findings with other uncharacterized proteins in Shigella .

• Host-pathogen interaction studies leveraging established infection models, including the human challenge model for S. sonnei, to evaluate potential contributions to virulence .

• Comparative analysis across Shigella species and other Enterobacteriaceae to identify evolutionary patterns that might suggest functional importance.