Recombinant Ureaplasma parvum serovar 3 Uncharacterized protein UU165.2 (UU165.2)

What is Ureaplasma parvum serovar 3 and how does it relate to other Ureaplasma species?

Ureaplasma parvum serovar 3 is one of the subtypes within the species Ureaplasma parvum, which was previously classified as Ureaplasma urealyticum biovar 1. The reclassification occurred after molecular evidence supported the division of U. urealyticum into two distinct species: U. parvum and U. urealyticum (previously biovar 2) . U. parvum comprises three subtypes represented by serovars 1, 3/14, and 6, while U. urealyticum contains ten serovars divided into three subtypes . Molecular studies have shown that serotype 3/14 is among the most prevalent (48%) of U. parvum isolates identified in clinical samples, suggesting its potential clinical significance .

What is currently known about the molecular structure of UU165.2 protein?

UU165.2 is a 122-amino acid uncharacterized protein with the following sequence: MNKIIKFHNERIKWLWILTAILAISFFVICFNNVKWIYTENTAKYELLTSSLEKIVKFYSFSLVDKPFARGVPNSIDVFSRAIIGVAFGLGFVGTMLIDYFIISKVAYIVKQKIKQSKKVGM . Preliminary sequence analysis suggests it likely contains hydrophobic regions consistent with membrane association, as indicated by sequences such as "TAILAISFFVICFNN" which show characteristics of transmembrane domains . The protein has been cataloged in the UniProt database with the accession number Q9PQX8, but functional studies and structural determinations remain limited .

How can researchers obtain recombinant UU165.2 for experimental studies?

Researchers can obtain recombinant UU165.2 as a full-length protein (1-122 amino acids) with an N-terminal His-tag expressed in E. coli expression systems . The purified protein is typically provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For optimal research use, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C . When handling the protein, researchers should avoid repeated freeze-thaw cycles and may store working aliquots at 4°C for up to one week .

What are the primary molecular detection methods for identifying Ureaplasma parvum serovar 3?

The identification of U. parvum serovar 3 relies primarily on PCR-based methods targeting several genomic regions. Key targets include the 16S rRNA gene, 16S rRNA-23S rRNA intergenic spacer regions, urease gene subunits, and the 5′ ends of the multiple-banded antigen (MBA) genes . For U. parvum serovar 3 specifically, primer pairs such as UMS-125–UMA269 have been developed to amplify only this serovar (and closely related serovar 14) . These molecular methods have largely replaced traditional serological techniques due to their higher specificity and the ability to work directly with clinical specimens. The development of these techniques has facilitated epidemiological studies showing that U. parvum is detected much more frequently (87% of isolates) than U. urealyticum (19%) in vaginal flora .

What is the predicted functional role of UU165.2 based on bioinformatic analyses?

While UU165.2 remains functionally uncharacterized, bioinformatic analyses of its amino acid sequence suggest it may function as a membrane protein. The presence of hydrophobic stretches typical of transmembrane domains (particularly "TAILAISFFVICFNN") indicates potential membrane insertion . Additional sequence motifs such as "VAYIVKQKIKQSKKVGM" at the C-terminus suggest possible interaction sites with other proteins or nucleic acids . Given the limited genome size of Ureaplasma species, uncharacterized proteins often play critical roles in basic cellular functions or virulence. Comparative genomic analyses with other bacterial species have not identified clear orthologs, suggesting UU165.2 may perform Ureaplasma-specific functions potentially related to its unique urease-dependent metabolism or host interaction capabilities.

How might UU165.2 contribute to the pathogenicity of Ureaplasma parvum serovar 3?

The potential role of UU165.2 in pathogenicity remains speculative but can be approached through several investigative angles. Serovar 3 represents 48% of U. parvum clinical isolates, suggesting potential adaptive advantages in colonization or persistence . As a putative membrane protein, UU165.2 could be involved in host-pathogen interactions, contributing to adhesion, immune evasion, or nutrient acquisition . The protein might also participate in phase variation mechanisms, similar to those observed in other Ureaplasma membrane proteins, which enable the organism to adapt to changing host environments . Research approaches to investigate pathogenicity contributions could include:

How does UU165.2 compare across different Ureaplasma serovars and species?

Comparative analysis of UU165.2 across Ureaplasma serovars could provide insights into its evolutionary conservation and functional importance. While specific comparative data for UU165.2 across serovars is not directly presented in the available literature, methodological approaches for such comparisons have been established. The PCR-based typing systems that differentiate U. parvum from U. urealyticum could be adapted to analyze the UU165.2 gene across different strains . Sequence divergence patterns might indicate whether the protein is under selective pressure, which would suggest functional importance. Additionally, expression analysis across different serovars could reveal whether UU165.2 expression correlates with specific phenotypic traits or virulence characteristics.

What are the optimal expression and purification conditions for recombinant UU165.2?

The optimal expression system for recombinant UU165.2 utilizes E. coli with an N-terminal His-tag to facilitate purification . When designing expression constructs, researchers should consider the following parameters:

For membrane-associated proteins like UU165.2, the addition of mild detergents such as 0.1% Triton X-100 or 0.5% CHAPS during lysis and purification may improve solubility and reduce aggregation .

What experimental approaches can determine the subcellular localization of UU165.2?

Determining the subcellular localization of UU165.2 is crucial for understanding its function. Several complementary approaches can be employed:

• Immunofluorescence microscopy: Using antibodies against the His-tag or the protein itself to visualize its location within Ureaplasma cells.

• Subcellular fractionation: Separating membrane, cytoplasmic, and periplasmic fractions followed by Western blotting to detect UU165.2.

• Membrane protein extraction: Using differential detergent extraction to distinguish between integral and peripheral membrane proteins.

• Protease protection assays: Determining the orientation of membrane-inserted proteins by assessing susceptibility to proteolytic degradation.

• Reporter fusion constructs: Creating fusions with fluorescent proteins like GFP to track localization in live cells.

Each of these methods has strengths and limitations, and a combination approach often provides the most reliable results for membrane protein localization .

How can researchers effectively design functional assays for an uncharacterized protein like UU165.2?

Designing functional assays for uncharacterized proteins requires a systematic approach:

• Start with bioinformatic predictions: Use tools like BLAST, Pfam, and TMHMM to identify potential functional domains, transmembrane regions, and structural motifs.

• Deploy structural characterization: Circular dichroism spectroscopy can provide secondary structure information, while more advanced techniques like X-ray crystallography or cryo-EM might be necessary for detailed structural insights.

• Conduct binding studies: Assess interaction with potential ligands including lipids, nucleic acids, or other proteins using techniques such as:

  • Surface plasmon resonance

  • Isothermal titration calorimetry

  • Pull-down assays

• Perform phenotypic assessments: Compare wild-type and UU165.2 mutant strains (if available) for changes in:

  • Growth characteristics

  • Stress responses

  • Host cell adhesion

  • Antibiotic susceptibility

• Utilize heterologous expression: Express UU165.2 in model organisms to observe phenotypic effects and potential functional complementation.

Given the membrane-associated nature of UU165.2, assays that examine membrane integrity, transport functions, or intercellular signaling would be particularly relevant .

What PCR-based approaches can be used to detect and quantify UU165.2 expression?

PCR-based approaches for detecting and quantifying UU165.2 expression should incorporate the following considerations:

• Primer design for UU165.2:

  • Target unique regions to avoid cross-amplification

  • Design primers with similar melting temperatures (±2°C)

  • Aim for amplicon sizes of 100-200 bp for qPCR applications

• Recommended PCR approaches:

  • Conventional PCR for detection

  • Real-time quantitative PCR (qPCR) for expression analysis

  • Reverse transcription qPCR (RT-qPCR) to quantify mRNA levels

• Reference genes for normalization:

  • 16S rRNA gene (though abundance may be an issue)

  • Housekeeping genes like DNA gyrase (gyrB) or elongation factor Tu

• Controls and validation:

  • Include positive controls (known U. parvum serovar 3 samples)

  • Include negative controls (no template, other Ureaplasma species)

  • Validate using multiple primer sets and sequencing of amplicons

The PCR-based typing systems developed for Ureaplasma species identification can serve as models for designing UU165.2-specific detection methods .

How can UU165.2 be utilized in serological studies of Ureaplasma infections?

The recombinant UU165.2 protein offers several applications in serological studies:

• Development of serological assays: Purified UU165.2 can be used as an antigen in ELISA, Western blot, or microarray-based assays to detect antibodies against U. parvum serovar 3 in patient sera.

• Seroprevalence studies: These assays can help determine the prevalence of exposure to U. parvum serovar 3 in different populations, providing epidemiological insights.

• Serological differentiation: If UU165.2 contains serovar-specific epitopes, it could help distinguish between different Ureaplasma serovars in serological tests.

• Antibody response characterization: Studies can examine whether antibodies against UU165.2 are present during active infection, their persistence after clearance, and their correlation with protective immunity.

• Vaccine development: If UU165.2 proves to be immunogenic and surface-exposed, it could be evaluated as a potential vaccine candidate.

For these applications, it's essential to establish the immunogenicity and serovar specificity of UU165.2 through careful validation studies comparing responses across different Ureaplasma species and serovars .

What is the potential role of UU165.2 in understanding phase variation mechanisms in Ureaplasma?

Phase variation mechanisms in Ureaplasma parvum serovar 3 involve site-specific DNA inversion events occurring at short inverted repeats, potentially mediated by tyrosine recombinases like XerC . While UU165.2 has not been directly implicated in these mechanisms, investigating its potential involvement could provide valuable insights:

• Expression analysis during phase variation: Determining whether UU165.2 expression changes during phase variation events could indicate regulation by this mechanism.

• Sequence analysis: Examining the UU165.2 gene and its flanking regions for the presence of inverted repeats similar to those found in known phase-variable loci.

• Interaction studies: Investigating whether UU165.2 interacts with known phase variation mediators like XerC (UU222) through co-immunoprecipitation or two-hybrid assays.

• Functional impact: Assessing whether UU165.2 influences the frequency or outcomes of phase variation events, possibly through structural roles or regulatory interactions.

Understanding these relationships could reveal important aspects of how U. parvum adapts to environmental changes and evades host immune responses .

How can comparative genomics approaches inform our understanding of UU165.2 function?

Comparative genomics approaches offer powerful tools for inferring UU165.2 function:

• Ortholog identification: Searching for UU165.2 orthologs across different bacterial species can reveal evolutionary relationships and potential functional conservation.

• Synteny analysis: Examining the genomic context of UU165.2 across related species may provide clues about functional associations and gene regulatory networks.

• Selection pressure analysis: Calculating dN/dS ratios (ratio of non-synonymous to synonymous substitutions) can indicate whether UU165.2 is under positive, negative, or neutral selection pressure.

• Structural homology modeling: Even in the absence of direct sequence homology, structural modeling might reveal similarities to proteins of known function.

• Co-evolution pattern analysis: Identifying genes that show correlated evolutionary patterns with UU165.2 might suggest functional relationships or physical interactions.

These approaches can generate testable hypotheses about UU165.2 function, particularly in the context of U. parvum serovar 3's unique ecological niche and pathogenic potential .

What technologies can be employed to investigate the role of UU165.2 in host-pathogen interactions?

Investigating UU165.2's potential role in host-pathogen interactions requires sophisticated experimental approaches:

• Cell culture infection models:

  • Epithelial cell adhesion/invasion assays with wild-type vs. UU165.2-deficient strains

  • Co-culture with immune cells to assess inflammatory responses

  • Barrier integrity measurements in polarized epithelial monolayers

• Protein interaction studies:

  • Yeast two-hybrid screening against host protein libraries

  • Pull-down assays using recombinant UU165.2 with host cell lysates

  • Surface plasmon resonance to quantify binding kinetics with candidate host receptors

• Advanced microscopy:

  • Super-resolution microscopy to visualize UU165.2 during host cell interaction

  • Live-cell imaging with fluorescently labeled UU165.2

  • Correlative light and electron microscopy to precisely localize UU165.2 at the host-pathogen interface

• Animal models:

  • Comparing colonization efficiency of wild-type vs. UU165.2-deficient strains

  • Measuring immune responses to recombinant UU165.2 immunization

  • Assessing protection conferred by anti-UU165.2 antibodies

These approaches can elucidate whether UU165.2 contributes to adhesion, invasion, immune evasion, or other aspects of U. parvum serovar 3 pathogenesis .