Recombinant Cronobacter sakazakii Spermidine export protein MdtI (mdtI)

What is Cronobacter sakazakii Spermidine export protein MdtI?

Spermidine export protein MdtI is a membrane protein expressed by Cronobacter sakazakii, a gram-negative opportunistic pathogen. It functions as part of a multidrug transport system involved in spermidine export. The full-length protein consists of 109 amino acids with the sequence: MSQVELQHILWLLLAIGLEIIANIWLKFSDGFRRPVYGVASLAAVLAAFSALGQAVEGIDLAVAYALWGGFGIAATVAAGWIMFGQRLNRKGWAGLGLLLVGMVIIKLA . MdtI is encoded by the mdtI gene (ordered locus name: ESA_01730) in the Cronobacter sakazakii genome, and its Uniprot accession number is A7MEJ5 .

What is the significance of Cronobacter sakazakii in food safety research?

Cronobacter sakazakii has gained significant attention in food safety research due to its association with severe infections, particularly in neonates and infants. It is an opportunistic pathogen linked to outbreaks in powdered infant formula (PIF), primarily causing meningitis and necrotizing enterocolitis . While traditionally considered a pathogen affecting primarily infants, recent research has demonstrated that it can also cause acute gastroenteritis in healthy adults, expanding its public health significance . The organism possesses various virulence factors, including genes for chemotaxis, flagellar motion, and heat shock proteins, which contribute to its pathogenicity and survival in food processing environments .

How does MdtI protein contribute to Cronobacter sakazakii pathogenicity?

MdtI protein plays a significant role in C. sakazakii's pathogenicity through several mechanisms:

• As a spermidine export protein, MdtI may contribute to polyamine homeostasis, which is crucial for bacterial survival and virulence.

• The protein structure suggests it functions as a membrane transporter, potentially contributing to antimicrobial resistance by effluxing compounds that would otherwise be toxic to the bacterium .

• C. sakazakii strains have been shown to adhere to and enter intestinal cells in vitro, with membrane proteins potentially playing a role in this host-pathogen interaction .

• The production of enterotoxin and the presence of bacterial outer membrane proteins (like OmpA) and lipopolysaccharide (LPS) play important roles in the host-pathogen interactions that lead to virulence .

What genomic variations exist in the mdtI gene across different Cronobacter sakazakii sequence types?

Genomic analysis of C. sakazakii isolates has revealed significant variations across different sequence types (STs). Whole-genome sequencing studies have shown that:

• Sequence Type 1 (ST1) is the dominant sequence type found in most C. sakazakii isolates, particularly those recovered from environmental samples in processing plants and raw materials .

• Other prominent sequence types include ST4 (26%) and ST99 (16%), suggesting genetic diversity within the species .

• Phylogenetic analysis based on whole-genome SNPs (single nucleotide polymorphisms) has demonstrated that C. sakazakii strains can be partitioned into distinct lineages separated by >25,000 SNPs .

• Within each lineage, hundreds to thousands of unique SNPs exist, providing high-resolution differentiation between isolates .

• Closely related isolates from the same outbreak sources may show as few as 5 SNPs difference, providing critical epidemiological information for source tracking .

These genomic variations can potentially affect the structure and function of numerous proteins, including MdtI, which may contribute to differences in virulence, antibiotic resistance, and survival capabilities among different strains.

How do post-translational modifications affect the functional properties of recombinant MdtI protein?

While the search results don't specifically address post-translational modifications (PTMs) of MdtI, research on recombinant membrane proteins suggests several considerations:

• Expression systems significantly impact PTMs - recombinant MdtI expressed in E. coli (as mentioned for the related Enterobacter protein ) may lack some PTMs present in the native Cronobacter protein.

• Potential PTMs for membrane transporters like MdtI could include phosphorylation, glycosylation, and lipidation, which would affect protein folding, stability, and function.

• Storage conditions for recombinant MdtI (-20°C to -80°C with 50% glycerol in a Tris-based buffer ) are optimized to preserve protein structure and function, suggesting that the native conformation is sensitive to environmental conditions.

• When designing experiments with recombinant MdtI, researchers should consider that the tag type (determined during the production process ) may influence protein folding and function.

What is the relationship between MdtI expression and antibiotic resistance in clinical Cronobacter sakazakii isolates?

Antibiotic resistance profiling of C. sakazakii isolates has revealed important connections to membrane transport systems:

• Genomic characterization has shown that C. sakazakii isolates carry multiple antibiotic resistance genes, with all isolates exhibiting resistance to Cephalosporins and Tetracycline .

• A significant correlation exists between genotypic and phenotypic antibiotic resistance, suggesting that resistance genes, including potentially those related to efflux systems like MdtI, are functionally expressed .

• Studies on clinical isolates have found varying resistance profiles, with all strains in one outbreak study showing resistance or intermediate resistance to cefazolin (CFZ) and erythromycin (ERY), while remaining sensitive to multiple other antibiotics .

• As part of the multidrug transport system, MdtI may contribute to antimicrobial resistance through efflux mechanisms, though its specific role in resistance to particular antibiotics requires further investigation.

What are the optimal methods for detecting Cronobacter sakazakii in research and clinical samples?

Research has optimized several methods for detecting C. sakazakii, with MALDI-TOF MS emerging as a particularly effective approach:

• An optimized liquid spotting pretreatment method for MALDI-TOF MS has been developed with the following parameters:

  • Volume of 70% formic acid: 25 μL

  • Ultrasound treatment: 350 W for 3 min

  • Volume of acetonitrile added: 75 μL

• The detailed procedure involves:

  • Transferring 500 μL of bacterial suspension (10^8 cfu/mL) into a 1.5 mL sterile centrifuge tube

  • Centrifuging at 12,000× g for 2 min

  • Removing supernatant and washing twice with 500 μL of sterile PBS

  • Adding formic acid and ultrasonicating

  • Adding acetonitrile, vortexing for 2 min, and centrifuging at 12,000× g for 2 min

  • Applying 1.5 μL of supernatant to the target plate and air drying

  • Adding 1.5 μL of MALDI matrix solution containing CHCA

• For environmental or clinical samples with low bacterial counts, pre-enrichment can be optimized using:

  • Different culture media (TSB, LB, and NB liquid medium)

  • Various media amounts (400, 600, 800, and 1000 μL)

  • Different pre-enrichment times (0, 2, 4, and 6 h)

This optimized approach allows for accurate and reproducible detection of C. sakazakii from various sample types.

How should recombinant MdtI protein be handled and stored to maintain optimal activity?

Proper handling and storage of recombinant MdtI protein is crucial for maintaining its structural integrity and functional activity:

• Storage recommendations:

  • Store at -20°C for regular use

  • For extended storage, conserve at -20°C or -80°C

  • Avoid repeated freezing and thawing as this can denature the protein

  • Store working aliquots at 4°C for up to one week

• Buffer composition:

  • Optimal storage buffer includes a Tris-based buffer with 50% glycerol, specifically optimized for this protein

  • The high glycerol concentration helps prevent freeze-thaw damage

• Handling precautions:

  • Thaw protein samples on ice when removing from frozen storage

  • Maintain cold chain during experiments

  • Prepare working aliquots to avoid repeated freeze-thaw cycles

  • When preparing dilutions, use the same buffer composition to maintain protein stability

How can recombinant MdtI be used in functional transport assays to study antimicrobial resistance?

Researchers can utilize recombinant MdtI in several experimental approaches to study its role in antimicrobial resistance:

• Reconstitution in liposomes:

  • Purified recombinant MdtI can be incorporated into artificial membrane systems

  • Transport assays with fluorescent-labeled spermidine or antimicrobial compounds can measure efflux activity

  • Comparison of transport rates in the presence of various antibiotics can elucidate the specificity of MdtI-mediated efflux

• Expression in heterologous systems:

  • The recombinant protein can be expressed in antibiotic-sensitive bacterial strains

  • Minimum inhibitory concentration (MIC) assays can determine if MdtI expression confers resistance

  • Comparison with strains expressing mutant MdtI variants can identify critical residues for transport function

• Correlation with clinical isolate data:

  • Studies have shown varying antibiotic resistance profiles in C. sakazakii clinical isolates, with all strains in one study showing resistance or intermediate resistance to cefazolin and erythromycin

  • Expression levels of MdtI can be correlated with resistance patterns to specific antibiotics

What are the implications of genomic variations in mdtI for strain-specific virulence and survival in food processing environments?

Genomic variations in mdtI may have significant implications for strain-specific differences in C. sakazakii:

• Sequence type correlations:

  • ST1 has been identified as the dominant sequence type in C. sakazakii isolates, particularly from environmental samples in processing plants

  • Different sequence types may contain variations in membrane transport proteins like MdtI that affect survival capabilities

• Transmission dynamics:

  • Phylogenetic analysis showing close relationships between clinical and food isolates (e.g., isolates with only 5 SNPs difference) suggests direct transmission routes

  • Variations in MdtI and other virulence factors may influence a strain's ability to survive in food processing environments and cause illness

• Environmental adaptations:

  • All C. sakazakii isolates in genomic studies carried virulence genes for chemotaxis, flagellar motion, and heat shock proteins

  • Membrane transporters like MdtI may contribute to stress responses that enable survival in challenging food processing environments

• Plasmid and phage contributions:

  • The plasmid Col(pHAD28) has been identified in isolates recovered from the same PIF environment

  • All isolates harbored at least one intact phage

  • Horizontal gene transfer via these elements may contribute to variations in antimicrobial resistance and virulence