A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors.

Acute systemic Gram-negative bacterial infections are accompanied by release of lipopolysaccharide (LPS) endotoxins into the bloodstream and an innate immune host response via the well-known toll like receptor 4 (TLR4) pathway. In this, LPS associates non-covalently with TLR4 to form an activated heterodimer (LPS/MD2/TLR4)2 complex in vivo, assisted by a coreceptor CD14. This complexation process has been illustrated ex vivo using indirect methods such as cytokine, interleukin, TNF-α measurements and by direct demonstration of sequential binding events on a surface using advanced optics. We are the first ones to carry out homogeneous self-assembly of LPS-rTLR4-MD2 conjugates in vitro in a single step, and further demonstrate the role of CD14 as a catalyst during this process. The assay comprises of LPS, MD2, CD14, and recombinant TLR4-conjugated magnetic particles co-incubated in a buffer at room temperature. The complexes are removed by magnetic separation and the extent of binding is estimated by quantifying the unbound biomolecules in the supernatant using standard biophysical techniques. Our results show that rTLR4-MD2-LPS complexes form in an hour and follow a 1:1:1 stoichiometry, in agreement with the in vivo/ex vivo studies. The assay is also highly specific; addition of known LPS-binding ligands decreased the LPS-rTLR4 complexation, allowing its use as a rapid tool for molecular inhibitor screening.

Mechanistic Evaluation of Lipopolysaccharide-Alexidine Interaction Using Spectroscopic and in Silico Approaches.

The increasing problem of multidrug resistance (MDR) in bacteria calls for discovery of new molecules and diagnostic methodologies that are effective against a wide range of microbial pathogens. We have studied the role of alexidine dihydrochloride (alex) as a bioaffinity ligand against lipopolysaccharide (LPS), a pathogen-associated surface marker universally present on all Gram-negative bacteria. While the activity of alex against bacteria is biologically known, little information exists on its mechanism of action or binding stoichiometry. We have used nuclear magnetic resonance (NMR), fluorescence, and surface plasmon resonance (SPR) spectroscopies to probe the binding characteristics of alex and LPS molecules. Our results indicate that LPS:alex stoichiometry lies between 1:2 and 1:4 and has a dissociation constant ( KD) of 38 µM that is mediated through electrostatic interactions between the negatively charged phosphate groups present on LPS and the positively charged guanidinium groups present in alex. Further, molecular dynamics (MD) simulations performed to determine the conformational interaction between the two molecules show good agreement with the experimental results, which substantiate the potential of alex molecule for LPS neutralization and hence, development of efficient in vitro diagnostic assays.

A Flowthrough Assay for Rapid Bedside Stratification of Bloodstream Bacterial Infection in Critically Ill Patients: a Pilot Study.

Bacterial infections affect more than 2 million people annually. Of these, systemic infections caused by bacteria in critically ill patients may lead to life-threatening conditions such as sepsis. We have developed a point-of-care (POC) device called Septiflo that can detect and stratify the Gram status of bloodstream bacterial infections in less than 10 min from a drop of human plasma. It works on the principle of identifying pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) and lipoteichoic acid (LTA) that are released into the bloodstream at the onset of Gram-negative and Gram-positive bacterial infections, respectively. The biomarkers are captured on a membrane without a receptor, and the Gram status specificity is conferred by the ligands attached to gold nanoparticles (AuNPs) used as signal amplification probes. The ultrasensitive colorimetric results are read by eye down to a 100-fg/ml detection limit without an instrument. No cross-interference between the PAMPs is seen during Gram stratification. Septiflo results also display better performance than commercial enzyme-linked immunosorbent assays (ELISAs). Tests performed on 60 clinical samples from patients showed a correlation accuracy of 70% against procalcitonin (PCT), an accepted surrogate biomarker for sepsis. A direct comparison with eubacterial PCR yielded up to 94% accuracy in 31 patients at a chosen cutoff level for LPS and LTA and area under the curve (AUC) values of 0.927 and 0.885, respectively, though blood culture was negative for most samples. The high sensitivity, low cost, and simple bedside utility of the assay may aid in better sepsis management apparently at the presymptomatic stage, lowering empirical therapy, medical costs, antimicrobial resistance, and mortality.

Nanotheranostic approaches for management of bloodstream bacterial infections.

Bloodstream bacterial infections are a serious threat to global public health and economy. The recent figures released by National Center for Health Statistics indicate that more than a million Americans get affected by it each year and the sepsis mortality alone is about 28%-50% Hall et al. (2011).1 Robust and affordable point-of-care medical technologies are, therefore, urgently needed for rapid decision-making to initiate appropriate line of treatment. Current techniques based on blood culture and serology do not have quick turnaround times or adequate sensitivities for early intervention. Moreover, antimicrobial resistance poses a great challenge in the fight towards effective bacterial infection management. Nanotheranostics is emerging as a novel strategy combining solutions for rapid diagnosis and treatment in a more personalized way. This review highlights the recent advances made in theranosis of bloodstream bacterial infections using different classes of nanomaterials and bioreceptors, and discusses present challenges and future way forward.