Development of novel antimicrobials with engineered endolysin LysECD7-SMAP to combat Gram-negative bacterial infections.

BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

Diagnostic Value of IgA Antibody Measurement in Tick-Borne Spotted Fever (Astrakhan Rickettsial Fever).

Tick-borne spotted fevers caused by Rickettsia occur worldwide. The symptoms of this bacterial infection are similar to those of viral infection, and thus, diagnostic accuracy has special clinical importance. One of the commonly used methods for the diagnosis of tick-borne spotted fever is enzyme-linked immunosorbent assay (ELISA), which is based on estimation of the presence of specific IgM antibodies in blood. However, IgA analysis has not been used for the diagnosis of rickettsial diseases thus far. We investigated the diagnostic value of IgA antibody determination using patient sera collected in the Astrakhan region of Russia, where an isolated site of Astrakhan rickettsial fever (ARF) caused by Rickettsia conorii subsp. caspia is located. Our investigation was performed on serum samples collected from 185 patients diagnosed with Astrakhan rickettsial fever from May to October 2019. Western blot analysis revealed that specific IgA antibodies, as well as IgM antibodies, from patient sera bind to high-molecular-weight pathogen proteins with similar masses. The obtained data show that the determination of IgM alone allows for serological confirmation of diagnosis in only 46.5% of cases but that the determination of both IgM and IgA increases this rate to 66.5%. Taken together, the findings show an important diagnostic value of IgA evaluation for tick-borne spotted fever rickettsiosis. IMPORTANCE Tick-borne spotted fevers caused by Rickettsia occur worldwide. The symptoms of this bacterial infection are similar to the symptoms of viral infection, and thus, diagnostic accuracy has special clinical importance. The most serious spotted fever group rickettsiosis is Rocky Mountain fever in the United States, which is caused by Rickettsia rickettsii, and disease complications can lead to hemiparesis, blindness, or amputation. Rickettsia conorii subsp. caspia causes a rickettsial spotted fever named Astrakhan rickettsial fever (ARF). One of the commonly used methods for the diagnosis of tick-borne spotted fevers is ELISA, which is based on estimation of the presence of specific IgM antibodies in blood, though IgA has not been used for the diagnosis of rickettsial diseases thus far. In this study, we showed that both IgA and IgM should be analyzed in the blood serum samples of patients to significantly enhance the accuracy of diagnostics of tick-borne spotted fever rickettsiosis.