Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and ß-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and ß-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Non-traditional approaches for control of antibiotic resistance.

INTRODUCTION: The drying up of antibiotic pipeline has necessitated the development of alternative therapeutic strategies to control the problem of antimicrobial resistance (AMR) that is expected to kill 10-million people annually by 2050. Newer therapeutic approaches address the shortcomings of traditional small-molecule antibiotics - the lack of specificity, evolvability, and susceptibility to mutation-based resistance. These 'non-traditional' molecules are biologicals having a complex structure and mode(s) of action that makes them resilient to resistance. AREAS COVERED: This review aims to provide information about the non-traditional drug development approaches to tackle the problem of antimicrobial resistance, from the pre-antibiotic era to the latest developments. We have covered the molecules under development in the clinic with literature sourced from reviewed scholarly articles, official company websites involved in innovation of concerned therapeutics, press releases from the regulatory bodies, and clinical trial databases. EXPERT OPINION: Formal introduction of non-traditional therapies in general practice can be quick and feasible only if supported with companion diagnostics and used in conjunction with established therapies. Owing to relatively higher development costs, non-traditional therapeutics require more funding as well as well as clarity in regulatory and clinical path. We are hopeful these issues are adequately addressed before AMR develops into a pandemic.

Identification of trypanosomatid PEX19: functional characterization reveals impact on cell growth and glycosome size and number.

Glycosomes are peroxisome-like organelles present in trypanosomatid pathogens. These organelles compartmentalize glycolysis, among other reactions, and are essential in both bloodstream and procyclic form Trypanosoma brucei. Peroxins (PEXs) are proteins necessary for biogenesis of peroxisomes and glycosomes. In each assembled trypanosomatid genome, we identified a predicted protein with approximately 20% sequence identity to human PEX19, a protein required for insertion of peroxisomal membrane proteins (PMPs) into the membrane. Functional analysis demonstrated that these proteins are indeed PEX19 orthologues. Like other PEX19s, T. brucei and Leishmania major PEX19 GFP fusion proteins are predominantly cytosolic. We further showed that LmPEX19 interacts with the glycosomal membrane protein PEX2 in the yeast two-hybrid system. Partial knockdown of TbPEX19 slowed parasite growth, particularly when glucose was present. Immunofluorescence and electron microscopic studies revealed biogenesis defect as evidenced by a sharp reduction in the number of glycosomes. Surprisingly, a four-fold increase in the size of the remaining glycosomes was observed. We propose that this phenotype of fewer but larger glycosomes results from the reduction in import of glycosomal membrane proteins.