Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens

AbstractAntibiotic resistance poses an increasingly grave threat to the public health. Of pressing concern, rapid spread of carbapenem-resistance among multidrug-resistant (MDR) Gram-negative rods (GNR) is associated with few treatment options and high mortality rates. Current antibiotic susceptibility testing guiding patient management is performed in a standardized manner, identifying minimum inhibitory concentrations (MIC) in bacteriologic media, but ignoring host immune factors. Lacking activity in standard MIC testing, azithromycin (AZM), the most commonly prescribed antibiotic in the U.S., is never recommended for MDR GNR infection. Here we report a potent bactericidal action of AZM against MDR carbapenem-resistant isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. This pharmaceutical activity is associated with enhanced AZM cell penetration in eukaryotic tissue culture media and striking multi-log-fold synergies with host cathelicidin antimicrobial peptide LL-37 or the last line antibiotic colistin. Finally, AZM monotherapy exerts clear therapeutic effects in murine models of MDR GNR infection. Our results suggest that AZM, currently ignored as a treatment option, could benefit patients with MDR GNR infections, especially in combination with colistin

New Approaches to Sensitize Multidrug-Resistant Gram-Negative Bacterial Pathogens to Host Innate Immune Clearance

Multidrug-resistant Gram-negative (MDRGN) bacterial pathogens pose a significant threat to public health in the US and worldwide. The lack of therapeutic options for MDRGN bacterial infection have forced clinicians to use older, once rarely used antibiotics that are associated with significant toxicity. An increasing number of immunocompromised individuals, including the elderly, premature neonates, surgical patients, organ transplant recipients, and those receiving cancer chemotherapy, represent an expanding reservoir for MDRGN bacteria in the hospital setting. Research to understand the molecular host-pathogen interactions involved in MDRGN bacterial infections and reveal novel therapeutic targets are imperative if we are to prevent the advent of a post-antibiotic era. For this PhD dissertation project, I began by searching for genes in the MDRGN bacterial pathogen Acinetobacter baumannii that are important in resisting the bactericidal action of the human cathelicidin antimicrobial peptide, LL-37, a critical front-line effector of host innate defense. A transposon insertion in vacJ, a component of the mla system conserved in all Gram-negative species, proved important in mediating resistance to LL-37, detergents, whole blood, complement, and neutrophils. A. baumannii lacking vacJ demonstrated reduced virulence compared to wild-type parent strain in a murine pneumonia model. To expand the general significance of this work, I examined the virulence capacity of multiple mla mutants in Pseudomonas aeruginosa, a leading MDRGN opportunistic human pathogen. P. aeruginosa lacking an mla system exhibited increased sensitivity to LL-37 and its murine homolog, as well as membrane active pharmaceuticals, serum, and whole blood. A murine pneumonia model revealed reduced bacterial burden and mortality when infected with bacteria lacking vacJ. Finally, I explored how a common antibiotic, azithromycin, traditionally considered ineffective against MDRGN bacteria in standard testing assays, gained entry to its ribosomal target when endogenous antimicrobial peptides modify the outer membrane, allowing it to provide clear efficacy in an in vivo model of A. baumannii pneumonia. Altogether, this dissertation furthered our understanding on the importance outer membrane integrity for Gram-negative bacteria to resist host immune clearance. This research points to a possible therapeutic target in the mla pathway to control MDRGN infections by sensitizing them to the host innate immune system

New Approaches to Sensitize Multidrug-Resistant Gram-Negative Bacterial Pathogens to Host Innate Immune Clearance

Multidrug-resistant Gram-negative (MDRGN) bacterial pathogens pose a significant threat to public health in the US and worldwide. The lack of therapeutic options for MDRGN bacterial infection have forced clinicians to use older, once rarely used antibiotics that are associated with significant toxicity. An increasing number of immunocompromised individuals, including the elderly, premature neonates, surgical patients, organ transplant recipients, and those receiving cancer chemotherapy, represent an expanding reservoir for MDRGN bacteria in the hospital setting. Research to understand the molecular host-pathogen interactions involved in MDRGN bacterial infections and reveal novel therapeutic targets are imperative if we are to prevent the advent of a post-antibiotic era. For this PhD dissertation project, I began by searching for genes in the MDRGN bacterial pathogen Acinetobacter baumannii that are important in resisting the bactericidal action of the human cathelicidin antimicrobial peptide, LL-37, a critical front-line effector of host innate defense. A transposon insertion in vacJ, a component of the mla system conserved in all Gram-negative species, proved important in mediating resistance to LL-37, detergents, whole blood, complement, and neutrophils. A. baumannii lacking vacJ demonstrated reduced virulence compared to wild-type parent strain in a murine pneumonia model. To expand the general significance of this work, I examined the virulence capacity of multiple mla mutants in Pseudomonas aeruginosa, a leading MDRGN opportunistic human pathogen. P. aeruginosa lacking an mla system exhibited increased sensitivity to LL-37 and its murine homolog, as well as membrane active pharmaceuticals, serum, and whole blood. A murine pneumonia model revealed reduced bacterial burden and mortality when infected with bacteria lacking vacJ. Finally, I explored how a common antibiotic, azithromycin, traditionally considered ineffective against MDRGN bacteria in standard testing assays, gained entry to its ribosomal target when endogenous antimicrobial peptides modify the outer membrane, allowing it to provide clear efficacy in an in vivo model of A. baumannii pneumonia. Altogether, this dissertation furthered our understanding on the importance outer membrane integrity for Gram-negative bacteria to resist host immune clearance. This research points to a possible therapeutic target in the mla pathway to control MDRGN infections by sensitizing them to the host innate immune system

Docking simulation and antibiotic discovery targeting the MlaC protein in Gram-negative bacteria.

To maintain the lipid asymmetry of the cell envelope in Gram-negative bacteria, the MlaC protein serves as a lipid transfer factor and delivers phospholipids from the outer to the inner membrane. A strategy of antibiotic discovery is to design a proper compound that can tightly bind to the MlaC protein and inhibit the MlaC function. In this study, we performed virtual screening on multiple MlaC structures obtained from molecular dynamics simulations to identify potential MlaC binders. Our results suggested that clorobiocin is a compound that could bind to the MlaC protein. Through the comparison of the bound geometry between clorobiocin and novobiocin, we pointed out that the methyl-pyrrole group of the noviose sugar in clorobiocin forms hydrophobic interactions with amino acids in the phospholipid binding pocket, which allows the compound to bind deep in the active site. This also explains why clorobiocin shows a tighter binding affinity than novobiocin. Our study highlights a practical path of antibiotic development against Gram-negative bacteria

Docking simulation and antibiotic discovery targeting the MlaC protein in Gram‐negative bacteria

To maintain the lipid asymmetry of the cell envelope in Gram-negative bacteria, the MlaC protein serves as a lipid transfer factor and delivers phospholipids from the outer to the inner membrane. A strategy of antibiotic discovery is to design a proper compound that can tightly bind to the MlaC protein and inhibit the MlaC function. In this study, we performed virtual screening on multiple MlaC structures obtained from molecular dynamics simulations to identify potential MlaC binders. Our results suggested that clorobiocin is a compound that could bind to the MlaC protein. Through the comparison of the bound geometry between clorobiocin and novobiocin, we pointed out that the methyl-pyrrole group of the noviose sugar in clorobiocin forms hydrophobic interactions with amino acids in the phospholipid binding pocket, which allows the compound to bind deep in the active site. This also explains why clorobiocin shows a tighter binding affinity than novobiocin. Our study highlights a practical path of antibiotic development against Gram-negative bacteria

Docking simulation and antibiotic discovery targeting the MlaC protein in Gram-negative bacteria.

To maintain the lipid asymmetry of the cell envelope in Gram-negative bacteria, the MlaC protein serves as a lipid transfer factor and delivers phospholipids from the outer to the inner membrane. A strategy of antibiotic discovery is to design a proper compound that can tightly bind to the MlaC protein and inhibit the MlaC function. In this study, we performed virtual screening on multiple MlaC structures obtained from molecular dynamics simulations to identify potential MlaC binders. Our results suggested that clorobiocin is a compound that could bind to the MlaC protein. Through the comparison of the bound geometry between clorobiocin and novobiocin, we pointed out that the methyl-pyrrole group of the noviose sugar in clorobiocin forms hydrophobic interactions with amino acids in the phospholipid binding pocket, which allows the compound to bind deep in the active site. This also explains why clorobiocin shows a tighter binding affinity than novobiocin. Our study highlights a practical path of antibiotic development against Gram-negative bacteria

Thermal and Desiccation Constraints Drive Territory Preference In Fiddler Crabs

Territory quality is often critical for reproductive success, survival and ultimately fitness. Holding a good quality territory can make a male more attractive to females but can also have several other advantages. It can decrease the effects of environmental stressors, for example, by reducing the need for behavioural thermoregulation or decreasing the risk of predation. We found that, in the fiddler crab Austruca mjoebergi, male territories differed in the shaded mangrove and unshaded open areas of the habitat. The lower temperatures and wetter sediments of the mangrove areas resulted in lower desiccation rates allowing males to spend longer periods of time on the surface than they did in the sun-exposed open areas. Males lived at higher densities in the mangrove areas, and preferentially selected shaded territories when given a choice. Male fights to attain/retain territories in the mangrove areas were longer than those in the open areas. Because females do not preferentially mate with males with territories in shaded mangrove habitats, the advantages to males in maintaining these territories is likely a reduction in thermal and/or desiccation stress

Molecular dynamic study of MlaC protein in Gram-negative bacteria: conformational flexibility, solvent effect and protein-phospholipid binding.

The composition of the outer membrane in Gram-negative bacteria is asymmetric, with the lipopolysaccharides found in the outer leaflet and phospholipids in the inner leaflet. The MlaC protein transfers phospholipids from the outer to inner membrane to maintain such lipid asymmetry in the Mla pathway. In this work, we have performed molecular dynamics simulations on apo and phospholipid-bound systems to study the dynamical properties of MlaC. Our simulations show that the phospholipid forms hydrophobic interactions with the protein. Residues surrounding the entrance of the binding site exhibit correlated motions to control the site opening and closing. Lipid binding leads to increase of the binding pocket volume and precludes entry of the water molecules. However, in the absence of the phospholipid, water molecules can freely move in and out of the binding site when the pocket is open. Dehydration occurs when the pocket closes. This study provides dynamic information of the MlaC protein and may facilitate the design of antibiotics against the Mla pathway of Gram-negative bacteria