Antibiotic 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.

Background: HIV-1 Vpu counteracts the cellular antiviral factor BST-2 via an interaction that maps to the transmembrane domains of each protein.Results: This interaction is detectable by NMR spectroscopy and involves conserved faces of each helix. Conclusion: HIV-1 avoids an innate host defense via a lipid-embedded helix-helix interface. Significance: Intermolecular interactions within the lipid bilayer can be highly specific and shape the host-pathogen relationship.

The HIV-1 protein Vpu counteracts the antiviral activity of the innate restriction factor BST-2/tetherin by a mechanism that partly depends on its interaction with ␤-TrCP, a substrate adaptor for an SCF (Skp-Cullin 1-F box) E3 ubiquitin ligase complex. This suggests that Vpu stimulates the ubiquitination of BST-2 and that this underlies the relief of restriction. Here, we show that Vpu stimulates ubiquitination of BST-2. Mutation of all potential ubiquitination sites in the cytoplasmic domain of BST-2, including lysines, cysteines, serines, and threonines, abrogates Vpu-mediated ubiquitination. However, a serine-threonine-serine sequence specifically mediates the downregulation of BST-2 from the cell surface and the optimal relief of restricted virion release. Serine-threonine ubiquitination of BST-2 is likely part of the mechanism by which Vpu counteracts innate defenses.

The rise of multidrug-resistant pathogens and the dearth of new antibiotic development place an existential strain on successful infectious disease therapy. Breakthrough strategies that go beyond classical antibiotic mechanisms are needed to combat this looming public health catastrophe. Reconceptualizing antibiotic therapy in the richer context of the host-pathogen interaction is required for innovative solutions. By defining specific virulence factors, the essence of a pathogen, and pharmacologically neutralizing their activities, one can block disease progression and sensitize microbes to immune clearance. Likewise, host-directed strategies to boost phagocyte bactericidal activity, enhance leukocyte recruitment, or reverse pathogen-induced immunosuppression seek to replicate the success of cancer immunotherapy in the field of infectious diseases. The answer to the threat of multidrug-resistant pathogens lies “outside-the-box” of current antibiotic paradigms.

We asked whether beta-lactamase inhibitors (BLIs) increased the activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA), the peptide antibiotic colistin (COL) against the emerging Gram-negative nosocomial pathogen Acinetobacter baumannii, and the human host defense peptide cathelicidin LL37 against either pathogen. DAP and LL37 kill curves were performed with or without BLIs against MRSA, vancomycin-intermediate S. aureus (VISA), and heterogeneous VISA (hVISA). COL and LL37 kill curves were performed against A. baumannii. Boron-dipyrromethene (BODIPY)-labeled DAP binding to MRSA grown with the BLI tazobactam (TAZ) was assessed microscopically. The combination of COL plus TAZ was studied in a murine model of A. baumannii pneumonia. TAZ alone lacked in vitro activity against MRSA or A. baumannii. The addition of TAZ to DAP resulted in a 2- to 5-log10 reduction in recoverable MRSA CFU at 24 h compared to the recoverable CFU with DAP alone. TAZ plus COL showed synergy by kill curves for 4 of 5 strains of A. baumannii tested. Growth with 20 mg/liter TAZ resulted in 2- to 2.5-fold increases in the intensity of BODIPY-DAP binding to MRSA and hVISA strains. TAZ significantly increased the killing of MRSA and A. baumannii by LL37 in vitro. TAZ increased the activity of COL in a murine model of A. baumannii pneumonia. Classical BLIs demonstrate synergy with peptide antibiotics. Since BLIs have scant antimicrobial activity on their own and are thus not expected to increase selective pressure toward antibiotic resistance, their use in combination with peptide antibiotics warrants further study.

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.

Pseudomonas aeruginosa is an important opportunistic pathogen that has become a serious problem due to increased rates of antibiotic resistance. Due to this along with a dearth in novel antibiotic development, especially against Gram-negative pathogens, new therapeutic strategies are needed to prevent a post-antibiotic era. Here we describe the importance of the vacJ/Mla pathway in resisting bactericidal actions of the host innate immune response. P. aeruginosa tn5 transposon mutants in genes from the VacJ/Mla pathway showed increased susceptibility to killing by the host cathelicidin antimicrobial peptide, LL-37 when compared to the wild-type parent strain. The P. aeruginosa vacJ− mutant demonstrated increased membrane permeability upon damage as well as sensitivity to killing in the presence of the detergent sodium dodecyl sulfate and the divalent cation chelator EDTA. When exposed to human whole blood and serum complement, the vacJ− mutant was killed more rapidly when compared to the wild-type parent strain and complemented mutant. Finally, in an in vivo mouse lung infection model, infection with the vacJ− mutant resulted in reduced mortality, lower bacterial burden, and reduced lung damage when compared to the wild-type strain. This study highlights the potential in therapeutically targeting the VacJ/Mla pathway in sensitizing P. aeruginosa to killing by the host innate immune response.

Salinipyrones and pacificanones are structurally related polyketides from Salinispora pacifica CNS-237 proposed to arise from the same modular polyketide synthase (PKS) assembly line. Genome sequencing revealed a large macrolide PKS gene cluster that codes for the biosynthesis of rosamicin A and a series of new macrolide antibiotics. Mutagenesis experiments unexpectedly correlated salinipyrone and pacificanone biosynthesis to the rosamicin octamodule Spr PKS. Remarkably, this bifurcated polyketide pathway illuminates a series of enzymatic domain and module skipping reactions that dictate natural polyketide product diversity. Our findings enlarge the growing knowledge of polyketide biochemistry and illuminate potential challenges in PKS bioengineering.