Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.

OBJECTIVES: Pseudomonas aeruginosa colonizes the cystic fibrosis (CF) airways causing chronic bacterial lung infections. CF patients are routinely treated with macrolides, however, P. aeruginosa is considered insusceptible as consequence of inadequate susceptibility testing leaving resistance mechanism completely overlooked. Here, we investigated a new mechanism of macrolide resistance caused by ribosomal protein mutations. METHODS: Investigating a longitudinal collection of 529 isolates from CF patients and analysing 5758 protein sequences from different sources, mutations in P. aeruginosa's ribosomal proteins connected to macrolide resistance were identified. Using a modified susceptibility testing protocol, isolates harbouring a mutated uL4 ribosomal protein were tested for resistance against macrolide antibiotics and macrolide-induced quorum sensing modulation. Proteome and ribosome profiling were applied to assess the impact of the mutations on the bacterial physiology. RESULTS: Five uL4 mutations were identified in isolates from different CF patients. Most mapped to the conserved loop region of uL4 and resulted in increased macrolide tolerance (>10-fold relative to wt strains). Greater concentrations (>10-fold) of macrolide antibiotic were needed to inhibit the growth, reduce swimming motility, and induce redox sensitivity of the uL4 mutants. 16 proteins involved in ribosome adaptation displayed altered expression possibly to compensate for the uL4 mutations, which changed the ribosome stoichiometry without negatively affecting bacterial physiology. CONCLUSIONS: Macrolide antibiotics should, therefore, be considered as active antimicrobial agents against P. aeruginosa and resistance development should be contemplated when patients are treated with prolonged courses of macrolides. Importantly, improved macrolide susceptibility testing is necessary for the detection of resistant bacteria.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

Antibiotic Potentiation in Multidrug-Resistant Gram-Negative Pathogenic Bacteria by a Synthetic Peptidomimetic.

The peptidomimetic H-[NLys-tBuAla]6-NH2 (CEP-136), which exhibits low inherent antimicrobial activity against Gram-negative bacteria (MIC = 16-64 µM), was shown to significantly potentiate the antibacterial activity of several clinically important antibiotics against the human pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Thus, the antibacterial spectrum of rifampicin, clarithromycin, and azithromycin could be extended to include also these Gram-negative bacteria. Additionally, the potentiation effect was demonstrated in a panel of clinically relevant multidrug-resistant isolates including extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing as well as colistin-resistant strains. For some peptidomimetic-antibiotic combinations, the strong synergy corresponded to a more than 50-fold reduction of the minimal inhibitory concentration of the antibiotic. Mechanistic studies indicate that the potentiation arises from a permeabilization effect exerted on the outer membrane lipopolysaccharide layer of the Gram-negative bacteria without significant disruption of the inner membrane. Furthermore, the peptidomimetic enhancer exhibited only a marginal effect on the viability of mammalian HepG2 cells even at concentrations 100-fold higher than that enabling the antibiotic enhancement. Also, a low hemolytic activity combined with limited in vivo acute toxicity of CEP-136 in healthy mice allowed in vivo validation of the potentiation effect on both rifampicin and azithromycin treatment in a murine peritonitis model. Thus, CEP-136 is an interesting hit compound for further development of effective adjuvants for repurposing antibiotics for use against infections by multidrug-resistant Gram-negative bacteria.

Uptake, Stability, and Activity of Antisense Anti-acpP PNA-Peptide Conjugates in Escherichia coli and the Role of SbmA.

PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)3K, targeting essential bacterial genes, such as acpP, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)3K-eg1-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)3K-eg1-PNA by showing that the peptide part of (KFF)3K-eg1-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture (t1/2 < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)3k-eg1-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)3K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)3K-eg1-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).

PNA Antisense Targeting in Bacteria: Determination of Antibacterial Activity (MIC) of PNA-Peptide Conjugates.

Antisense PNA-peptide conjugates targeting essential bacterial genes have shown interesting potential for discovery of novel precision antibiotics. In this context, the minimal inhibitory concentration (MIC) assay is used to assess and compare the antimicrobial activity of natural as well as synthetic antimicrobial compounds. Here, we describe the determination of the minimal inhibitory concentration of peptide-PNA conjugates against Escherichia coli. This method can be expanded to include minimal bactericidal concentration (MBC) determination and kill-curve kinetics.

PNA Length Restriction of Antibacterial Activity of Peptide-PNA Conjugates in Escherichia coli Through Effects of the Inner Membrane.

Peptide Nucleic Acid (PNA)-peptide conjugates targeting essential bacterial genes are showing promise as antisense antimicrobials in drug discovery. Optimization has focused on selection of target genes and exact localization around the ribosome binding site, but surprisingly a length optimum around 10-12 nucleobases has been found. Addressing this observation, we have investigated the relationship between PNA-length, PNA-RNA duplex stability and antimicrobial activity in E. coli in more detail. For PNAs of identical length of ten nucleobases the expected reverse correlation between the thermal stability (Tm) of the PNA-RNA duplex and the MIC for single mismatched PNAs was found. Also the expected direct correlation between the length of the PNA and the PNA-RNA duplex stability was found. Nonetheless, 10-mer PNAs [in a 6-18 mer extension series of (KFF)3K- and (RXR)4 conjugates] were the most active as antisense antimicrobials in both wild type E. coli MG1655 and AS19, suggesting that the size constraint is related to the bacterial uptake of PNA-peptide conjugates. This conclusion was supported by flow cytometry data showing higher bacterial uptake of shorter PNA fluorophore labeled conjugates. Interestingly, the size-limited uptake seems independent on outer membrane integrity (AS19), and thus the results suggest that the inner membrane limits the molecular size for peptide-PNA passage.

Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates.

Pseudomonas aeruginosa is an opportunistic pathogen that can infect the lungs of cystic fibrosis (CF) patients and persist in the form of antibiotic-tolerant aggregates in the mucus. It has recently been suggested that such aggregates are formed due to restricted bacterial motility independent of the production of extracellular matrix components, and that they do not rely on an extracellular matrix for antimicrobial tolerance. However, we show here that biofilm matrix overexpression, as displayed by various clinical isolates, significantly protects P. aeruginosa aggregates against antimicrobial treatment. Alginate-overproducing mucA mutant bacteria growing in aggregates showed highly increased antibiotic tolerance compared to wild-type bacteria in aggregates. Deletion of algD in the mucA mutant strain abrogated alginate production and reversed the antibiotic tolerance displayed by the aggregates to a level similar to that observed for aggregates formed by the wild type. The P. aeruginosa ΔwspF and ΔyfiR mutant strains both overproduce Pel and Psl exopolysaccharide, and when these bacteria grew in aggregates, they showed highly increased antibiotic tolerance compared to wild-type bacteria growing in aggregates. However, the ΔwspF and ΔyfiR mutant strains, deficient in Pel/Psl production due to additional ΔpelA ΔpslBCD deletions, formed aggregates that displayed antibiotic tolerance levels close to those of wild-type aggregates. These results suggest that biofilm matrix components, such as alginate, Pel, and Psl, do play a role in the tolerance toward antimicrobials when bacteria grow as aggregates.

The bactericidal activity of ß-lactam antibiotics is increased by metabolizable sugar species.

Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to ß-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on ß-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to ß-lactam-mediated inhibition of cell division, rapidly appeared upon ß-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of ß-lactam treatment. Interestingly, the effect of sugar on ß-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in ß-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.

Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses.

Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance.

Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli.

For almost half of a century, we have known that aminoglycoside antibiotics corrupt ribosomes, causing translational misreading, yet it remains unclear whether or not misreading triggers protein misfolding, and possible effects of chaperone action on drug susceptibilities are poorly understood. Here, we show that aminoglycosides cause cytosolic protein misfolding and that chaperonin GroEL/GroES overexpression counters this defect. During aminoglycoside exposure to exponential cultures, chaperonin overexpression protected the bacterial membrane potential, rescued cell growth, and facilitated survival, whereas inhibition of chaperonin expression sensitized bacteria. Overexpression of the DnaK/DnaJ/GrpE chaperone system similarly facilitated survival but did not promote growth of aminoglycoside-treated bacteria. Inhibition of chaperonin expression sensitized bacteria to aminoglycosides as measured by reduced minimum inhibitory concentrations, whereas GroEL/GroES overexpression did not increase minimum inhibitory concentrations. Our observations establish misfolding of cytosolic proteins as an effect of aminoglycoside action and reveal that chaperones, chaperonins in particular, help bacteria cope during early exposure to these drugs.

Tuning protein expression using synonymous codon libraries targeted to the 5' mRNA coding region.

In bacteria, the 5' mRNA coding region plays an important role in determining translation output. Here, we report synthetic sequences that when placed in the 5'-mRNA coding region, leading to recombinant proteins containing short N-terminal extensions, virtually abolish, enhance or produce intermediate expression levels of green fluorescent protein in Escherichia coli. At least in one case, no apparent effect on protein stability was observed, pointing to RNA level effects as the principal reason for the observed expression differences. Targeting a synonymous codon library to the 5' coding sequence allowed tuning of protein expression over ~300-fold with preservation of amino acid identity. This approach is simple and should be generally applicable in bacteria. The data support that features in the 5' mRNA coding region near the AUG start codon are key in determining translation output and hence is important to recombinant and, most certainly, endogenous gene expression.

Protein evolution via amino acid and codon elimination.

BACKGROUND: Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles. METHODOLOGY/PRINCIPAL FINDINGS: The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance. CONCLUSIONS/SIGNIFICANCE: The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.

Mega-cloning and the advent of synthetic genomes.

Molecular biology owes its prominent role in the biological sciences to the tools of recombinant DNA. While the foundations of recombinant DNA were laid in the 1970s with the discovery of type II restriction endonucleases,1,2 development of robust sequencing technology3 and pioneering work on gene synthesis,4,5 it was not until the turn of the new millennium before the first complete synthetic viral genomes saw the light of day including that of hepatitis C,6 poliovirus,7 and bacteriophage PhiX174.8 Recombinant DNA has come of age as entire cellular genomes are sequenced and stored as digitized information. So what's next? One novel branch of recombinant DNA, referred to as synthetic genomics,9 is occupied with (re)construction of entire cellular genomes from virtual sequence information and using chemical components. Here we look at the most recent developments in such de novo construction. For a broader and more extensive review on genome engineering, the reader is referred to the excellent paper by Carr and Church.10.