Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

Abstract

bGram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens. With emerging multidrug resistance and a limited number of treatment options, bacterial infections pose an ever growing threat to human health. Gram-negative bacteria are particularly recalcitrant to antimicrobial intervention due to their cell envelope structure. Possessing two membranes with different chemical properties (1) and containing an arsenal of efflux pumps (2), Gram-negative bacteria are capable of both excluding and expelling molecules, rendering them resistant to many drugs. Despite major Gram-negative pathogens being classified as urgent or serious threats by the CDC (3), no Gram-negative-active antibiotic with a new mechanism of action has been introduced in over 40 years. Of the 28 new antibiotics approved since 2000, only 18 are indicated for treatment of Gram-negative bacteria (4), and all of those are derived from the four legacy classes ␤-lactams, tetracyclines, macrolides, and fluoroquinolones, which were first introduced in 1942 (5), 1948 (6), 1952 (7), and 1967 (8), respectively. Novel derivatives of old antibiotics often have limited spectra of coverage and can only do so much to overcome existing mechanisms of resistance; therefore, introduction of novel classes of antibiotics is critical.There is no shortage of potential targets in the antibacterial space given that the essential gene set for several Gram-negative pathogens has been defined (9-12). However, despite genetically demonstrating target essentiality, cognate inhibitors with exquisite in vitro activity often have no measurable cellular activity (13,14). The reasons for this can be attributed to a lack of penetration of compounds into bacteria, efflux of molecules out of bacteria, insufficient inhibition of the target, metabolism within the cells, or a combination of the aforementioned factors. Understanding the reason for failure to translate enzymatic 50% inhibitory concentrations (IC 50 s) into cellular MICs is paramou...

Muropeptides in Pseudomonas aeruginosa and their Role as Elicitors of β‐Lactam‐Antibiotic Resistance

“…The strain P. aeruginosa Z61 has the full complement of the genes necessary for induction of β-lactamases, but it expresses a mutant version of the β-lactamase with diminished activity. [22] Nonetheless, using the nitrocefin assay we observed a 4.7 fold increase of induction of β-lactamase at quarter-MIC level of cefoxitin. The same experiment performed with the bacteria exposed to 100 μg/mL muropeptides 2e or 4e resulted in 1.4 or 1.7 induction, respectively (Table S1).…”

Targeting intracellular p-aminobenzoic acid production potentiates the anti-tubercular action of antifolates

“…However, enzymes capable of inactivating drugs are only one factor contributing to intrinsic drug resistance in bacteria. Other factors including membrane penetration, efflux mechanisms, and endogenous native substrate competition contribute to intrinsic drug resistance262728. The ability to improve susceptibility of M .…”

LptD is a promising vaccine antigen and potential immunotherapeutic target for protection against Vibrio species infection

“…The OMP LptD, formerly known as OstA or Imp, has been subjected to in-depth investigation in E. coli (see reviews of refs 28 and 43), and it has also been subjected to studies in several other Gram-negative bacteria, such as Neisseria gonorrhoeae 36, Acinetobacter baumannii 26, N. meningitidis 44 and Pseudomonas aeruginosa 45, in recent years. In these studies, LptD was revealed to be one a component of the LPS transport complex, which consists of 7 essential proteins (LptA-LptG), and its physiological function has been suggested to mediate the final translocation of fully synthesized LPS to the cell surface, thus being involved in cell envelope biogenesis46.…”

“…More importantly, the LPS that surrounds bacteria forms a permeation barrier, which prevents hydrophobic antibiotics from entering the cells, thus conferring antibiotic resistance252854. It has been shown previously that deletion of lptD caused a significant decrease in cell LPS253645, and the lack of LPS is commonly accompanied by a decreasing growth rate and increasing sensitivity to antibiotics26455556. The LPS transport proteins have accordingly been considered attractive targets for new drug development5758.…”