Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Abstract

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-

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

“…7b). These results were in agreement with previous findings obtained in lptD deletion mutants263738.…”

“…There have been several previous studies finding that the deletion of lptD impacted the growth rate and reduced LPS levels in Gram-negative bacteria263536. However, whether deficiency in LptD by targeting specific LptD antibodies on the bacterial surface would have similar impacts on bacteria remains unknown.…”

“…Various studies have shown that the lack of LPS could increase outer membrane permeability and susceptibility to hydrophobic antibiotics263738 because LPS provide a barrier against hydrophobic antibiotics38. Owing to the dramatically decreased LPS in bacteria by targeting of the LptD antibody that was observed, we next assayed the impact of antibiotic susceptibility.…”

“…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.…”

“…LptD and other 6 proteins – LptA, B, C, E, F and G – constitute a trans-membrane complex responsible for transporting lipopolysaccharide (LPS)222324. Through interaction with the lipoprotein component LptE, which adopts a roll-like structure embedded in the LptD barrel, LptD mediates the final translocation of fully synthesized LPS from periplasm to the outer leaflet of the outer membrane252627282930. LPS, a glycolipid consisting of several fatty acyl chains and perhaps hundreds of sugars, is synthesized in the cytoplasm and finally assembled in inner membrane.…”