PBP1A Directly Interacts with the Divisome Complex to Promote Septal Peptidoglycan Synthesis in Acinetobacter baumannii.

The class A penicillin-binding proteins (aPBPs), PBP1A and PBP1B, are major peptidoglycan synthases that synthesize more than half of the peptidoglycan per generation in Escherichia coli. Whereas aPBPs have distinct roles in peptidoglycan biosynthesis during growth (i.e., elongation and division), they are semiredundant; disruption of either is rescued by the other to maintain envelope homeostasis and promote proper growth. Acinetobacter baumannii is a nosocomial pathogen that has a high propensity to overcome antimicrobial treatment. A. baumannii contains both PBP1A and PBP1B (encoded by mrcA and mrcB, respectively), but only mrcA deletion decreased fitness and contributed to colistin resistance through inactivation of lipooligosaccharide biosynthesis, indicating that PBP1B was not functionally redundant with the PBP1A activity. While previous studies suggested a distinct role for PBP1A in division, it was unknown whether its role in septal peptidoglycan biosynthesis was direct. Here, we show that A. baumannii PBP1A has a direct role in division through interactions with divisome components. PBP1A localizes to septal sites during growth, where it interacts with the transpeptidase PBP3, an essential division component that regulates daughter cell formation. PBP3 overexpression was sufficient to rescue the division defect in ΔmrcA A. baumannii; however, PBP1A overexpression was not sufficient to rescue the septal defect when PBP3 was inhibited, suggesting that their activity is not redundant. Overexpression of a major dd-carboxypeptidase, PBP5, also restored the canonical A. baumannii coccobacilli morphology in ΔmrcA cells. Together, these data support a direct role for PBP1A in A. baumannii division and highlights its role as a septal peptidoglycan synthase. IMPORTANCE Peptidoglycan biosynthesis is a validated target of ß-lactam antibiotics, and it is critical that we understand essential processes in multidrug-resistant pathogens such as Acinetobacter baumannii. While model systems such as Escherichia coli have shown that PBP1A is associated with side wall peptidoglycan synthesis, we show herein that A. baumannii PBP1A directly interacts with the divisome component PBP3 to promote division, suggesting a unique role for the enzyme in this highly drug-resistant nosocomial pathogen. A. baumannii demonstrated unanticipated resistance and tolerance to envelope-targeting antibiotics, which may be driven by rewired peptidoglycan machinery and may underlie therapeutic failure during antibiotic treatment.

Peptidoglycan Recycling Promotes Outer Membrane Integrity and Carbapenem Tolerance in Acinetobacter baumannii.

ß-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. Carbapenem ß-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections, but treatment failure is increasingly prevalent. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate tolerance to high-level meropenem treatment, form spheroplasts upon exposure to the antibiotic, and revert to normal growth after antibiotic removal. Using transcriptomics and genetic screens, we show that several genes associated with outer membrane integrity maintenance and efflux promote tolerance, likely by limiting entry into the periplasm. Genes associated with peptidoglycan homeostasis in the periplasm and cytoplasm also answered our screen, and their disruption compromised cell envelope barrier function. Finally, we defined the enzymatic activity of the tolerance determinants penicillin-binding protein 7 (PBP7) and ElsL (a cytoplasmic ld-carboxypeptidase). These data show that outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to A. baumannii meropenem tolerance. IMPORTANCE Carbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting the urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote the acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well understood. Here, we characterized the role of peptidoglycan recycling in outer membrane integrity maintenance and meropenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlight physiological processes that could be targeted to improve antimicrobial treatment.

Septal Class A Penicillin-Binding Protein Activity and ld-Transpeptidases Mediate Selection of Colistin-Resistant Lipooligosaccharide-Deficient Acinetobacter baumannii.

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several Acinetobacter baumannii clinical isolates produced LOS- colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS-A. baumannii, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS-A. baumannii PBP1A contributes to proper cell division in A. baumannii, and its absence induced cell chaining. Only isolates producing three or more septa supported selection of colistin-resistant LOS-A. baumannii PBP1A was enriched at the midcell, where the divisome complex facilitates daughter cell formation, and its localization was dependent on glycosyltransferase activity. Transposon mutagenesis showed that genes encoding two putative ld-transpeptidases (LdtJ and LdtK) became essential in the PBP1A mutant. Both LdtJ and LdtK were required for selection of LOS-A. baumannii, but each had distinct enzymatic activities in the cell. Together, these findings demonstrate that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity remodel the cell envelope to support selection of colistin-resistant LOS-A. baumanniiIMPORTANCE The increasing prevalence of antibiotic treatment failure associated with Gram-negative bacterial infections highlights an urgent need to develop new alternative therapeutic strategies. The last-line antimicrobial colistin (polymyxin E) targets the ubiquitous outer membrane lipopolysaccharide (LPS)/LOS membrane anchor, lipid A, which is essential for viability of most diderms. However, several LOS-Acinetobacter baumannii clinical isolates were recovered after colistin selection, suggesting a conserved resistance mechanism. Here, we characterized a role for penicillin-binding protein 1A in A. baumannii septation and intrinsic ß-lactam susceptibility. We also showed that defects in PBP1A glycosyltransferase activity and ld-transpeptidase activity support isolation of colistin-resistant LOS-A. baumannii.

Discovery and characterization of New Delhi metallo-ß-lactamase-1 inhibitor peptides that potentiate meropenem-dependent killing of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: Metallo-ß-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade ß-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent ß-lactamase inhibitors are formulated with commonly prescribed ß-lactams, no MBL inhibitors are currently approved for combinatorial therapies. New compounds that target MBLs to restore carbapenem activity against CPE are therefore urgently needed. Herein we identified and characterized novel synthetic peptide inhibitors that bound to and inhibited NDM-1, which is an emerging ß-lactam resistance mechanism in CPE. METHODS: We leveraged Surface Localized Antimicrobial displaY (SLAY) to identify and characterize peptides that inhibit NDM-1, which is a primary carbapenem resistance mechanism in CPE. Lead inhibitor sequences were chemically synthesized and MBCs and MICs were calculated in the presence/absence of carbapenems. Kinetic analysis with recombinant NDM-1 and select peptides tested direct binding and supported NDM-1 inhibitor mechanisms of action. Inhibitors were also tested for cytotoxicity. RESULTS: We identified approximately 1700 sequences that potentiated carbapenem-dependent killing against NDM-1 Escherichia coli. Several also enhanced meropenem-dependent killing of other CPE. Biochemical characterization of a subset indicated the peptides penetrated the bacterial periplasm and directly bound NDM-1 to inhibit enzymatic activity. Additionally, each demonstrated minimal haemolysis and cytotoxicity against mammalian cell lines. CONCLUSIONS: Our approach advances a molecular platform for antimicrobial discovery, which complements the growing need for alternative antimicrobials. We also discovered lead NDM-1 inhibitors, which serve as a starting point for further chemical optimization.

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ-dependent 4-amino-4-deoxy-l-arabinose addition to lipid A.

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram-negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine-containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two-component system (TCS) directly activates 4-amino-4-deoxy-l-arabinose (l-Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l-Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrABEcl TCS. Instead, PhoPEcl binds to the arnBEcl promoter to induce l-Ara4N biosynthesis and PmrAB-independent addition to the lipid A disaccharolipid. Therefore, PhoPQEcl contributes to regulation of CAMP heteroresistance in some ECC clusters.