Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

Degradation of MinD oscillator complexes by Escherichia coli ClpXP.

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase-dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation.

Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after completion of successful antibiotic treatment. Many clinical isolates, including the well characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex IHF and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two hybrid assays. We report direct interactions between succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions likely enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility.

The Stress-Active Cell Division Protein ZapE Alters FtsZ Filament Architecture to Facilitate Division in Escherichia coli.

During pathogenic infections, bacterial cells experience environmental stress conditions, including low oxygen and thermal stress. Bacterial cells proliferate during infection and divide by a mechanism characterized by the assembly of a large cytoskeletal structure at the division site called the Z-ring. The major protein constituting the Z-ring is FtsZ, a tubulin homolog and GTPase that utilizes the nucleotide to assemble into dynamic polymers. In Escherichia coli, many cell division proteins interact with FtsZ and modulate Z-ring assembly, while others direct cell wall insertion and peptidoglycan remodeling. Here, we show that ZapE, an ATPase that accumulates during late constriction, directly interacts with FtsZ and phospholipids in vitro. In the presence of adenosine triphosphate (ATP), ZapE induces bundling of GTP-induced FtsZ polymers; however, ZapE also binds FtsZ in the absence of GTP. The ZapE mutant protein ZapE(K84A), which is defective for ATP hydrolysis, also interacts with FtsZ and induces FtsZ filament bundling. In vivo, cultures of zapE deletion cells contain a low percentage of filamentous cells, suggesting that they have a modest division defect; however, they are able to grow when exposed to stress, such as high temperature and limited oxygen. When combined with the chromosomal deletion of minC, which encodes an FtsZ disassembly factor, ΔzapE ΔminC cells experience growth delays that slow proliferation at high temperature and prevent recovery. This synthetic slow growth phenotype after exposure to stress suggests that ZapE may function to ensure proliferation during and after stress, and this is exacerbated when cells are also deleted for minC. Expression of either ZapE or ZapE(K84A) complements the aberrant growth phenotypes in vivo suggesting that the division-associated role of ZapE does not require ZapE ATP hydrolysis. These results support that ZapE is a stress-regulated cell division protein that interacts directly with FtsZ and phospholipids, promoting growth and division after exposure to environmental stress.