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

Abstract

Uropathogenic Escherichia coli (UPEC) are 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, non-proliferative 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 adhere to and infiltrate bladder epithelial cells where they may form intracellular bacterial communities (IBCs) or enter into a non-growing 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.

Bacterial virulence regulation through soluble peptidoglycan fragments sensing and response: knowledge gaps and therapeutic potential

“…Beyond GlcNAc, other PGN-derived fragments have been reported to display a clear capacity for bacterial behavior modulation. This would be the case of the stem tetra- and penta-peptides (P4 and P5) cleaved from the glycan chains of PGN, which have been recently demonstrated to prevent the quiescent nonproliferative status that appears at low population density in uropathogenic E. coli (DiBiasio et al 2020 ). Interestingly, this metabolic response is obtained when providing these fragments exogenously, both obtained from E. coli and from Gram-positive PGN, indicating that the nature of the third amino acid in the chain (DAP vs. l -lysine, respectively) is not important to activate this quorum sensing-independent growth.…”

“…Meanwhile, the known mechanism for the uptake of these peptides into the cytosol, namely the oligo-peptide permease system Opp, seems dispensable for the response, from which it is deduced that there exists either an additional unknown transporter for the aforementioned P4 and P5, or a sensor responsible for their detection in the periplasm. Moreover, shorter stem peptides or larger PGN fragments including sugar moieties did not exert the abovementioned effects on the bacterium, suggesting quite a tight array of PGN fragments to which the unidentified sensor/regulator could respond (DiBiasio et al 2020 ). Identification of this sensor/regulator as well as the downstream signaling events causing the prevention of quiescence is a very interesting topic worth delving into, because of the clear bacterial fitness-related implications.…”

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenicEscherichia coli

“…We introduced a mini-Tn5 transposon in the UPEC genome (CFT073) by conjugative transfer from a donor strain (ATM161), and then screened a culture of recipients (10 4 CFU ml -1 ) for growth on glucose minimal agar supplemented with kanamycin. We previously reported that UPEC cells (CFT073) become quiescent on glucose minimal agar at low plating density (<10 6 CFU ml -1 ) but proliferate rapidly at high plating density (>10 6 CFU ml -1 ) (28,29). We isolated 21 mutant strains that formed colonies on glucose minimal agar when plated at low cells density (10 4 CFU ml -1 ) and identified the mini-Tn5 insertion site by arbitrarily-primed PCR and sequencing (34).…”

“…Next, we tested if known proliferants could prevent quiescence and promote proliferation of CFT073 ∆ihfB::kan (JM028) and CFT073 ∆zapE::kan (JM023) strains complemented with expression plasmids. Proliferant cues that were previously identified include peptidoglycan (PG) tetra-and pentapeptides and the amino acids L-lysine (1 mM) and L-methionine (1 mM) (28,29).…”

“…At low inoculation density (<10 6 CFU ml -1 ), many clinical UPEC isolates, including the widely studied ST-73 strain CFT073, are non-proliferative, or quiescent, on minimal medium containing glucose as the sole carbon source (28,29). At high inoculation density, i.e., > 10 6 CFU ml -1 , the quiescence-capable UPEC cells are proliferative on glucose minimal medium, suggesting that proliferation on glucose is quorum-dependent.…”