From Modules to Networks: a Systems-Level Analysis of the Bacitracin Stress Response in Bacillus subtilis

Abstract

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor. IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

BceAB-Type Antibiotic Resistance Transporters Appear To Act by Target Protection of Cell Wall Synthesis

“…However, any attempts at changing the concentration of lipid II in the cell, e.g., by deletion of penicillin-binding proteins (PBPs), have so far been unsuccessful. This is presumably due to redundancy between multiple PBPs and is also consistent with our mathematical model, which showed that the large UPP pool in the cycle can act as an effective buffering reservoir to compensate for interference with the much smaller lipid II pool (26,27).…”

“…It also explains the observations on reduced resistance upon overproduction of HPP in the cell, where some of BceAB's transport activity is likely invested in the futile removal of bacitracin from HPP rather than UPP (14). It is consistent with the data reported here and previously (9,27) that the factor determining the transport activity of BceAB is the concentration of UPP-BAC complexes in the cell. Target protection of cell wall synthesis also offers a plausible explanation for the use of transporters in resistance against cell wall-active antibiotics in Gram-positive bacteria.…”

“…3A and B). This is because the total amount of lipid carrier is homeostatically increased in a bcrC deletion strain to ensure a close-to-optimal rate of peptidoglycan synthesis (27). The model therefore confirms that decreasing the UPP dephosphorylation rate in the ΔbcrC strain specifically causes accumulation of UPP but does not affect other cycle intermediates.…”

“…There was no detectable BceAB activity in either of the tested strains, which suggested that accumulation of UPP alone was not The rate of peptidoglycan (PG) synthesis is shown in molecules of precursor incorporated per minute. The thickness of the arrow for de novo UPP synthesis reflects the previously described homeostatic increase in lipid carrier synthesis upon bcrC deletion (27). Wild type (A) and bcrC deletion mutant (B).…”

“…Based on the computational description of the lipid II cycle (26), we recently developed a mathematical model that describes the protective effect of the bacitracin resistance determinants BceAB and BcrC of B. subtilis on the progression of the lipid II cycle (27). This model can predict changes to the pool levels of lipid II cycle intermediates under different conditions and has been verified by comparing predictions of growth inhibition by antibiotics targeting different steps of the cycle to corresponding experimental data (26,27). The model suggests that reducing the rate of UPP dephosphorylation increases the level of UPP displayed on the extracellular face of the membrane.…”