Unravelling the collateral damage of antibiotics on gut bacteria.

Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease1. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species2. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for ß-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal3-5 and human6,7 gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.

The functional proteome landscape of Escherichia coli.

Recent developments in high-throughput reverse genetics1,2 have revolutionized our ability to map gene function and interactions3-6. The power of these approaches depends on their ability to identify functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out7-9. However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts10. Here we use a high-content biochemical readout, thermal proteome profiling11, to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes12 that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.

How the assembly and protection of the bacterial cell envelope depend on cysteine residues.

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria.

Stress-induced chaperones: a first line of defense against the powerful oxidant hypochlorous acid.

Hypochlorous acid (HOCl; bleach) is a powerful weapon used by our immune system to eliminate invading bacteria. Yet the way HOCl actually kills bacteria and how they defend themselves from its oxidative action have only started to be uncovered. As this molecule induces both protein oxidation and aggregation, bacteria need concerted efforts of chaperones and antioxidants to maintain proteostasis during stress. Recent advances in the field identified several stress-activated chaperones, like Hsp33, RidA, and CnoX, which display unique structural features and play a central role in protecting the bacterial proteome during HOCl stress.

The Chaperone and Redox Properties of CnoX Chaperedoxins Are Tailored to the Proteostatic Needs of Bacterial Species.

Hypochlorous acid (bleach), an oxidizing compound produced by neutrophils, turns the Escherichia coli chaperedoxin CnoX into a powerful holdase protecting its substrates from bleach-induced aggregation. CnoX is well conserved in bacteria, even in non-infectious species unlikely to encounter this oxidant, muddying the role of CnoX in these organisms. Here, we found that CnoX in the non-pathogenic aquatic bacterium Caulobacter crescentus functions as a holdase that efficiently protects 50 proteins from heat-induced aggregation. Remarkably, the chaperone activity of Caulobacter CnoX is constitutive. Like E. coli CnoX, Caulobacter CnoX transfers its substrates to DnaK/J/GrpE and GroEL/ES for refolding, indicating conservation of cooperation with GroEL/ES. Interestingly, Caulobacter CnoX exhibits thioredoxin oxidoreductase activity, by which it controls the redox state of 90 proteins. This function, which E. coli CnoX lacks, is likely welcome in a bacterium poorly equipped with antioxidant defenses. Thus, the redox and chaperone properties of CnoX chaperedoxins were fine-tuned during evolution to adapt these proteins to the specific needs of each species.IMPORTANCE How proteins are protected from stress-induced aggregation is a crucial question in biology and a long-standing mystery. While a long series of landmark studies have provided important contributions to our current understanding of the proteostasis network, key fundamental questions remain unsolved. In this study, we show that the intrinsic features of the chaperedoxin CnoX, a folding factor that combines chaperone and redox protective function, have been tailored during evolution to fit to the specific needs of their host. Whereas Escherichia coli CnoX needs to be activated by bleach, a powerful oxidant produced by our immune system, its counterpart in Caulobacter crescentus, a bacterium living in bleach-free environments, is a constitutive chaperone. In addition, the redox properties of E. coli and C. crescentus CnoX also differ to best contribute to their respective cellular redox homeostasis. This work demonstrates how proteins from the same family have evolved to meet the needs of their hosts.

CnoX Is a Chaperedoxin: A Holdase that Protects Its Substrates from Irreversible Oxidation.

Bleach (HOCl) is a powerful oxidant that kills bacteria in part by causing protein aggregation. It inactivates ATP-dependent chaperones, rendering cellular proteins mostly dependent on holdases. Here we identified Escherichia coli CnoX (YbbN) as a folding factor that, when activated by bleach via chlorination, functions as an efficient holdase, protecting the substrates of the major folding systems GroEL/ES and DnaK/J/GrpE. Remarkably, CnoX uniquely combines this function with the ability to prevent the irreversible oxidation of its substrates. This dual activity makes CnoX the founding member of a family of proteins, the "chaperedoxins." Because CnoX displays a thioredoxin fold and a tetratricopeptide (TPR) domain, two structural motifs conserved in all organisms, this investigation sets the stage for the discovery of additional chaperedoxins in bacteria and eukaryotes that could cooperate with proteins from both the Hsp60 and Hsp70 families.

An essential thioredoxin is involved in the control of the cell cycle in the bacterium Caulobacter crescentus.

Thioredoxins (Trxs) are antioxidant proteins that are conserved among all species. These proteins have been extensively studied and perform reducing reactions on a broad range of substrates. Here, we identified Caulobacter crescentus Trx1 (CCNA_03653; CcTrx1) as an oxidoreductase that is involved in the cell cycle progression of this model bacterium and is required to sustain life. Intriguingly, the abundance of CcTrx1 varies throughout the C. crescentus cell cycle: although the expression of CcTrx1 is induced in stalked cells, right before DNA replication initiation, CcTrx1 is actively degraded by the ClpXP protease in predivisional cells. Importantly, we demonstrated that regulation of the abundance of CcTrx1 is crucial for cell growth and survival as modulating CcTrx1 levels leads to cell death. Finally, we also report a comprehensive biochemical and structural characterization of this unique and essential Trx. The requirement to precisely control the abundance of CcTrx1 for cell survival underlines the importance of redox control for optimal cell cycle progression in C. crescentus.

A new role for Escherichia coli DsbC protein in protection against oxidative stress.

We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the L-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing L-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.