Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Abstract

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROSdependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ 72-47 protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.A gents or conditions that inhibit important biological processes can kill cells. However, physiological processes metabolically downstream of the initial inhibition can also contribute to cell death (1). For example, β-lactam antibiotics not only inhibit penicillin-binding proteins leading to lysis; they also induce a futile cycle of cell wall synthesis and degradation that contributes to their killing (2). In another example, lethal attacks on Escherichia coli mediated by the type VI secretion system P1vir phage and the antimicrobial peptide polymyxin B elicit the production of reactive oxygen species (ROS) that contribute to cell death (3). In bacteria, ROS production has been associated with the lethal effects of diverse stresses (4). In most cases, the detailed mechanisms responsible for ROS production are poorly understood; however, a variety of futile metabolic cycles can elicit H 2 O 2 production, illustrating the breadth of possible metabolic perturbations that could potentially induce oxidative stress (5).Despite widespread evidence that endogenous ROS produced as a consequence of metabolic stress can be lethal to bacteria and eukaryotes, the application of this concept to antibiotic lethality has been complicated. Evidence from multiple investiga...

Post-stress bacterial cell death mediated by reactive oxygen species

“…It has been argued that potential off-target effects are difficult to rule out for perturbations with chemical agents, such as thiourea and bipyridyl (23,24); thus, statements of causality are questionable when such agents are used. Off-target effects are unlikely with enzymatic suppression of ROS, as when catalase is added to agar or when a deficiency of katG (catalase) increases killing (20,26,28). A causal role for ROS in stress-mediated death has also emerged from the effects of mutations in a variety of other genes, most notably genes involved in the processing of 8-oxoguanine in .…”

“…both quinolone and ROS are known to generate DNA breaks (11,26,28,30,42,43). Treatment with the quinolone nalidixic acid led to rapid accumulation of DSBs; after 1 h treatment, 97% of cells scored positive (Fig.…”

“…However, an ROS contribution to antimicrobial killing became controversial when the original observation (9) was challenged (22)(23)(24). Subsequent work countered many of the challenges (13)(14)(15)25) and extended the phenomenon to thymineless death (26), phage infection (27), the type VI secretion system (27), and overexpression of a MalE-LacZ fusion (28). Moreover, nitric oxide and hydrogen sulfide interfere with antimicrobial killing by suppressing ROS generation/accumulation (17)(18)(19).…”

Antibiotic efficacy — context matters

“…It may be possible to distinguish between these biochemically, as the lethality driven by secondary processes can be interrupted by inhibiting protein translation and cell respiration [34] or by metabolic shunting [37]. Cell death emerging from stress-induced death processes was recently studied in an antibiotic-free system using a historically significant fusion protein [64]. The authors demonstrated that the primary consequence of jamming the SecY protein translocation machinery was cell stasis and that cell death instead emerged from downstream events shared with antibiotic lethality, including ROS accumulation and double-stranded DNA damage.…”

Antibiotic killing through oxidized nucleotides

“…However, regardless of the primary cellular target, different bactericidal antibiotics all induce a similar cascade of events that contribute to their lethality in Escherichia coli: accelerated respiration (7); changes in iron and redox metabolism (8); formation of reactive oxygen species (9); oxidative damage of DNA (10), RNA, and proteins (11); and cell death. Interestingly, the same killing cascade is seen in a number of antibiotic-independent killing phenomena, for example, T6SS-effector dependent killing (12) and expression of a toxic fusion protein (13).…”