Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics.

Rod-shaped bacteria frequently localize proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the roles of polar factors in responses to extracellular stimuli have been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal resistance of cell envelope-directed antibiotics, including vancomycin which does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin sensitivity. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive ΔtipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in ΔtipN cells relative to the wild type under some, but not all, cell wall stress conditions that this strain was sensitized to, in particular cefixime and detergent exposure. Together, these results indicate that TipN contributes to cell envelope stress resistance in addition to its roles in intracellular development, and its loss influences signaling through the ChvIG two-component system which has been co-opted as a sensor of cell wall stress in CaulobacterIMPORTANCE Maintenance of an intact cell envelope is essential for free-living bacteria to protect themselves against their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and reform the cell envelope at the division plane as the cells divide. We have found that TipN, a factor required for correct division and cell pole development in Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilization of these weak points of the cell envelope as well as its other roles inside the cell.

Development of a semi-automated image-based high-throughput drug screening system.

We previously reported that the innate sensing of the endosymbiont Leishmania RNA virus 1 (LRV1) within Leishmania (Viannia) guyanensis through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing -L. guyanensis and -L. braziliensis parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing L. guyanensis. The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against Leishmania. Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number.

Growth control switch by a DNA-damage-inducible toxin-antitoxin system in Caulobacter crescentus.

Bacterial toxin-antitoxin systems (TASs) are thought to respond to various stresses, often inducing growth-arrested (persistent) sub-populations of cells whose housekeeping functions are inhibited. Many such TASs induce this effect through the translation-dependent RNA cleavage (RNase) activity of their toxins, which are held in check by their cognate antitoxins in the absence of stress. However, it is not always clear whether specific mRNA targets of orthologous RNase toxins are responsible for their phenotypic effect, which has made it difficult to accurately place the multitude of TASs within cellular and adaptive regulatory networks. Here, we show that the TAS HigBA of Caulobacter crescentus can promote and inhibit bacterial growth dependent on the dosage of HigB, a toxin regulated by the DNA damage (SOS) repressor LexA in addition to its antitoxin HigA, and the target selectivity of HigB's mRNA cleavage activity. HigB reduced the expression of an efflux pump that is toxic to a polarity control mutant, cripples the growth of cells lacking LexA, and targets the cell cycle circuitry. Thus, TASs can have outcome switching activity in bacterial adaptive (stress) and systemic (cell cycle) networks.