SUMMARY We describe a mechanism of flagellar motor control by the bacterial signaling molecule c-di-GMP, which regulates several cellular behaviors. E. coli and Salmonella have multiple c-di-GMP cyclases and phosphodiesterases, yet absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di-GMP-binding protein, for motility inhibition. We demonstrate that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, most strongly in the presence of c-di-GMP. This interaction reduces the efficiency of torque generation and induces CCW motor bias. We present a “backstop brake” model showing how both effects can result from disrupting the organization of the FliG C-terminal domain, which interacts with the stator protein MotA to generate torque. Inhibition of motility and chemotaxis may represent a strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.

The bacterial flagellum is driven by a bidirectional rotary motor, which propels bacteria to swim through liquids or swarm over surfaces. While the functions of the major structural and regulatory components of the flagellum are known, the function of the well-conserved FliL protein is not. In Salmonella and Escherichia coli, the absence of FliL leads to a small defect in swimming but complete elimination of swarming. Here, we tracked single motors of these bacteria and found that absence of FliL decreases their speed as well as switching frequency. We demonstrate that FliL interacts strongly with itself, with the MS ring protein FliF, and with the stator proteins MotA and MotB and weakly with the rotor switch protein FliG. These and other experiments show that FliL increases motor output either by recruiting or stabilizing the stators or by increasing their efficiency and contributes additionally to torque generation at higher motor loads. The increased torque enabled by FliL explains why this protein is essential for swarming on an agar surface expected to offer increased resistance to bacterial movement.

UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram‐negative bacteria. ACHN‐975 (N‐((S)‐3‐amino‐1‐(hydroxyamino)‐3‐methyl‐1‐oxobutan‐2‐yl)‐4‐(((1R,2R)‐2‐(hydroxymethyl)cyclopropyl)buta‐1,3‐diyn‐1‐yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose‐limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN‐975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC‐516, (S)‐N‐(2‐(hydroxyamino)‐1‐(3‐methoxy‐1,1‐dioxidothietan‐3‐yl)‐2‐oxoethyl)‐4‐(6‐hydroxyhexa‐1,3‐diyn‐1‐yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL−1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

No abstract

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa. We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.

Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes.

Pseudomonas aeruginosa is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for P. aeruginosa to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s−1 while dividing intracellularly. Similar results were obtained with flagellin (fliC) and flagellum assembly (flhA) mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, pilA mutants (lacking T4P) and pilT mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. IMPORTANCE Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Pseudomonas aeruginosa. Previously, we showed that the type III secretion system (T3SS) of invasive P. aeruginosa strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology in vivo, and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular P. aeruginosa can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.