Adaptivity and dynamics in type III secretion systems.

The type III secretion system is the common core of two bacterial molecular machines: the flagellum and the injectisome. The flagellum is the most widely distributed prokaryotic locomotion device, whereas the injectisome is a syringe-like apparatus for inter-kingdom protein translocation, which is essential for virulence in important human pathogens. The successful concept of the type III secretion system has been modified for different bacterial needs. It can be adapted to changing conditions, and was found to be a dynamic complex constantly exchanging components. In this review, we highlight the flexibility, adaptivity, and dynamic nature of the type III secretion system.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.

Life After Secretion-Yersinia enterocolitica Rapidly Toggles Effector Secretion and Can Resume Cell Division in Response to Changing External Conditions.

Many pathogenic bacteria use the type III secretion system (T3SS) injectisome to manipulate host cells by injecting virulence-promoting effector proteins into the host cytosol. The T3SS is activated upon host cell contact, and its activation is accompanied by an arrest of cell division; hence, many species maintain a T3SS-inactive sibling population to propagate efficiently within the host. The enteric pathogen Yersinia enterocolitica utilizes the T3SS to prevent phagocytosis and inhibit inflammatory responses. Unlike other species, almost all Y. enterocolitica are T3SS-positive at 37°C, which raises the question, how these bacteria are able to propagate within the host, that is, when and how they stop secretion and restart cell division after a burst of secretion. Using a fast and quantitative in vitro secretion assay, we have examined the initiation and termination of type III secretion. We found that effector secretion begins immediately once the activating signal is present, and instantly stops when this signal is removed. Following effector secretion, the bacteria resume division within minutes after being introduced to a non-secreting environment, and the same bacteria are able to re-initiate effector secretion at later time points. Our results indicate that Y. enterocolitica use their type III secretion system to promote their individual survival when necessary, and are able to quickly switch their behavior toward replication afterwards, possibly gaining an advantage during infection.

Evaluation of Mitoquinone for Protecting Against Amikacin-Induced Ototoxicity in Guinea Pigs.

HYPOTHESIS: Mitoquinone (MitoQ) attenuates amikacin ototoxicity in guinea pigs. BACKGROUND: MitoQ, a mitochondria-targeted derivative of the antioxidant ubiquinone, has improved bioavailability and demonstrated safety in humans. Thus, MitoQ is a promising therapeutic approach for protecting against amikacin-induced ototoxicity. METHODS: Both oral and subcutaneous administrations of MitoQ were tested. Amikacin-treated guinea pigs (n = 12-18 per group) received water alone (control) or MitoQ 30 mg/l-supplemented drinking water; or injected subcutaneously with 3 to 5 mg/kg MitoQ or saline (control). Auditory brainstem responses and distortion product otoacoustic emissions were measured before MitoQ or control solution administration and after amikacin injections. Cochlear hair cell damage was assessed using scanning electron microscopy and Western blotting. RESULTS: With oral administration, animals that received 30 mg/l MitoQ had better hearing than controls at only 24 kHz at 3-week (p = 0.017) and 6-week (p = 0.027) post-amikacin. With subcutaneous administration, MitoQ-injected guinea pigs had better hearing than controls at only 24 kHz, 2-week post-amikacin (p = 0.013). Distortion product otoacoustic emission (DPOAE) amplitudes were decreased after amikacin injections, but were not different between treatments (p > 0.05). Electron microscopy showed minor difference in outer hair cell loss between treatments. Western blotting demonstrated limited attenuation of oxidative stress in the cochlea of MitoQ-supplemented guinea pigs. CONCLUSIONS: Oral or subcutaneous MitoQ provided limited protection against amikacin-induced hearing loss and cochlear damage in guinea pigs. Other strategies for attenuating aminoglycoside-induced ototoxicity should be explored.

Collagen and α-Tubulin of Mouse Tympanic Membrane Fibroblasts Treated with Quinolones and Aminoglycosides.

Objective To assess collagen and α-tubulin levels of mouse tympanic membrane fibroblasts treated with quinolone and aminoglycoside antibiotics at concentrations found in eardrops. Study Design Prospective controlled cell culture study. Setting Academic tertiary medical center. Subjects Mouse tympanic membrane fibroblasts. Methods In experiment 1, fibroblasts were treated with the following for 24 or 48 hours: phosphate-buffered saline (negative control), dilute hydrochloric acid (positive control), 0.5% gatifloxacin, or commercially available 0.3% ciprofloxacin, 0.3% ciprofloxacin + 0.1% dexamethasone, 0.3% ofloxacin, 0.5% moxifloxacin, 0.3% gentamicin, or 3.5 mg/mL of neomycin + polymyxin B sulfate + hydrocortisone. In experiment 2, cells were treated with the pure form of gatifloxacin, gentamicin, ofloxacin, or ciprofloxacin. Cells were observed with phase-contrast microscope until harvested. Proteins were extracted for Western blotting with antibodies against collagen α1 type I (collagen 1A1) and α-tubulin, and for densitometry to quantify levels. Results Collagen and tubulin levels in fibroblasts treated with ofloxacin, moxifloxacin, gatifloxacin, or gentamicin for 24 hours were not different from the saline control. Fibroblasts treated with neomycin + polymyxin B + hydrocortisone, ciprofloxacin + dexamethasone, or ciprofloxacin for 24 hours had lower collagen 1A1 and α-tubulin levels (all P < .001) than the negative control. After 48 hours, fibroblasts treated with neomycin + polymyxin B sulfate + hydrocortisone, ciprofloxacin + dexamethasone, ciprofloxacin, or moxifloxacin had lower collagen 1A1 ( P ≤ .007) and α-tubulin ( P < .0001; except ciprofloxacin, P = .033) as compared with control. In experiment 2, only cells treated with ciprofloxacin had lower collagen 1A1 and α-tubulin levels and cell viability (all P < .0001) than control. Cytotoxicity assay and phase-contrast images mirrored the protein findings. Conclusion The adverse impact of topical antibiotic exposure on tympanic membrane collagen and tubulin protein levels is drug specific. This may be important for selection of ototopical therapy.

Ciprofloxacin Decreases Collagen in Mouse Tympanic Membrane Fibroblasts.

OBJECTIVES: To determine how collagen production by tympanic membrane fibroblasts is affected by ciprofloxacin at levels found in eardrops. STUDY DESIGN: Prospective, controlled, and blinded cell culture study. SETTING: Academic tertiary medical center. SUBJECTS: Cell culture of mouse fibroblasts. METHODS: A primary fibroblast culture was established from mouse tympanic membranes. Fibroblasts were cultured until they were 75% confluent, then treated with dilute hydrochloric acid (control) or ciprofloxacin (0.01% or 0.3%) for 24 or 72 hours for Western blotting and for 24 or 48 hours for cytotoxicity assay. Cells were observed with phase-contrast microscope. Western blotting was performed for collagen type 1 α1 (collagen 1A1) and α-tubulin. RESULTS: Fibroblasts treated with 0.01% and 0.3% ciprofloxacin for 24 hours had lower levels of collagen 1A1 (P = .0005 and P < .0001, respectively) and α-tubulin (both P < .0001) than control fibroblasts. Collagen 1A1 and α-tubulin levels were lower in fibroblasts treated with 0.3% than with 0.01% ciprofloxacin (P = .02 and P = .014). After 72 hours, 0.3% ciprofloxacin completely eliminated collagen 1A1 and α-tubulin (P < .001). Cells treated with 0.01% ciprofloxacin for 72 hours also had lower collagen 1A1 (P < .0001) and α-tubulin (P = .005) as compared with the control. Seventy-two-hour incubation in 0.01% or 0.3% ciprofloxacin resulted in lower levels of collagen 1A1 (P = .009 and P < .0001, respectively) and α-tubulin (P = .007 and P < .0001, respectively) than 24-hour incubation. Cytotoxicity assay and phase-contrast microscopy mirrored these findings. CONCLUSIONS: Treatment of tympanic membrane fibroblasts with 0.3% ciprofloxacin, as found in eardrops, reduces fibroblast viability and collagen and α-tubulin protein levels. These findings could explain tympanic membrane healing problems associated with quinolone eardrops.