Identification of novel toxins associated with the extracellular contractile injection system using machine learning.

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.

Perspectives of Circadian-Based Music Therapy for the Pathogenesis and Symptomatic Treatment of Neurodegenerative Disorders.

Music therapy (MT) and other rhythmic-based interventions for the treatment of neurodegeneration (ND) have been successful in improving the quality of life of affected individuals. Music therapy and rhythm-based stimuli affect patients with Alzheimer's disease (AD) and Parkinson's disease (PD) respectively not only through cognitive channels and subjective qualifications but also through altered brain structures and neural systems. Often implicated in the pathogenesis and resulting symptoms of these diseases is the role of aberrant circadian rhythmicity (CR), namely disrupted sleep. Recent literature suggests that proper maintenance of this timekeeping framework may be beneficial for patients with neurodegenerative disorders and serve a neuroprotective role. While music therapy can improve the quality of life for neurodegenerative patients, longitudinal studies analyzing sleep patterns of affected individuals and possible mechanisms of intervention remain sparse. Furthermore, the role of music therapy in the context of circadian rhythmicity has not been adequately explored. By analyzing the links between circadian rhythmicity, neurodegeneration, and music therapy, a more comprehensive picture emerges, suggesting that possible uses of non-pharmacological circadian-based music therapy to target mechanisms involved in the pathogenesis of Alzheimer's disease and Parkinson's disease may enhance clinical treatment and potentially indicate neuroprotection as a preventative measure.

Coupling between energy and phase in hollow-core fiber based f-to-2f interferometers.

The dependence of the carrier-envelope (CE) phase of the pulses from a hollow-core fiber on the input laser energy was studied using two f-to- 2f interferometers. The CE phase in the in-loop f-to-2f interferometer was measured with the octave spanning white-light spectrum from the hollow-core fiber, whereas the out-of-loop interferometer was based on a sapphire plate. By modulating the input power of the in-loop interferometer and measuring the out-of-loop CE phase at the same time, the coupling coefficient between the measured CE phase and the laser energy for the hollow-core fiber was determined to be 128 mrad per 1% energy change .

Mechanism of phase-energy coupling in f-to-2f interferometry.

White-light generation has been used widely in single-shot f-to-2f interferometers for stabilizing the carrier-envelope (CE) phase of laser amplifiers. The accuracy of the relative phase values measured by such an interferometer is affected by fluctuations in the laser pulse energy. A simple two-step model is proposed to explain the mechanism that couples the laser energy and the CE phase. The model explains the experimentally observed dependence of the group delay between the f and the 2f pulses on the laser energy, as well as the CE phase shift caused by the pulse energy variation.

Precision control of carrier-envelope phase in grating based chirped pulse amplifiers.

It is demonstrated that the carrier-envelope (CE) phase of pulses from a high power ultrafast laser system with a grating-based stretcher and compressor can be stabilized to a root mean square (rms) value of 180 mrad over almost 2 hours, excluding a brief re-locking period. The stabilization was accomplished via feedback control of the grating separation in the stretcher. It shows that the long term CE phase stability of a grating based chirped pulse amplification system can be as good as that of lasers using a glass-block stretcher and a prism pair compressor. Moreover, by adjusting the grating separation to preset values, the relative CE phase could be locked to an arbitrary value in the range of 2pi. This method is better than using a pair of wedge plates to adjust the phase after the hollow-core fiber compressor. The CE phase stabilization after a hollow-core fiber compressor was confirmed by a CE-phase meter based on the measurement of the left-to-right asymmetry of electrons produced by above-threshold ionization.

Double optical gating of high-order harmonic generation with carrier-envelope phase stabilized lasers.

We demonstrated a novel optical switch to control the high-order harmonic generation process so that single attosecond pulses can be generated with multiple-cycle pulses. The technique combines two powerful optical gating methods: polarization gating and two-color gating. An extreme ultraviolet supercontinuum supporting 130 as was generated with neon gas using 9 fs laser pulses. We discovered a unique dependence of the harmonic spectra on the carrier-envelope phase of the laser fields, which repeats every 2 pi radians.

Determining the phase-energy coupling coefficient in carrier-envelope phase measurements.

For f-to-2f interferometers based on white-light generation in sapphire plates, the accuracy of the carrier-envelope (CE) phase measurement and stabilization is affected by the laser energy fluctuation. The coupling coefficient between the CE phase and the laser energy has been determined by modulating the pulse energy in an in-loop f-to-2f interferometer while measuring the CE phase variation with an out-loop interferometer. When the total spectral phase measured by the in-loop interferometer was locked, a 1% change in laser energy caused a 160 mrad shift in the CE phase of the output pulses.

Carrier-envelope phase shift caused by variation of grating separation.

The effects of variation of the grating separation in a stretcher on the carrier-envelope (CE) phase of amplified pulses are investigated. By translating one of the telescope mirrors in the stretcher with a piezoelectric transducer, it is found that a 1 mum change of the distance causes a 3.7+/-1.2 rad shift of the CE phase, which is consistent with theoretical estimations. The results indicate that optical mounts used for gratings and telescope mirrors must be interferometrically stable; otherwise their vibration and thermal drift will cause significant phase error. The CE phase drift was corrected by feedback controlling the grating separation.