Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus.

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular ß sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its ß region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Structural basis of Acinetobacter type IV pili targeting by an RNA virus.

Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics.

Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

Rapid and noninvasive diagnostics of Huanglongbing and nutrient deficits on citrus trees with a handheld Raman spectrometer.

Huanglongbing (HLB) or citrus greening is a devastating disease of citrus trees that is caused by the gram-negative Candidatus Liberibacter spp. bacteria. The bacteria are phloem limited and transmitted by the Asian citrus psyllid, Diaphorina citri, and the African citrus psyllid, Trioza erytreae, which allows for a wider dissemination of HLB. Infected trees exhibit yellowing of leaves, premature leaf and fruit drop, and ultimately the death of the entire plant. Polymerase chain reaction (PCR) and antibody-based assays (ELISA and/or immunoblot) are commonly used methods for HLB diagnostics. However, they are costly, time-consuming, and destructive to the sample and often not sensitive enough to detect the pathogen very early in the infection stage. Raman spectroscopy (RS) is a noninvasive, nondestructive, analytical technique which provides insight into the chemical structures of a specimen. In this study, by using a handheld Raman system in combination with chemometric analyses, we can readily distinguish between healthy and HLB (early and late stage)-infected citrus trees, as well as plants suffering from nutrient deficits. The detection rate of Raman-based diagnostics of healthy vs HLB infected vs nutrient deficit is ~ 98% for grapefruit and ~ 87% for orange trees, whereas the accuracy of early- vs late-stage HLB infected is 100% for grapefruits and ~94% for oranges. This analysis is portable and sample agnostic, suggesting that it could be utilized for other crops and conducted autonomously. Graphical abstract.