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1. ssRNA phage penetration triggers detachment of the F-pilus

   https://doi.org/10.1073/pnas.2011901117  

   Harb, Laith; Chamakura, Karthik; Khara, Pratick; Christie, Peter J.; Young, Ry; Zeng, Lanying (September 2020, Proceedings of the National Academy of Sciences)

   Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili. 

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2. A Phage‐Based Approach to Identify Antivirulence Inhibitors of Bacterial Type IV Pili

   https://doi.org/10.1111/1751-7915.70081  

   Shimozono, Tori_M; Vogelaar, Nancy_J; O'Hara, Megan_T; Yang, Zhaomin (January 2025, Microbial Biotechnology)

   ABSTRACT The increasing threat of antibiotic resistance underscores the urgent need for innovative strategies to combat infectious diseases, including the development of antivirulants. Microbial pathogens rely on their virulence factors to initiate and sustain infections. Antivirulants are small molecules designed to target virulence factors, thereby attenuating the virulence of infectious microbes. The bacterial type IV pilus (T4P), an extracellular protein filament that depends on the T4P machinery (T4PM) for its biogenesis, dynamics and function, is a key virulence factor in many significant bacterial pathogens. While the T4PM presents a promising antivirulence target, the systematic identification of inhibitors for its multiple protein constituents remains a considerable challenge. Here we report a novel high‐throughput screening (HTS) approach for discovering T4P inhibitors. It usesPseudomonas aeruginosa,a high‐priority pathogen, in combination with its T4P‐targeting phage, φKMV. Screening of a library of 2168 compounds using an optimised protocol led to the identification of tuspetinib, based on its deterrence of the lysis ofP. aeruginosaby φKMV. Our findings show that tuspetinib also inhibits two additional T4P‐targeting phages, while having no effect on a phage that recognises lipopolysaccharides as its receptor. Additionally, tuspetinib impedes T4P‐mediated motility inP. aeruginosaandAcinetobacterspecies without impacting growth or flagellar motility. This bacterium‐phage pairing approach is applicable to a broad range of virulence factors that are required for phage infection, paving ways for the development of advanced chemotherapeutics against antibiotic‐resistant infections. 

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

   https://doi.org/10.1126/sciadv.adl4450  

   Wang, Yuhang; Theodore, Matthew; Xing, Zhongliang; Narsaria, Utkarsh; Yu, Zihao; Zeng, Lanying; Zhang, Junjie (May 2024, Science Advances)

   Caulobacter crescentusTad (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 β 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 β 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 inEscherichia coli, maintaining infectivity againstC. crescentus, which presents promising applications, including RNA delivery and phage display. 

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4. Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

   https://doi.org/10.1073/pnas.2108349119  

   Hughes, Hannah Q.; Floyd, Kyle A.; Hossain, Sajjad; Anantharaman, Sweta; Kysela, David T.; Zöldi, Miklόs; Barna, Lászlό; Yu, Yuanchen; Kappler, Michael P.; Dalia, Triana N.; et al (February 2022, Proceedings of the National Academy of Sciences)

   Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae , which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)–sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species. 

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5. Surface hydrophilicity promotes bacterial twitching motility

   https://doi.org/10.1128/msphere.00390-24  

   O'Hara, Megan T; Shimozono, Tori M; Dye, Keane J; Harris, David; Yang, Zhaomin (September 2024, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Twitching motility is a form of bacterial surface translocation powered by the type IV pilus (T4P). It is frequently analyzed by interstitial colony expansion between agar and the polystyrene surfaces of petri dishes. In such assays, the twitching motility ofAcinetobacter nosocomialiswas observed with MacConkey but not Luria-Bertani (LB) agar media. One difference between these two media is the presence of bile salts as a selective agent in MacConkey but not in LB. Here, we demonstrate that the addition of bile salts to LB allowedA. nosocomialisto display twitching. Similarly, bile salts enhanced the twitching ofAcinetobacter baumanniiandPseudomonas aeruginosain LB. These observations suggest that there is a common mechanism, whereby bile salts enhance bacterial twitching and promote interstitial colony expansion. Bile salts disrupt lipid membranes and apply envelope stress as detergents. Surprisingly, their stimulatory effect on twitching appears not to be related to a bacterial physiological response to stressors. Rather, it is due to their ability to alter the physicochemical properties of a twitching surface. We observed that while other detergents promoted twitching like bile salts, stresses applied by antibiotics, including the outer membrane-targeting polymyxin B, did not enhance twitching motility. More importantly, bacteria displayed increased twitching on hydrophilic surfaces such as those of glass and tissue culture-treated polystyrene plastics, and bile salts no longer stimulated twitching on these surfaces. Together, our results show that altering the hydrophilicity of a twitching surface significantly impacts T4P functionality. IMPORTANCEThe bacterial type IV pilus (T4P) is a critical virulence factor for many medically important pathogens, some of which are prioritized by the World Health Organization for their high levels of antibiotic resistance. The T4P is known to propel bacterial twitching motility, the analysis of which provides a convenient assay for T4P functionality. Here, we show that bile salts and other detergents augment the twitching of multiple bacterial pathogens. We identified the underlying mechanism as the alteration of surface hydrophilicity by detergents. Consequently, hydrophilic surfaces like those of glass or plasma-treated polystyrene promote bacterial twitching, bypassing the requirement for detergents. The implication is that surface properties, such as those of tissues and medical implants, significantly impact the functionality of bacterial T4P as a virulence determinant. This offers valuable insights for developing countermeasures against the colonization and infection by bacterial pathogens of critical importance to human health on a global scale. 

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