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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. Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa

   https://doi.org/10.1111/mmi.15202  

   Schwartzkopf, Caleb M. ; Taylor, Véronique L. ; Groleau, Marie‐Christine ; Faith, Dominick R. ; Schmidt, Amelia K. ; Lamma, Tyrza L. ; Brooks, Diane M. ; Déziel, Eric ; Maxwell, Karen L. ; Secor, Patrick R. ( December 2023 , Molecular Microbiology)

   Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogenPseudomonas aeruginosais a model system for the study of quorum sensing.P. aeruginosais also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor ofPseudomonasQuinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protectingP. aeruginosafrom infection by type IV pili‐dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

    

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3. Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile

   https://doi.org/10.1128/spectrum.02120-23  

   Sidner, Ben ; Lerma, Armando ; Biswas, Baishakhi ; Do, Thi Van ; Yu, Yafan ; Ronish, Leslie A. ; McCullough, Hugh ; Auchtung, Jennifer M. ; Piepenbrink, Kurt H. ( December 2023 , Microbiology Spectrum)

   Atack, John M. (Ed.)

   Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others.Clostridioides difficileis an anaerobic bacterium, which colonizes the mammalian gastrointestinal (GI) tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. AlthoughC. difficiletoxicity stems from secreted toxins, colonization is a prerequisite forC. difficiledisease. WhileC. difficileis known to associate with the mucous layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well understood. To understand the molecular mechanisms by whichC. difficileinteracts with mucins, we usedex vivomucosal surfaces to test the ability ofC. difficileto bind to mucins from different mammalian tissues. We found significant differences inC. difficileadhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed defects in adhesion by mutants deficient in flagella but not type IV pili. These results imply that interactions between host mucins andC. difficileflagella facilitate the initial host attachment ofC. difficileto host cells and secreted mucus.

   Clostridioides difficileis one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by whichC. difficilecolonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.

    

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4. Subcellular localization of type IV pili regulates bacterial multicellular development

   https://doi.org/10.1038/s41467-022-33564-7  

   Ellison, Courtney K. ; Fei, Chenyi ; Dalia, Triana N. ; Wingreen, Ned S. ; Dalia, Ankur B. ; Shaevitz, Joshua W. ; Gitai, Zemer ( December 2022 , Nature Communications)

   Abstract In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi . This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities. 

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5. Identification of a hormogonium polysaccharide‐specific gene set conserved in filamentous cyanobacteria

   https://doi.org/10.1111/mmi.14566  

   Zuniga, Esthefani G. ; Boateng, Kelvin K. A. ; Bui, Nhi U. ; Kurnfuli, Shadi ; Muthana, Sawsan M. ; Risser, Douglas D. ( July 2020 , Molecular Microbiology)

   Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst‐forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P‐driven mechanism. Here, several novelhpsloci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild‐type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co‐occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy‐type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P‐mediated mechanism.

    

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