Background: Type IV pili (T4P) are virulence factors composed of major and minor pilins. Results: Minor pilins prime pilus assembly and traffic anti-retraction protein PilY1 to the surface; FimU and GspH are structurally similar. Conclusion: Minor pilins are essential for pilus assembly and function. Significance: This work expands the structural and functional similarities between the T4P and T2S systems.

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The Pseudomonas aeruginosa inner membrane protein FimV is among several proteins of unknown function required for type IV pilus-mediated twitching motility, arising from extension and retraction of pili from their site of assembly in the inner membrane. The pili transit the periplasm and peptidoglycan (PG) layer, ultimately exiting the cell through the PilQ secretin. Although fimV mutants are nonmotile, they are susceptible to killing by pilus-specific bacteriophage, a hallmark of retractable surface pili. Here we show that levels of recoverable surface pili were markedly decreased in fimV pilT retraction-deficient mutants compared with levels in the pilT control, demonstrating that FimV acts at the level of pilus assembly. Levels of inner membrane assembly subcomplex proteins PilM/N/O/P were decreased in fimV mutants, but supplementation of these components in trans did not restore pilus assembly or motility. Loss of FimV dramatically reduced the levels of the PilQ secretin multimer through which pili exit the cell, in part due to decreased levels of PilQ monomers, while PilF pilotin levels were unchanged. Expression of pilQ in trans in the wild type or fimV mutants increased total PilQ monomer levels but did not alter secretin multimer levels or motility. PG pulldown assays showed that the N terminus of FimV bound PG in a LysM motif-dependent manner, and a mutant with an in-frame chromosomal deletion of the LysM motif had reduced motility, secretin levels, and surface piliation. Together, our data show that FimV's role in pilus assembly is to promote secretin formation and that this function depends upon its PG-binding domain.Type IV pili (T4P) are thin, long, flexible, and retractable protein filaments. The broad distribution of T4P among bacterial genera correlates with their ability to mediate a wide range of functions, including attachment to surfaces, DNA uptake, and twitching motility. During twitching motility, pili extend from the cell and attach to a surface, and pilus retraction occurs, moving the bacteria forward toward the point of adhesion (41,51). T4P have been classified into two distinct subtypes, T4aP and T4bP, based on differences in structure of the major pilin subunit and in architecture of the assembly systems (3,16,43). T4aP have been characterized extensively in species such as Pseudomonas aeruginosa, Neisseria spp., and Myxococcus xanthus (10,22,23,39,40,46,49,52). T4bP are found predominately in enteric pathogens, where they promote adhesion (37,58) and bacterial aggregation (6).In P. aeruginosa, a large number of genes involved in the regulation, assembly, and dynamics of T4aP function have been identified. These include the major pilin subunit PilA, as well as a conserved set of minor pilin-like proteins (21); the N-methyltransferase/peptidase PilD, essential for processing prepilins into their mature form (53); an inner membrane assembly subcomplex composed of PilM/N/O/P (4, 47); an outer membrane PilQ secretin pore oligomerized with the assistance of the lipoprotein PilF (7, 29); a...

Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulated P. aeruginosa biofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active against P. aeruginosa. Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR) P. aeruginosa clinical isolates at low-micromolar concentrations. TS also had activity against Acinetobacter baumannii clinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producer Streptomyces azureus, in trans conferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity against P. aeruginosa and A. baumannii was potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening of P. aeruginosa mutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR-Cas, restriction-modification and abortive infection systems . Such strategies are effective, but the phage genome-which encodes potentially inhibitory gene products-is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P) , two of which are glycosylated with O-antigen units or polymers of D-arabinofuranose . We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.