Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

Michel, Gérard; Aguzzi, Anthony; Ball, Geneviève; Soscia, Chantal; Bleves, Sophie; Voulhoux, Romé

doi:10.1099/mic.0.045849-0

Cited by 17 publications

(18 citation statements)

References 39 publications

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“…We tested whether FimV 689 could complement defects in T2S by assessing casein hydrolysis using skim milk agar. fimV was defective for protease secretion, an observation consistent with previous findings (19). Complementation with full-length FimV restored ϳ48% of T2S relative to the wild type, whereas FimV 689 failed to restore secretion.…”

Section: Resultssupporting

confidence: 79%

“…5). Transcription of T2S genes is cAMP dependent (16), and FimV regulates T2S at least in part through the cAMP-binding protein, Vfr (19). We tested whether FimV 689 could complement defects in T2S by assessing casein hydrolysis using skim milk agar.…”

Section: Resultsmentioning

confidence: 99%

“…FimV is involved in a number of virulence mechanisms, including twitching motility, type II secretion (T2S) of lipases and proteases, and regulation of cAMP production, and thus Vfr function (15,17,19). FimV is a 919-residue protein consisting of a large periplasmic domain with a peptidoglycan-binding lysin motif (LysM) (20), a single transmembrane segment, and a highly acidic cytoplasmic domain that is predicted to contain three discontinuous tetratricopeptide repeat (TPR) protein-protein interaction motifs (21,22).…”

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confidence: 99%

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The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

et al. 2016

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FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV 861-919 , containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV 689 , a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV 689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV 689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCEFimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs. T ype IV pili (T4P) are filamentous surface appendages produced by a wide range of bacteria and archaea (1, 2), where they assist in DNA uptake, surface attachment, and twitching motility (3-5). There are two major subfamilies of T4P: T4aP and T4bP. T4aP are typically associated with twitching (2), a process in which pili undergo repeated cycles of extension, adhesion, and retraction, thus acting as molecular grappling hooks to pull cells along a surface.The T4aP machinery is composed of four structural subcomplexes (6). In the model bacterium Pseudomonas aeruginosa, an inner membrane motor subcomplex consisting of the platform protein PilC and three hexameric ATPases-PilB, PilT, and PilU-provide energy for T4aP extension and retraction (7-9). A second inner membrane al...

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Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

et al. 2016

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“…It has been previously shown that the cAMP-dependent operon PaQa is up-regulated in colonies growing on solid medium (13). We show that the PaQa operon is directly regulated by contact between the cell and the substratum, independently of surface chemistry.…”

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confidence: 65%

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

Persat

Inclán

Engel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

343

398

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Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.surface sensing | mechanotransduction | type IV pili | virulence N umerous bacterial species exhibit a range of behaviors that are specific to life on surfaces. For many pathogens, surfacespecific phenotypes are often associated with virulent activity, because many infection strategies require contact with a host (1). For example, in Pseudomonas aeruginosa, the type III secretion system, which infects by injecting toxins, requires attachment of individual cells to host-cell membranes (2). The genetic pathways that regulate P. aeruginosa secretion systems have been well characterized (3), but the environmental signals that activate these pathways remain poorly defined (4). Given the requirement for host-cell contact for efficient infection, P. aeruginosa could leverage surface contact as a signal to properly activate and coordinate pathogenicity. Indeed, our laboratory recently demonstrated that prolonged association with a solid surface contributes to P. aeruginosa pathogenicity when bacteria and host cells are forced together (5). Here we address the fundamental question of how bacteria rapidly sense surface contact and transduce this input into a cellular response over short timescales.Surface-specific behaviors require intimate contact between cells and substrate. In P. aeruginosa, contact is mediated by several adhesins, particularly type IV pili (TFP). TFP are long, motorized fimbriae that also provide cells with surface-specific twitching motility and are essential to virulence and biofilm formation (6, 7). Successive TFP extension, attachment, and retraction promote intimate association with surfaces and motility along them. Because TFP dynamically int...

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“…Mutants for three genes showed defects in both biofilm assays: the pilus assembly protein FimV and hypothetical proteins PA14_44460 and PA14_69770. FimV and PA14_44460 have previously been implicated as contributors to type II secretion-a process known to be important for biofilm formation (22). In contrast, PA14_69770 has no homology to any characterized proteins or domains and has not been investigated to date.…”

Section: Boncat Enables Enrichment and Identification Of Proteins Synmentioning

confidence: 99%

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

Babin

Bergkessel

Sweredoski

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteomelabeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally upregulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).Pseudomonas aeruginosa | slow growth | transcription | proteomics | BONCAT T he cosmopolitan bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen of burn wounds, medical devices, and the lungs of cystic fibrosis (CF) patients. The bacterium's genome is large and encodes an unusually high proportion of regulators (1). Compared with Escherichia coli, P. aeruginosa possesses more σ factors that direct RNA polymerase (RNAP) to promoter regions (24 vs. 7), more DNAbinding activators and repressors that enhance or prevent RNAP binding and transcription (∼550 vs. 150) (2, 3) and more small, noncoding RNAs (ncRNAs) that modulate the stability or translation of target transcripts (200 vs. 100) (4, 5). Much effort has been directed toward understanding the mechanisms by which this regulatory capacity governs the behaviors-such as quorum sensing, protein secretion, secondary metabolite production, and biofilm formation-that contribute to P. aeruginosa virulence.The physiological states of bacteria involved in chronic infections are substantially different from those most often studied in standard laboratory experiments; chronic infections are characterized by slow growth rates imposed by limited nutrients or oxidants or by host immune responses. Direct measurements of in situ microbial growth rates in the context of lung infections in CF patients have revealed doubling times of several days (6). Measurements of expectorated sputum show that hypoxic and anoxic zones exist within infected CF airways and can experience dramatic fluctuations in redox potential (7); P. aeruginosa strains isolated from the CF lung show gene expression patterns consistent with adaptations to hypoxia (8), suggesting ...

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Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

Cited by 17 publications

References 39 publications

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

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