Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

ABSTRACTThe intestinal pathogenClostridium difficileis an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States.C. difficilerates of recurrence and mortality have increased in recent years due to the emergence of so-called “hypervirulent” epidemic strains. A great deal of the basic biology ofC. difficilehas not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger forC. difficilegene regulation. However, the function(s) of TFP inC. difficileremains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains ofC. difficile. We demonstrate that TFP contribute toC. difficilebiofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described inC. difficile. The expression and regulation of thepilA1pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences inpilA1expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemicC. difficilestrains may contribute to the different infection outcomes presented by these strains.IMPORTANCEHowClostridium difficileestablishes and maintains colonization of the host bowel is poorly understood. Surface behaviors ofC. difficileare likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation byC. difficile. TFP are also required for surface motility, which has not previously been demonstrated forC. difficile. Furthermore, an epidemic-associatedC. difficilestrain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

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Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽

10.1128/msphere.00423-18 ◽

2018 ◽

Vol 3 (5) ◽

Cited By ~ 11

Author(s):

Robert W. McKee ◽

Carissa K. Harvest ◽

Rita Tamayo

Keyword(s):

Gene Expression ◽

Clostridium Difficile ◽

Biofilm Formation ◽

Toxin Production ◽

Intestinal Colonization ◽

Cyclic Diguanylate ◽

Signaling Molecule ◽

Content Type ◽

Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

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Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin

Applied and Environmental Microbiology ◽

10.1128/aem.00753-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6158-6165 ◽

Cited By ~ 13

Author(s):

Tiffany C. Williams ◽

Mesrop Ayrapetyan ◽

James D. Oliver

Keyword(s):

Quorum Sensing ◽

Vibrio Vulnificus ◽

The United States ◽

Type Iv Pili ◽

Negative Regulator ◽

Physical Factors ◽

Wound Infections ◽

Environmental Persistence ◽

Content Type ◽

Type Iv

ABSTRACTThe human pathogenVibrio vulnificusis the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains ofV. vulnificusdisplay different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence ofV. vulnificus. With the increasing incidence of wound infections caused byV. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment inV. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.

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Non-motile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pili- and Pel-dependent mechanism

10.1101/2021.10.21.465385 ◽

2021 ◽

Author(s):

Kimberley A. Lewis ◽

Danielle M Vermilyea ◽

Shanice S Webster ◽

Jaime de Anda ◽

Gerard Wong ◽

...

Keyword(s):

Biofilm Formation ◽

Total Population ◽

Type Iv Pili ◽

External Factors ◽

Flagellar Motility ◽

Wild Type ◽

Type Iv ◽

Motile Cells ◽

Flagellum Biosynthesis ◽

Dependent Mechanism

The downregulation of P. aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-motile cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-motile cells required functional type IV pili and the ability to produce the Pel exopolysaccharide to suppress swarming by the motile wild type. In contrast, motile cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-motile cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species.

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Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

Journal of Bacteriology ◽

10.1128/jb.02340-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 819-832 ◽

Cited By ~ 77

Author(s):

Eric Bordeleau ◽

Erin B. Purcell ◽

Daniel A. Lafontaine ◽

Louis-Charles Fortier ◽

Rita Tamayo ◽

...

Keyword(s):

Clostridium Difficile ◽

Cell Aggregation ◽

Flagellar Motility ◽

Transcription Terminator ◽

Content Type ◽

Type Iv ◽

Intestinal Infections ◽

Diguanosine Monophosphate ◽

Primary Type

Clostridium difficileis an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration inC. difficilewas recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes,pilA1(CD3513) andpilB1(CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions.pilA1is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-roundin vitrotranscription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation inC. difficile.

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Imipenem Treatment Induces Expression of Important Genes and Phenotypes in a Resistant Acinetobacter baumannii Isolate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01696-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1370-1376 ◽

Cited By ~ 12

Author(s):

Ghulam Nasser Dhabaan ◽

Sazaly AbuBakar ◽

Gustavo Maia Cerqueira ◽

Mohammed Al-Haroni ◽

Sui Ping Pang ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Clinical Success ◽

Multidrug Resistant ◽

Type Iv Pili ◽

Control Measures ◽

Content Type ◽

Type Iv ◽

Abiotic Surfaces ◽

Colonization Factors

Acinetobacter baumanniihas emerged as a notorious multidrug-resistant pathogen, and development of novel control measures is of the utmost importance. Understanding the factors that play a role in drug resistance may contribute to the identification of novel therapeutic targets. Pili are essential forA. baumanniiadherence to and biofilm formation on abiotic surfaces as well as virulence. In the present study, we found that biofilm formation was significantly induced in an imipenem-resistant (Impr) strain treated with a subinhibitory concentration of antibiotic compared to that in an untreated control and an imipenem-susceptible (Imps) isolate. Using microarray and quantitative PCR analyses, we observed that several genes responsible for the synthesis of type IV pili were significantly upregulated in the Imprbut not in the Impsisolate. Notably, this finding is corroborated by an increase in the motility of the Imprstrain. Our results suggest that the ability to overproduce colonization factors in response to imipenem treatment confers biological advantage toA. baumanniiand may contribute to clinical success.

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Mucin Promotes Rapid Surface Motility in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00073-12 ◽

2012 ◽

Vol 3 (3) ◽

Cited By ~ 38

Author(s):

Amy T. Y. Yeung ◽

Alicia Parayno ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iv Pili ◽

Transcriptional Analysis ◽

Bacterial Cells ◽

Content Type ◽

Type Iv ◽

Sensing Systems ◽

Bright Green ◽

Surface Motility

ABSTRACTAn important environmental factor that determines the mode of motility adopted byPseudomonas aeruginosais the viscosity of the medium, often provided by adjusting agar concentrationsin vitro. However, the viscous gel-like property of the mucus layer that overlays epithelial surfaces is largely due to the glycoprotein mucin.P. aeruginosais known to swim within 0.3% (wt/vol) agar and swarm on the surface at 0.5% (wt/vol) agar with amino acids as a weak nitrogen source. When physiological concentrations or as little as 0.05% (wt/vol) mucin was added to the swimming agar, in addition to swimming,P. aeruginosawas observed to undergo highly accelerated motility on the surface of the agar. The surface motility colonies in the presence of mucin appeared to be circular, with a bright green center surrounded by a thicker white edge. While intact flagella were required for the surface motility in the presence of mucin, type IV pili and rhamnolipid production were not. Replacement of mucin with other wetting agents indicated that the lubricant properties of mucin might contribute to the surface motility. Based on studies with mutants, the quorum-sensing systems (lasandrhl) and the orphan autoinducer receptor QscR played important roles in this form of surface motility. Transcriptional analysis of cells taken from the motility zone revealed the upregulation of genes involved in virulence and resistance. Based on these results, we suggest that mucin may be promoting a new or highly modified form of surface motility, which we propose should be termed “surfing.”IMPORTANCEAn important factor that dictates the mode of motility adopted byP. aeruginosais the viscosity of the medium, often provided by adjusting agar concentrationsin vitro. However, the gel-like properties of the mucous layers that overlay epithelial surfaces, such as those of the lung, a major site ofPseudomonasinfection, are contributed mostly by the production of the glycoprotein mucin. In this study, we added mucin to swimming media and found that it promoted the ability ofP. aeruginosato exhibit rapid surface motility. These motility colonies appeared in a circular form, with a bright green center surrounded by a thicker white edge. Interestingly, bacterial cells at the thick edge appeared piled up and lacked flagella, while cells at the motility center had flagella. Our data from various genetic and phenotypic studies suggest that mucin may be promoting a modified form of swarming or a novel form of surface motility inP. aeruginosa.

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Bundle-Forming Pilus Locus of Aeromonas veronii bv. Sobria

Infection and Immunity ◽

10.1128/iai.06304-11 ◽

2012 ◽

Vol 80 (4) ◽

pp. 1351-1360 ◽

Cited By ~ 16

Author(s):

Nahal Hadi ◽

Qin Yang ◽

Timothy C. Barnett ◽

S. Mohammed B. Tabei ◽

Sylvia M. Kirov ◽

...

Keyword(s):

Biofilm Formation ◽

Content Type ◽

Aeromonas Veronii ◽

Colonization Factor ◽

Type Iv ◽

Transcriptional Units ◽

Pilin Gene ◽

A Minor ◽

Pilin Protein

ABSTRACTLittle is known about the colonization mechanisms ofAeromonasspp. Previous work has suggested that the type IV bundle-forming pilus (Bfp) is an aeromonad intestinal colonization factor. This study provides the first genetic characterization of this structure. To define the role of Bfp inAeromonas veroniibv. Sobria adherence, a 22-kb locus encoding the bundle-forming pilus was isolated; this contained 17 pilus-related genes similar to the mannose-sensitive hemagglutinin (MSHA) ofVibrio cholerae. Reverse transcriptase PCR (RT-PCR) demonstrated that the locus had two major transcriptional units,mshItomshFandmshBtomshQ. Transcriptional fusion experiments demonstrated the presence of two strong promoters upstream ofmshIandmshB. The locus encoded four putative prepilin proteins, one of which (MshA) corresponded to the N-terminal sequence of the previously isolated major pilin protein. All the pilin genes were inactivated, mutation of each minor or major pilin gene greatly reduced the bacterium's ability to adhere and form biofilms, and complementation of each mutant intransrescued this phenotype. Mutation of the major pilin MshA and MshB, a minor pilin, resulted in their loss. The position of themshHgene is conserved within a number of bacteria, and we have shown it is not transcriptionally linked to the othermshgenes; moreover, its mutation did not have a dramatic effect on either adhesion or biofilm formation. We conclude that the bundle-forming pilus is required forA. veroniibv. Sobria adherence and biofilm formation; furthermore, both the major and minor pilin proteins are essential for this process.

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Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors

Journal of Bacteriology ◽

10.1128/jb.00716-16 ◽

2017 ◽

Vol 199 (8) ◽

Cited By ~ 11

Author(s):

Lubna V. Richter ◽

Ashley E. Franks ◽

Robert M. Weis ◽

Steven J. Sandler

Keyword(s):

Electron Transfer ◽

Amino Acid ◽

Posttranslational Modification ◽

Biofilm Formation ◽

Electron Acceptors ◽

Type Iv Pili ◽

Geobacter Sulfurreducens ◽

Surface Attachment ◽

Content Type ◽

Type Iv

ABSTRACT Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces. IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells.

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Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics

Journal of Bacteriology ◽

10.1128/jb.00859-16 ◽

2017 ◽

Vol 199 (8) ◽

Cited By ~ 12

Author(s):

Jan Ribbe ◽

Amy E. Baker ◽

Sebastian Euler ◽

George A. O'Toole ◽

Berenike Maier

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Molecular Motor ◽

Type Iv Pili ◽

Force Generation ◽

Surface Attachment ◽

Content Type ◽

Type Iv ◽

Critical Components ◽

Planktonic State

ABSTRACT For Pseudomonas aeruginosa, levels of cyclic di-GMP (c-di-GMP) govern the transition from the planktonic state to biofilm formation. Type IV pili (T4P) are crucial determinants of biofilm structure and dynamics, but it is unknown how levels of c-di-GMP affect pilus dynamics. Here, we scrutinized how c-di-GMP affects molecular motor properties and adhesive behavior of T4P. By means of retraction, T4P generated forces of ∼30 pN. Deletion mutants in the proteins with known roles in biofilm formation, swarming motility, and exopolysaccharide (EPS) production (specifically, the diguanylate cyclases sadC and roeA or the c-di-GMP phosphodiesterase bifA) showed only modest effects on velocity or force of T4P retraction. At high levels of c-di-GMP, the production of exopolysaccharides, particularly of Pel, is upregulated. We found that Pel production strongly enhances T4P-mediated surface adhesion of P. aeruginosa, suggesting that T4P-matrix interactions may be involved in biofilm formation by P. aeruginosa. Finally, our data support the previously proposed model of slingshot-like “twitching” motility of P. aeruginosa. IMPORTANCE Type IV pili (T4P) play various important roles in the transition of bacteria from the planktonic state to the biofilm state, including surface attachment and surface sensing. Here, we investigate adhesion, dynamics, and force generation of T4P after bacteria engage a surface. Our studies showed that two critical components of biofilm formation by Pseudomonas aeruginosa, T4P and exopolysaccharides, contribute to enhanced T4P-mediated force generation by attached bacteria. These data indicate a crucial role for the coordinated impact of multiple biofilm-promoting factors during the early stages of attachment to a surface. Our data are also consistent with a previous model explaining why pilus-mediated motility in P. aeruginosa results in characteristic “twitching” behavior.

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Motor-independent retraction of type IV pili is governed by an inherent property of the pilus filament

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2102780118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2102780118

Author(s):

Jennifer L. Chlebek ◽

Rémi Denise ◽

Lisa Craig ◽

Ankur B. Dalia

Keyword(s):

Biofilm Formation ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Ii Secretion ◽

Secretion Systems ◽

Acinetobacter Baylyi ◽

Dynamic Activity ◽

Dynamic Surface ◽

Type Iv ◽

Pilin Gene

Type IV pili (T4P) are dynamic surface appendages that promote virulence, biofilm formation, horizontal gene transfer, and motility in diverse bacterial species. Pilus dynamic activity is best characterized in T4P that use distinct ATPase motors for pilus extension and retraction. Many T4P systems, however, lack a dedicated retraction motor, and the mechanism underlying this motor-independent retraction remains a mystery. Using the Vibrio cholerae competence pilus as a model system, we identify mutations in the major pilin gene that enhance motor-independent retraction. These mutants likely diminish pilin–pilin interactions within the filament to produce less-stable pili. One mutation adds a bulky residue to α1C, a universally conserved feature of T4P. We found that inserting a bulky residue into α1C of the retraction motor–dependent Acinetobacter baylyi competence T4P enhances motor-independent retraction. Conversely, removing bulky residues from α1C of the retraction motor–independent, V. cholerae toxin-coregulated T4P stabilizes the filament and diminishes pilus retraction. Furthermore, alignment of pilins from the broader type IV filament (T4F) family indicated that retraction motor–independent T4P, gram-positive Com pili, and type II secretion systems generally encode larger residues within α1C oriented toward the pilus core compared to retraction motor–dependent T4P. Together, our data demonstrate that motor-independent retraction relies, in part, on the inherent instability of the pilus filament, which may be a conserved feature of diverse T4Fs. This provides evidence for a long-standing yet previously untested model in which pili retract in the absence of a motor by spontaneous depolymerization.

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