Pili: the microbes' Swiss army knifes

Surface attachment is the crucial first step for a single cell transitions from a planktonic to a surface associated state, which can lead to the development of multicellular communities called biofilms. Microbes extensively use pili for initial surface attachment. Pili are filamentous appendages that extend into the extracellular environment and can attach to a wide range of surfaces. This Thesis contributes to the understanding of how pili work and how bacteria transition from a planktonic to a surface bound life style. This will aid future development in creating new ways to prevent bacterial attachment and biofilm formation and thereby avoid the necessity for the removal of fully developed biofilms which often requires harsh physical and chemical treatments which can be impractical in a biomedical context. We used single cell studies, microfluidic methods and quantitative computational analysis to study in detail the mechanism of pili-mediated attachment in Caulobacter crescentus and Pseudomonas aeruginosa. In C. crescentus we confirm the recently described ability of pili to retract, which was previously considered not possible for this type of pili. We characterized this functionality in greater detail and our results highlight the importance of pili in reorienting cells and bringing the cell body closer to surfaces, whereby cells can promote long term attachment by secreting a glue-like substance called holdfast. We also investigated the role of the second messenger c-di-GMP during pilimediated cell attachment and biofilm formation. We show a novel role for c-di-GMP in directly regulating elongation and retraction of pili in C. crescentus and P. aeruginosa. In P aeruginosa a novel c-di-GMP effector, FimW, regulates surface attachment and walking behaviour, and how its asymmetric distribution drives surface colonization. In C. crescentus we show that c-di-GMP positively regulates attachment. We manipulated a key component of the secretion machinery, HfsK, and show that c-di-GMP not only regulates the timing of holdfast synthesis, but also its cohesion and adhesion properties. Lastly, we report a novel protein, PdeL, which is both a phosphodiesterase and a transcriptional factor that regulates the expression of biofilm related genes in Escherichia coli. In the appendixes we describe in detail the process for creating microfluidic devices, extensively used in the studies described in this thesis. Moreover, we include a manual for the use of WHISIT, a custom-made software program for the analysis of bacterial fluorescent signals in an automated and quantitative approach

Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation

Surface attachment of bacteria is the first step of biofilm formation and is often mediated and coordinated by the extracellular appendages, flagellum and pili. The model organism Caulobacter crescentus undergoes an asymmetric division cycle, giving rise to a motile "swarmer cell" and a sessile "stalked cell", which is attached to the surface. In the highly polarized predivisional cell, pili and flagellum, which are assembled at the pole opposite the stalk, are both activated before and during the process of cell separation. We explored the interplay of flagellum and active pili by growing predivisional cells on colloidal beads, creating a bacteria-on-a-bead system. Using this set-up, we were able to simultaneously visualize the bacterial motility and analyze the dynamics of the flagellum and pili during cell separation. The observed activities of flagellum and pili at the new cell pole of the predivisional cell result in a cooperating interplay of the appendages during approaching and attaching to a surface. Even in presence of a functioning flagellum, pili are capable of surface attachment and keeping the cell in position. Moreover, while flagellar rotation decreases the average attachment time of a single pilus, it increases the overall attachment rate of pili in a synergetic manner

In situ structure of the Caulobacter crescentus flagellar motor and visualization of binding of a CheY-homolog

Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity

Tad pili play a dynamic role in Caulobacter crescentus surface colonization

Bacterial surface attachment is mediated by rotary flagella and filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions as a mimic of natural environments, we demonstrate that Tad pili undergo repeated cycles of extension and retraction. Within seconds after establishing surface contact, pili reorient cells into an upright position promoting walking-like movements against the medium flow. Pili-mediated positioning of the flagellated pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pili dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model, in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells towards permanent surface attachment

Cohesive Properties of the Caulobacter crescentus Holdfast Adhesin Are Regulated by a Novel c-di-GMP Effector Protein

When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression.IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems

Tad Pili Play a Dynamic Role in Caulobacter crescentus Surface Colonization

Bacteria are able to colonize surfaces in environmental, industrial, and medical settings, where they form resilient communities called biofilms. In order to control bacterial surface colonization, microbiologists need to gain a detailed understanding of the processes that bacteria use to live at the liquid-surface interface and that allow them to adhere to and move on surfaces and eventually grow and persist on solid media. To facilitate these processes, bacteria are equipped with adhesive structures such as flagella and pili and with matrix components such as exopolysaccharides. How these cellular organelles are coordinated to optimize surface processes is currently subject to intense investigations. Here we used the model organism Caulobacter crescentus to demonstrate that polar pili are highly dynamic structures that are functionally interconnected with the flagellar motor to mediate surface sensing, thereby enforcing rapid and permanent surface attachment. These studies provide an entry point for an in-depth molecular analysis of bacterial surface colonization.Bacterial surface attachment is mediated by filamentous appendages called pili. Here, we describe the role of Tad pili during surface colonization of Caulobacter crescentus. Using an optical trap and microfluidic controlled flow conditions to mimic natural environments, we demonstrated that Tad pili undergo repeated dynamic cycles of extension and retraction. Within seconds after establishing surface contact, pilus retraction reorients cells into an upright position, promoting walking-like movements against the medium flow. Pilus-mediated positioning of the flagellate pole close to the surface facilitates motor-mediated mechanical sensing and promotes anchoring of the holdfast, an adhesive substance that affords long-term attachment. We present evidence that the second messenger c-di-GMP regulates pilus dynamics during surface encounter in distinct ways, promoting increased activity at intermediate levels and retraction of pili at peak concentrations. We propose a model in which flagellum and Tad pili functionally interact and together impose a ratchet-like mechanism that progressively drives C. crescentus cells toward permanent surface attachment

Digital control of c-di-GMP in E. coli balances population-wide developmental transitions and phage sensitivity

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness

Paediatric arterial ischaemic stroke and cerebral sinovenous thrombosis: First report from the Italian registry of pediatric thrombosis (R. I. T. I., Registro Italiano Trombosi Infantili)

reserved55noData from large case series of children with cerebral thrombotic events are pivotal to improve prevention, early recognition and treatment of these conditions. The Italian Registry of Pediatric Thrombosis (R. I. T. I.) was established in 2007 by a multidisciplinary team, aiming for a better understanding of neonatal and paediatric thrombotic events in Italy and providing a preliminary source of data for the future development of specific clinical trials and diagnostic-therapeutic protocols. We analysed data relative to the paediatric cerebral thrombotic events of the R. I. T. I. which occurred between January 2007 and June 2012. In the study period, 79 arterial ischaemic stroke (AIS) events (49 in males) and 91 cerebral sinovenous thrombosis (CSVT) events (65 in males) were enrolled in the R. I. T. I. Mean age at onset was 4.5 years in AIS, and 7.1 years in CSVT. Most common modes of presentation were hemiparesis, seizures and speech disturbances in AIS, and headache, seizures and lethargy in CSVT. Most common etiologies were underlying chronic diseases, vasculopathy and cardiopathy in AIS, and underlying chronic diseases and infection in CSVT. Time to diagnosis exceeded 24 hours in 46% AIS and 59% CSVT. Overall data from the Italian Registry are in substantial agreement with those from the literature, despite small differences. Among these, a longer time to diagnosis compared to other registries and case series poses the accent to the need of an earlier recognition of paediatric cerebrovascular events in Italy, in order to enable prompt and effective treatment strategies.mixedSuppiej A.; Gentilomo C.; Saracco P.; Sartori S.; Agostini M.; Bagna R.; Bassi B.; Giordano P.; Grassi M.; Guzzetta A.; Lasagni D.; Luciani M.; Molinari A.C.; Palmieri A.; Putti M.C.; Ramenghi L.A.; Rota L.L.; Sperli D.; Laverda A.M.; Simioni P.; Angriman M.; Aru A.B.; Barisone E.; Bartalena L.; Berta M.; Bertoni E.; Cancarini P.; Cavaliere E.; Celle M.E.; Cerbone A.M.; Cesaroni E.; Via L.D.; Dell'Oro M.G.; Di Rosa G.; Ferrari G.M.; Fiori S.; Gaffuri M.; Gallina M.R.; Gimmillaro A.; Grandone E.; Ladogana S.; Laforgia N.; La Piana R.; Maschio F.; Miniero R.; Nosadini M.; Panzeri D.; Petrucci A.; Piersigilli F.; Sala D.; Sangermani R.; Santoro N.; Tufano A.; Ventura G.; Vittorini R.Suppiej, A.; Gentilomo, C.; Saracco, P.; Sartori, S.; Agostini, M.; Bagna, R.; Bassi, B.; Giordano, P.; Grassi, M.; Guzzetta, A.; Lasagni, D.; Luciani, M.; Molinari, A. C.; Palmieri, A.; Putti, M. C.; Ramenghi, L. A.; Rota, L. L.; Sperli, D.; Laverda, A. M.; Simioni, P.; Angriman, M.; Aru, A. B.; Barisone, E.; Bartalena, L.; Berta, M.; Bertoni, E.; Cancarini, P.; Cavaliere, E.; Celle, M. E.; Cerbone, A. M.; Cesaroni, E.; Via, L. D.; Dell'Oro, M. G.; Di Rosa, G.; Ferrari, G. M.; Fiori, S.; Gaffuri, M.; Gallina, M. R.; Gimmillaro, A.; Grandone, E.; Ladogana, S.; Laforgia, N.; La Piana, R.; Maschio, F.; Miniero, R.; Nosadini, M.; Panzeri, D.; Petrucci, A.; Piersigilli, F.; Sala, D.; Sangermani, R.; Santoro, N.; Tufano, A.; Ventura, G.; Vittorini, R