Structure and Dynamics of Type 4a Pili and Type 2 Secretion System Endopili.

Within the highly diverse type four filament (TFF or T4F) superfamily, the machineries of type IVa pili (T4aP) and the type 2 secretion system (T2SS) in diderm bacteria exhibit a substantial sequence similarity despite divergent functions and distinct appearances: T4aP can extend micrometers beyond the outer membrane, whereas the endopili in the T2SS are restricted to the periplasm. The determination of the structure of individual components and entire filaments is crucial to understand how their structure enables them to serve different functions. However, the dynamics of these filaments poses a challenge for their high-resolution structure determination. This review presents different approaches that have been used to study the structure and dynamics of T4aP and T2SS endopili by means of integrative structural biology, cryo-electron microscopy (cryo-EM), and molecular dynamics simulations. Their conserved features and differences are presented. The non-helical stretch in the long-conserved N-terminal helix which is characteristic of all members of the TFF and the impact of calcium on structure, function, and dynamics of these filaments are discussed in detail.

CryoEM reveals the structure of an archaeal pilus involved in twitching motility.

Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales, member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius. We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility.

Microbiology: Murder as a solution to promiscuity?

Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.

Interactions between pili affect the outcome of bacterial competition driven by the type VI secretion system.

The bacterial type VI secretion system (T6SS) is a widespread, kin-discriminatory weapon capable of shaping microbial communities. Due to the system's dependency on contact, cellular interactions can lead to either competition or kin protection. Cell-to-cell contact is often accomplished via surface-exposed type IV pili (T4Ps). In Vibrio cholerae, these T4Ps facilitate specific interactions when the bacteria colonize natural chitinous surfaces. However, it has remained unclear whether and, if so, how these interactions affect the bacterium's T6SS-mediated killing. In this study, we demonstrate that pilus-mediated interactions can be harnessed by T6SS-equipped V. cholerae to kill non-kin cells under liquid growth conditions. We also show that the naturally occurring diversity of pili determines the likelihood of cell-to-cell contact and, consequently, the extent of T6SS-mediated competition. To determine the factors that enable or hinder the T6SS's targeted reduction of competitors carrying pili, we developed a physics-grounded computational model for autoaggregation. Collectively, our research demonstrates that T4Ps involved in cell-to-cell contact can impose a selective burden when V. cholerae encounters non-kin cells that possess an active T6SS. Additionally, our study underscores the significance of T4P diversity in protecting closely related individuals from T6SS attacks through autoaggregation and spatial segregation.

Data-driven modelling makes quantitative predictions regarding bacteria surface motility.

In this work, we quantitatively compare computer simulations and existing cell tracking data of P. aeruginosa surface motility in order to analyse the underlying motility mechanism. We present a three dimensional twitching motility model, that simulates the extension, retraction and surface association of individual Type IV Pili (TFP), and is informed by recent experimental observations of TFP. Sensitivity analysis is implemented to minimise the number of model parameters, and quantitative estimates for the remaining parameters are inferred from tracking data by approximate Bayesian computation. We argue that the motility mechanism is highly sensitive to experimental conditions. We predict a TFP retraction speed for the tracking data we study that is in a good agreement with experimental results obtained under very similar conditions. Furthermore, we examine whether estimates for biologically important parameters, whose direct experimental determination is challenging, can be inferred directly from tracking data. One example is the width of the distribution of TFP on the bacteria body. We predict that the TFP are broadly distributed over the bacteria pole in both walking and crawling motility types. Moreover, we identified specific configurations of TFP that lead to transitions between walking and crawling states.

The type IVc pilus: just a Tad different.

Bacteria utilize type IV pili (T4P) to interact with their environment, where they facilitate processes including motility, adherence, and DNA uptake. T4P require multisubunit, membrane-spanning nanomachines for assembly. The tight adherence (Tad) pili are an Archaea-derived T4P subgroup whose machinery exhibits significant mechanistic and architectural differences from bacterial type IVa and IVb pili. Most Tad biosynthetic genes are encoded in a single locus that is widespread in bacteria due to facile acquisition via horizontal gene transfer. These loci experience extensive structural rearrangements, including the acquisition of novel regulatory or biosynthetic genes, which fine-tune their function. This has permitted their integration into many different bacterial lifestyles, including the Caulobacter crescentus cell cycle, Myxococcus xanthus predation, and numerous plant and mammalian pathogens and symbionts.

Significance of a minor pilin PilV in biofilm cohesion of Geobacter sulfurreducens.

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at -0.1 V and 37.7 % at -0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm.

Different Group A Streptococcus pili lead to varying proinflammatory cytokine responses and virulence.

The human pathogen Streptococcus pyogenes, or Group A Streptococcus (GAS), is associated with a variety of diseases ranging from mild skin and soft tissue infections to invasive diseases and immune sequelae such as rheumatic heart disease. We have recently reported that one of the virulence factors of this pathogen, the pilus, has inflammatory properties and strongly stimulates the innate immune system. Here we used a range of nonpathogenic Lactococcus lactis gain-of-function mutants, each expressing one of the major pilus types of GAS, to compare the immune responses generated by various types of fully assembled pili. In vitro assays indicated variability in the inflammatory response induced by different pili, with the fibronectin-binding, collagen-binding, T antigen (FCT)-1-type pilus from GAS serotype M6/T6 inducing significantly stronger cytokine secretion than other pili. Furthermore, we established that the same trend of pili-mediated immune response could be modeled in Galleria mellonella larvae, which possess a similar innate immune system to vertebrates. Counterintuitively, across the panel of pili types examined in this study, we observed a negative correlation between the intensity of the immune response demonstrated in our experiments and the disease severity observed clinically in the GAS strains associated with each pilus type. This observation suggests that pili-mediated inflammation is more likely to promote bacterial clearance instead of causing disruptive damages that intensify pathogenesis. This also indicates that pili may not be the main contributor to the inflammatory symptoms seen in GAS diseases. Rather, the immune-potentiating properties of the pilus components could potentially be exploited as a vaccine adjuvant.

Two antagonistic response regulators control Pseudomonas aeruginosa polarization during mechanotaxis.

The opportunistic pathogen Pseudomonas aeruginosa adapts to solid surfaces to enhance virulence and infect its host. Type IV pili (T4P), long and thin filaments that power surface-specific twitching motility, allow single cells to sense surfaces and control their direction of movement. T4P distribution is polarized to the sensing pole by the chemotaxis-like Chp system via a local positive feedback loop. However, how the initial spatially resolved mechanical signal is translated into T4P polarity is incompletely understood. Here, we demonstrate that the two Chp response regulators PilG and PilH enable dynamic cell polarization by antagonistically regulating T4P extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not strictly required for twitching reversals, it becomes activated upon phosphorylation and breaks the local positive feedback mechanism established by PilG, allowing forward-twitching cells to reverse. Chp thus uses a main output response regulator, PilG, to resolve mechanical signals in space and employs a second regulator, PilH, to break and respond when the signal changes. By identifying the molecular functions of two response regulators that dynamically control cell polarization, our work provides a rationale for the diversity of architectures often found in non-canonical chemotaxis systems.

Suicidal chemotaxis in bacteria.

Bacteria commonly live in surface-associated communities where steep gradients of antibiotics and other chemical compounds can occur. While many bacterial species move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here, we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of several antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that bacteria use pili-based ('twitching') motility to navigate towards antibiotics. Our analyses suggest that this behaviour is driven by a general response to the effects of antibiotics on cells. Migrating bacteria reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration within hours and remain highly motile. However, isolating cells - using fluid-walled microfluidic devices - reveals that these bacteria are terminal and unable to reproduce. Despite moving towards their death, migrating cells are capable of entering a suicidal program to release bacteriocins that kill other bacteria. This behaviour suggests that the cells are responding to antibiotics as if they come from a competing colony growing nearby, inducing them to invade and attack. As a result, clinical antibiotics have the potential to lure bacteria to their death.

Pseudomonas aeruginosa distinguishes surfaces by stiffness using retraction of type IV pili.

The ability of eukaryotic cells to differentiate surface stiffness is fundamental for many processes like stem cell development. Bacteria were previously known to sense the presence of surfaces, but the extent to which they could differentiate stiffnesses remained unclear. Here we establish that the human pathogen Pseudomonas aeruginosa actively measures surface stiffness using type IV pili (TFP). Stiffness sensing is nonlinear, as induction of the virulence factor regulator is peaked with stiffness in a physiologically important range between 0.1 kPa (similar to mucus) and 1,000 kPa (similar to cartilage). Experiments on surfaces with distinct material properties establish that stiffness is the specific biophysical parameter important for this sensing. Traction force measurements reveal that the retraction of TFP is capable of deforming even stiff substrates. We show how slow diffusion of the pilin PilA in the inner membrane yields local concentration changes at the base of TFP during extension and retraction that change with substrate stiffness. We develop a quantitative biomechanical model that explains the transcriptional response to stiffness. A competition between PilA diffusion in the inner membrane and a loss/gain of monomers during TFP extension/retraction produces substrate stiffness-dependent dynamics of the local PilA concentration. We validated this model by manipulating the ATPase activity of the TFP motors to change TFP extension and retraction velocities and PilA concentration dynamics, altering the stiffness response in a predictable manner. Our results highlight stiffness sensing as a shared behavior across biological kingdoms, revealing generalizable principles of environmental sensing across small and large cells.

Adhesion properties of cell surface proteins in Lactobacillus strains in the GIT environment.

As a broadly defined member of lactic acid bacteria (LAB), the Lactobacillus strain is well characterized in food fermentation and specific strains can enhance the intestinal barrier function and be recognized as the probiotic strain. In recent years, many molecules of the cell surface are thought to be related to the adhesion property in the gastrointestinal mucosa. Mucus layer-related proteins, extracellular matrix proteins, and immunoglobulins also exhibit immunity regulation and protection of the intestinal epithelial barrier function. Meanwhile, the effects of bile and the low pH of the gastrointestinal tract (GIT) on Lactobacillus colonization are also needed to be considered. Furthermore, LAB can adhere and aggregate in the GIT to promote the maturity of biofilm and the extracellular matrix secreting through the signal molecules in the quorum sensing (QS) system. Therefore, it is of great interest to use the QS system to regulate the initial adhesion ability of Lactobacillus and further enhance the probiotic effect of the biofilm formation of beneficial bacteria. This review summarizes the adhesion properties of cell surface proteins derived from Lactobacillus strains in recent studies and provides valuable information on the QS effect on the adhesion property of Lactobacillus strains in the GIT environment.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Type IV Pilus Shapes a 'Bubble-Burst' Pattern Opposing Spatial Intermixing of Two Interacting Bacterial Populations.

Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be important determinants of community-level properties. Spatial intermixing emerging from microbial interaction is one of the best-studied characteristics of spatial patterns. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two mutualistic Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (here referred to as a 'bubble-burst' pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments. IMPORTANCE When growing on surfaces, multispecies microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, enabling otherwise impermissible metabolic functions to occur as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells, and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganisms, but its role on pattern formation within microbial communities is unclear. Here, we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy for engineering high-performance synthetic microbial communities.

Bacterial motility: machinery and mechanisms.

Bacteria have developed a large array of motility mechanisms to exploit available resources and environments. These mechanisms can be broadly classified into swimming in aqueous media and movement over solid surfaces. Swimming motility involves either the rotation of rigid helical filaments through the external medium or gyration of the cell body in response to the rotation of internal filaments. On surfaces, bacteria swarm collectively in a thin layer of fluid powered by the rotation of rigid helical filaments, they twitch by assembling and disassembling type IV pili, they glide by driving adhesins along tracks fixed to the cell surface and, finally, non-motile cells slide over surfaces in response to outward forces due to colony growth. Recent technological advances, especially in cryo-electron microscopy, have greatly improved our knowledge of the molecular machinery that powers the various forms of bacterial motility. In this Review, we describe the current understanding of the physical and molecular mechanisms that allow bacteria to move around.

The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

Phototaxis in Cyanobacteria: From Mutants to Models of Collective Behavior.

Cyanobacteria rely on photosynthesis, and thus have evolved complex responses to light. These include phototaxis, the ability of cells to sense light direction and move towards or away from it. Analysis of mutants has demonstrated that phototaxis requires the coordination of multiple photoreceptors and signal transduction networks. The output of these networks is relayed to type IV pili (T4P) that attach to and exert forces on surfaces or other neighboring cells to drive "twitching" or "gliding" motility. This, along with the extrusion of polysaccharides or "slime" by cells, facilitates the emergence of group behavior. We evaluate recent models that describe the emergence of collective colony-scale behavior from the responses of individual, interacting cells. We highlight the advantages of "active matter" approaches in the study of bacterial communities, discussing key differences between emergent behavior in cyanobacterial phototaxis and similar behavior in chemotaxis or quorum sensing.

Tad pilus-mediated twitching motility is essential for DNA uptake and survival of Liberibacters.

Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Mechanotaxis directs Pseudomonas aeruginosa twitching motility.

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.