Biology subjects

Gitai, Z.

Publications and source records attributed to Gitai, Z..

33 records · Page 2Linked to original sources

bioRxiv2021

Pseudomonas aeruginosa differentiates substrate rigidity using retraction of type IV pili

The ability of eukaryotic cells to differentiate substrate stiffness is fundamental for many processes such as the development of stem cells into mature tissue. Here, we establish that bacteria feel their microenvironment in a similar manner. We show that Pseudomonas aeruginosa actively probes and measures substrate stiffness using type IV pili (TFP). The activity of the major virulence factor regulator Vfr is peaked with stiffness in a physiologically important range between 0.1 kPa (mucus) and 1000 kPa (cartilage). The local concentration of PilA at the base of dynamic TFP changes during extension and retraction in a surface dependent manner due to slow PilA diffusion in the cell membrane. Traction force measurements reveal that TFP retraction deforms even stiff substrates. Modeling of the measured substrate deformation and optical tweezers experiments suggest that TFP adhere at the tip only. Informed by these experimental results, we developed a model that describes substrate stiffness dependent dynamics of the polar PilA concentration which are quantitatively consistent with the transcriptional response to stiffness. Manipulating the ATPase activity of the TFP motors changes the TFP extension and retraction velocities and consequently the PilA concentration dynamics in a manner that is predictive of the experimental stiffness response. This work points to the use of a competition between PilA diffusion and TFP extension-retraction as a molecular shear rheometer. Our results highlight that stiffness sensing is a conserved property between the kingdoms of life.

microbiology↗

bioRxiv2021

Bacterial DNA on the skin surface overrepresents the viable skin microbiome

The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria as compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in-vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation, as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it. Significance statementThis study provides a crucial update to the skin microbiome paradigm by showing that viable bacteria of the skin microbiome are primarily localized to hair follicles and other sub-cutaneous structures rather than the skin surface. The native distribution of skin-associated bacteria has not been previously evaluated, and here we show that the skin surface has few intact bacteria while deeper structures are replete with bacterial contents. We used orthogonal approaches to evaluate the source of bacterial DNA and the dynamics of bacterial repopulation on the skin surface to develop an updated model of the skin microbiome. By adjusting the current understanding of the skin microbiome to match this model, we will be able to address outstanding questions in the field.

microbiology↗

bioRxiv2020

Horizontal and vertical transmission of transgenerational memories via the Cer1 transposon

Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within. Previously, we discovered that C. elegans protects itself from pathogens by "reading" bacterial small RNAs and using this information to both induce avoidance and transmit memories for several generations. Here we found that these memories can be transferred to naive animals via Cer1 retrotransposon-encoded capsids. Cer1 functions at the step of transmission of information from the germline to neurons, and is required for C. elegans learned avoidance ability and for mothers to pass this information on to progeny. The presence of the Cer1 retrotransposon in wild C. elegans strains correlates with the ability to learn and inherit small RNA-induced pathogen avoidance. Together, these results suggest that C. elegans has co-opted a potentially dangerous retrotransposon to instead protect itself and its progeny from a common pathogen through its inter-tissue signaling ability, hijacking this genomic element for its own adaptive immunity benefit.

genetics↗

bioRxiv2020

Novel mechanisms of type IV pilus regulation in Acinetobacter baylyi

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and twitching motility1. Dynamic extension and retraction of T4P is essential for their function, yet little is known about the mechanisms controlling these dynamics or the extent to which their regulation is conserved across bacterial species. Here, we develop Acinetobacter baylyi as a new model to study T4P by employing a recently developed pilus labeling method2,3. Our findings overturn the current dogma that T4P extension occurs through the action of a single, highly conserved motor, PilB, by showing that T4P synthesis in A. baylyi is dependent on an additional, phylogenetically distinct motor, TfpB. Furthermore, we uncover an inhibitor of T4P extension that specifically binds to and inhibits PilB but not TfpB. These results expand our understanding of T4P regulation and highlight how inhibitors might be exploited to inhibit T4P synthesis.

microbiology↗

bioRxiv2020

Pseudomonas aeruginosa detachment from surfaces via a self-made small molecule

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and biofilm formation, which hamper later antibiotic treatments. Previous efforts have searched for biofilm dispersal agents, but there are no known factors that specifically disperse surface-attached P. aeruginosa. In this study we develop a quantitative surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, Mass Spectrometry, and Nuclear Magnetic Resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl-quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal. Significance StatementWe discovered that the clinically relevant human bacterial pathogen P. aeruginosa, typically associated with surface-based infections, is dispersed by a small molecule that the bacteria themselves produce. We elucidate the chemical structure of this molecule and find that mechanistically it functions to inhibit the activity of the P. aeruginosa extra cellular surface motility appendage, the type IV pilus.

microbiology↗

bioRxiv2020

A dual-mechanism antibiotic targets Gram-negative bacteria and avoids drug resistance

The rise of antibiotic resistance and declining discovery of new antibiotics have created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. In an animal host model, SCH-79797 reduces pathogenesis of Acinetobacter baumannii, a drug-resistant Gram-negative pathogen. To characterize the MoA of SCH-79797 we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline shows that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments with other antifolates and membrane disruptors in killing MRSA persisters. Thus, SCH-79797 represents a promising lead antibiotic and suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to target challenging bacterial pathogens.

microbiology↗

bioRxiv2020

The evolution of bacterial shape complexity by a curvature-inducing module

Bacteria can achieve a staggering diversity of cell shapes that promote critical functions like growth, motility, and virulence1-4. Previous studies suggested that bacteria establish complex shapes by co-opting the core machineries essential for elongation and division5,6. In contrast, we discovered a two-protein module, CrvAB, that can curve bacteria autonomously of the major elongation and division machinery by forming a dynamic, asymmetrically-localized structure in the periplasm. CrvAB is essential for curvature in its native species, Vibrio cholerae, and is sufficient to curve multiple heterologous species spanning 2.5 billion years of evolution. Thus, modular shape determinants can promote the evolution of morphological complexity independently of existing cell shape regulation.

microbiology↗

bioRxiv2020

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

The functions of type IV pili (TFP) are mediated by cycles of extension and retraction. The coordination of these cycles remains mysterious due to poor quantification of TFP dynamics. Here we fluorescently label the TFP in the opportunistic pathogen Pseudomonas aeruginosa and track the full extension and retraction cycles of individual TFP to quantify their dynamics. We test several models for the switch between extension and retraction using quantitative experiments, biophysical modeling and genetics. We invalidate the prominent hypothesis that this switch is triggered by surface contact. Instead, we show that the entire repetitive cycle of extension and retraction of individual TFP is governed by the stochastic binding of antagonistic extension and retraction motors and explain how this mechanism quantitatively defines physiologically-important features like TFP length and their production rate. Interestingly, our results suggest that the major throttle of TFP production is the unbinding of the retraction motor.

microbiology↗

bioRxiv2020

C. elegans reads bacterial non-coding RNAs to learn pathogenic avoidance

C. elegans is exposed to many different bacteria in its environment, and must distinguish pathogenic from nutritious bacterial food sources. Here, we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance, both in the treated animals and in four subsequent generations of progeny. The RNA interference and piRNA pathways, the germline, and the ASI neuron are required for bacterial small RNA-induced avoidance behavior and transgenerational inheritance. A single non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. A natural microbiome Pseudomonas isolate, GRb0427, can induce avoidance via its small RNAs, and the wild C. elegans strain JU1580 responds similarly to bacterial sRNA. Our results suggest that this ncRNA-dependent mechanism evolved to survey the worms microbial environment, use this information to make appropriate behavioral decisions, and pass this information on to its progeny.

genetics↗

bioRxiv2019

Surface association induces cytotoxic alkyl-quinolones in Pseudomonas aeruginosa

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the signaling pathways and downstream toxins specifically induced by surface association to stimulate P. aeruginosa virulence are not fully understood. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and represent a major class of surface-dependent cytotoxins. AQ cytotoxicity is direct and independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. Furthermore, the regulation of AQ production can explain the surface-dependent virulence regulation of the quorum sensing receptor, LasR, and the pilin-associated candidate mechanosensor, PilY1. PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AQs also contribute to the known cytotoxicity of secreted outer membrane vesicles. These findings collectively explain previously mysterious aspects of virulence regulation and provide new avenues for the development of anti-infectives.

microbiology↗

bioRxiv2019

Modeling microbial diversity with metabolic trade-offs

Microbes face intense competition in the natural world, and so need to wisely allocate their resources to multiple functions, in particular to metabolism. Understanding competition among metabolic strategies that are subject to trade-offs is therefore crucial for deeper insight into the competition, cooperation, and community assembly of microorganisms. In this work, we evaluate competing metabolic strategies within an ecological context by considering not only how the environment influences cell growth, but also how microbes shape their chemical environment. Utilizing chemostat-based resource-competition models, we exhibit a set of intuitive and general procedures for assessing metabolic strategies. Using this framework, we are able to relate and unify multiple metabolic models, and to demonstrate how the fitness landscape of strategies becomes intrinsically dynamic due to species-environment feedback. Such dynamic fitness landscapes produce rich behaviors, and prove to be crucial for ecological and evolutionary stable coexistence in all the models we examined.

systems biology↗

bioRxiv2019

AimB is a small protein regulator of cell size and MreB assembly

The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeletons, few MreB interacting proteins have been well characterized. Here we identify a small protein from Caulobacter crescentus, AimB, as an Assembly Inhibitor of MreB. AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft. We validate this model through functional analysis of point mutants in both AimB and MreB, photo-crosslinking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings indicate that AimB promotes MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor.

microbiology↗

bioRxiv2019

Both clinical and environmental Caulobacter species act as opportunistic pathogens

The Caulobacter genus, including the widely-studied model organism Caulobacter crescentus, has been thought to be non-pathogenic and thus proposed as a bioengineering vector for various environmental remediation and medical purposes. However, Caulobacter species have been implicated as the causative agents of several hospital-acquired infections, raising the question of whether these clinical isolates represent an emerging pathogenic species or whether Caulobacters on whole possess previously-unappreciated virulence capability. Given the proposed environmental and medical applications for C. crescentus, understanding the potential pathogenicity and human health implications of this bacterium is crucial. Consequently, we sequenced a clinical Caulobacter isolate to determine if it has acquired novel virulence determinants. We found that the clinical isolate represents a new species, Caulobacter mirare. C. mirare phylogenetically resembles both C. crescentus and the related C. segnis, which was also thought to be non-pathogenic. The similarity to other Caulobacters and lack of obvious pathogenesis markers suggested that C. mirare is not unique amongst Caulobacters and that consequently other Caulobacters may also have the potential to be virulent. We tested this hypothesis by characterizing the ability of Caulobacters to infect the model animal host Galleria mellonella. In this context, two different lab strains of C. crescentus proved to be as pathogenic as C. mirare, while lab strains of E. coli were non-pathogenic. Further characterization showed that Caulobacter pathogenesis is mediated by a dose-dependent, cell-associated toxic factor that does not require active bacterial cells or host cellular innate immunity to elicit its toxic effects. Finally, we show that C. crescentus does not grow well in standard clinical culture conditions, suggesting that Caulobacter infections may be more common than generally appreciated but rarely cultured. Taken together, our findings redefine Caulobacters as opportunistic pathogens and highlight the importance of broadening our methods for identifying and characterizing pathogens.\n\nAUTHOR SUMMARYBacterial species have historically been classified as either capable of causing disease in an animal (pathogenic) or not. Caulobacter species represent a class of bacteria that were thought to be non-pathogenic. Caulobacters have been widely studied and proposed to be used for various industrial and medical applications due to their presumed safety. However, recent reports of human Caulobacter infections raised the question of whether disease-causing Caulobacters have acquired special factors that help them cause disease or whether the ability to infect is a more general feature of most Caulobacters. By combining genomic sequencing and animal infection studies we show that a clinical Caulobacter strain is similar to lab Caulobacters and that all Caulobacters studied can infect a model host. We explore the mechanism of this infectivity and show that it is due to a toxic factor that associates with Caulobacter cells. We also provide a possible explanation for why Caulobacters have not traditionally been isolated from human patients, owing to their inability to tolerate the salt levels used in most medical culturing systems.

microbiology↗

bioRxiv2018

E. coli translation strategies differ across nutrient conditions

For cells to grow faster they must increase their protein production rate. Microorganisms have traditionally been thought to accomplish this increase by producing more ribosomes to enhance protein synthesis capacity, leading to the linear relationship between ribosome level and growth rate observed under most growth conditions previously examined. Past studies have suggested that this linear relationship represents an optimal resource allocation strategy for each growth rate, independent of any specific nutrient state. Here we investigate protein production strategies in continuous cultures limited for carbon, nitrogen, and phosphate, which differentially impact substrate supply for protein versus nucleic acid metabolism. Unexpectedly, we find that at slow growth rates, E. coli achieves the same protein production rate using three different strategies under the three different nutrient limitations. Upon phosphate (P) limitation, translation is slow due to a particularly low abundance of ribosomes, which are RNA-rich and thus particularly costly for phosphorous-limited cells. In nitrogen (N) limitation, translation is slowed by limited glutamine and stalling at glutamine codons, resulting is slow elongation. In carbon (C) limitation, translation is slowed by accumulation of inactive ribosomes not bound to mRNA. These extra ribosomes enable rapid growth acceleration upon nutrient upshift. Thus, bacteria tune ribosome usage across different limiting nutrients to enable balanced nutrient-limited growth while also preparing for future nutrient upshifts.

microbiology↗

bioRxiv2017

RodZ promotes MreB polymer formation and curvature localization to determine the cylindrical uniformity of E. coli shape

Cell shape in bacteria is determined by the cell wall, which is synthesized by a variety of proteins whose actions are coordinated by the actin-like MreB protein. MreB uses local geometric cues of envelope curvature to avoid the cell poles and localize to specific regions of the cell body. However, it remains unclear whether MreBs curvature preference is regulated by additional factors, and which features of MreB are essential for specific aspects of rod shape growth, such as cylindrical uniformity. Here we show that in addition to its previously-described role in mediating MreB motion, RodZ also modulates MreB polymer number and curvature preference. MreB polymer number and curvature localization can be regulated independently. Quantitative 3D measurements and a series of mutant strains show that among various properties of MreB, polymer number, total length of MreB polymers, and MreB curvature preference are the key determinants of cylindrical uniformity, a measure of the variability in radius within a single cell. Changes in the values of these parameters are highly predictive of the resulting changes in cell shape (r2=0.93). Our data suggest a model for rod shape in which RodZ promotes the assembly of multiple long MreB polymers that ensure the growth of a uniform cylinder.

microbiology↗