Continuum Theory of Active Phase Separation in Cellular Aggregates.

Dense cellular aggregates are common in biology, ranging from bacterial biofilms to organoids, cell spheroids, and tumors. Their dynamics, driven by intercellular forces, is intrinsically out of equilibrium. Motivated by bacterial colonies as a model system, we present a continuum theory to study dense, active, cellular aggregates. We describe the process of aggregate formation as an active phase separation phenomenon, while the merging of aggregates is rationalized as a coalescence of viscoelastic droplets where the key timescales are linked to the turnover of the active force. Our theory provides a general framework for studying the rheology and nonequilibrium dynamics of dense cellular aggregates.

Type IV Pilin Post-Translational Modifications Modulate Material Properties of Bacterial Colonies.

Bacterial type 4 pili (T4P) are extracellular polymers that initiate the formation of microcolonies and biofilms. T4P continuously elongate and retract. These pilus dynamics crucially affect the local order, shape, and fluidity of microcolonies. The major pilin subunit of the T4P bears multiple post-translational modifications. By interfering with different steps of the pilin glycosylation and phosphoform modification pathways, we investigated the effect of pilin post-translational modification on the shape and dynamics of microcolonies formed by Neisseria gonorrhoeae. Deleting the phosphotransferase responsible for phosphoethanolamine modification at residue serine 68 inhibits shape relaxations of microcolonies after perturbation and causes bacteria carrying the phosphoform modification to segregate to the surface of mixed colonies. We relate these mesoscopic phenotypes to increased attractive forces generated by T4P between cells. Moreover, by deleting genes responsible for the pilin glycan structure, we show that the number of saccharides attached at residue serine 63 affects the ratio between surface tension and viscosity and cause sorting between bacteria carrying different pilin glycoforms. We conclude that different pilin post-translational modifications moderately affect the attractive forces between bacteria but have severe effects on the material properties of microcolonies.

The distinctive cell division interactome of Neisseria gonorrhoeae.

BACKGROUND: Bacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW. RESULTS: Using a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsANg were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW. CONCLUSIONS: Results from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.

Population-Dynamic Modeling of Bacterial Horizontal Gene Transfer by Natural Transformation.

Natural transformation is a major mechanism of horizontal gene transfer (HGT) and plays an essential role in bacterial adaptation, evolution, and speciation. Although its molecular underpinnings have been increasingly revealed, natural transformation is not well characterized in terms of its quantitative ecological roles. Here, by using Neisseria gonorrhoeae as an example, we developed a population-dynamic model for natural transformation and analyzed its dynamic characteristics with nonlinear tools and simulations. Our study showed that bacteria capable of natural transformation can display distinct population behaviors ranging from extinction to coexistence and to bistability, depending on their HGT rate and selection coefficient. With the model, we also illustrated the roles of environmental DNA sources-active secretion and passive release-in impacting population dynamics. Additionally, by constructing and utilizing a stochastic version of the model, we examined how noise shapes the steady and dynamic behaviors of the system. Notably, we found that distinct waiting time statistics for HGT events, namely a power-law distribution, an exponential distribution, and a mix of the both, are associated with the dynamics in the regimes of extinction, coexistence, and bistability accordingly. This work offers a quantitative illustration of natural transformation by revealing its complex population dynamics and associated characteristics, therefore advancing our ecological understanding of natural transformation as well as HGT in general.

Neisseria gonorrhoeae Crippled Its Peptidoglycan Fragment Permease To Facilitate Toxic Peptidoglycan Monomer Release.

Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency. IMPORTANCE: Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease.

Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC physiology, which may represent promising targets for designing new therapeutic interventions.

The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC.

UNLABELLED: Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-d-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE: N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a "superbug" by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.

[ABOUT UNIFICATION OF LABORATORY CRITERIA OF DIFFERENTIATION OF BACTERIAL VAGINOSIS].

The article presents analysis of laboratory criteria and classifcations used to interpret results of laboratory analysis by technique of microscopy on bacterial vaginosis or dysbacteriosis of vagina. Their advantages and restrictions are demonstrated The unified criteria of evaluation are proposed concerning results of microscopy of mucosal discharge of vagina and corresponding classification. Thereafter, three degrees of bacterial vaginosis (dysbacteriosis of vagina) are differentiated: first degree--compensated dysbacteriosis of vagina, second degree--sub compensated dysbacteriosis of vagina and third degree--decompensated dysbacteriosis of vagina. The corresponding laboratory report of physician is formulated. The proposals are presented concerning development of common unified requirements to stages (pre-analytical, analytical, post-analytical) of laboratory diagnostic of bacterial vaginosis (dysbacteriosis of vagina) with purpose of their unambiguous understanding by clinicians and hence their decision making concerning necessity and tactics of management of patient.

Uncovering the mechanism of trapping and cell orientation during Neisseria gonorrhoeae twitching motility.

Neisseria gonorrheae bacteria are the causative agent of the second most common sexually transmitted infection in the world. The bacteria move on a surface by means of twitching motility. Their movement is mediated by multiple long and flexible filaments, called type IV pili, that extend from the cell body, attach to the surface, and retract, thus generating a pulling force. Moving cells also use pili to aggregate and form microcolonies. However, the mechanism by which the pili surrounding the cell body work together to propel bacteria remains unclear. Understanding this process will help describe the motility of N. gonorrheae bacteria, and thus the dissemination of the disease which they cause. In this article we track individual twitching cells and observe that their trajectories consist of alternating moving and pausing intervals, while the cell body is preferably oriented with its wide side toward the direction of motion. Based on these data, we propose a model for the collective pili operation of N. gonorrheae bacteria that explains the experimentally observed behavior. Individual pili function independently but can lead to coordinated motion or pausing via the force balance. The geometry of the cell defines its orientation during motion. We show that by changing pili substrate interactions, the motility pattern can be altered in a predictable way. Although the model proposed is tangibly simple, it still has sufficient robustness to incorporate further advanced pili features and various cell geometries to describe other bacteria that employ pili to move on surfaces.

Mating pair formation homologue TraG is a variable membrane protein essential for contact-independent type IV secretion of chromosomal DNA by Neisseria gonorrhoeae.

Neisseria gonorrhoeae uses a type IV secretion system (T4SS) to secrete chromosomal DNA into the surrounding milieu. The DNA is effective in transforming gonococci in the population, and this mechanism of DNA donation may contribute to the high degree of genetic diversity in this species. Similar to other F-like T4SSs, the gonococcal T4SS requires a putative membrane protein, TraG, for DNA transfer. In F-plasmid and related systems, the homologous protein acts in pilus production, mating pair stabilization, and entry exclusion. We characterized the localization, membrane topology, and variation of TraG in N. gonorrhoeae. TraG was found to be an inner-membrane protein with one large periplasmic region and one large cytoplasmic region. Each gonococcal strain carried one of three different alleles of traG. Strains that carried the smallest allele of traG were found to lack the peptidoglycanase gene atlA but carried a peptidoglycan endopeptidase gene in place of atlA. The purified endopeptidase degraded gonococcal peptidoglycan in vitro, cutting the peptide cross-links. Although the other two traG alleles functioned for DNA secretion in strain MS11, the smallest traG did not support DNA secretion. Despite the requirement for a mating pair stabilization homologue, static coculture transformation experiments demonstrated that DNA transfer was nuclease sensitive and required active uptake by the recipient, thus demonstrating that transfer occurred by transformation and not conjugation. Together, these results demonstrate the TraG acts in a process of DNA export not specific to conjugation and that different forms of TraG affect what substrates can be transported.

Neisseria gonorrhoeae virulence factor NG1686 is a bifunctional M23B family metallopeptidase that influences resistance to hydrogen peroxide and colony morphology.

Symptomatic gonococcal infection, caused exclusively by the human-specific pathogen Neisseria gonorrhoeae (the gonococcus), is characterized by the influx of polymorphonuclear leukocytes (PMNs) to the site of infection. Although PMNs possess a potent antimicrobial arsenal comprising both oxidative and non-oxidative killing mechanisms, gonococci survive this interaction, suggesting that the gonococcus has evolved many defenses against PMN killing. We previously identified the NG1686 protein as a gonococcal virulence factor that protects against both non-oxidative PMN-mediated killing and oxidative killing by hydrogen peroxide. In this work, we show that deletion of ng1686 affects gonococcal colony morphology but not cell morphology and that overexpression of ng1686 does not confer enhanced survival to hydrogen peroxide on gonococci. NG1686 contains M23B endopeptidase active sites found in proteins that cleave bacterial cell wall peptidoglycan. Strains of N. gonorrhoeae expressing mutant NG1686 proteins with substitutions in many, but not all, conserved metallopeptidase active sites recapitulated the hydrogen peroxide sensitivity and altered colony morphology of the Δng1686 mutant strain. We showed that purified NG1686 protein degrades peptidoglycan in vitro and that mutations in many conserved active site residues abolished its degradative activity. Finally, we demonstrated that NG1686 possesses both dd-carboxypeptidase and endopeptidase activities. We conclude that the NG1686 protein is a M23B peptidase with dual activities that targets the cell wall to affect colony morphology and resistance to hydrogen peroxide and PMN-mediated killing.

Studies on gonococcus infection. II. Freeze-fracture, freeze-etch studies on gonocci.

Gonococci have been studied by electron microscopy after freeze-cleavage, freeze-etching and the findings correlated with those obtainable through thin sectioning and negative staining. The outer membrane of the cell wall is composed of round to hexagonal subunits 80 A in diameter. This membrane is also punctuated by 80-A holes visible on the exterior of the organism and extending into the substance or through the outer membrane. Pili coursing over the surface of the organisms appear to maintain a close anatomical relationship with the cell wall. In some instances, the surfaces of the organisms are virtually covered by a layer of pili.

Studies on gonococcus infection. I. Pili and zones of adhesion: their relation to gonococcal growth patterns.

The four colony types of several different strains of gonococci were isolated by selective transfers on agar. These colony variants differed in the degree of autoagglutination which occurred when they were grown in fluid medium. It was found that this clumping behavior was related to the colonial type, with type 2 isolates exhibiting the greatest autoagglutination followed by types 3, 1, and 4. Electron microscopic examination of thin sections indicated that the clumping in fluid medium was mediated by peculiar zones of adherence of the outer membranes of gonococci. These resembled the gap junctions seen in animal cell systems but differed in that the gonococcal membranes involved in the zone of adherence did not bear typical surface modifications. Electron microscopic study of negatively stained specimens of gonococci revealed that pili with a diameter of approximately 85 A and a length up to 4 micro were present on the surfaces of all type 1 and type 2 gonococci examined, and were not seen on any type 3 or 4 gonococci. The consistent presence of pili on type 1 and type 2 gonococci which are virulent colony forms and the lack of pili on avirulent colony types 3 and 4 suggests a relationship between the gonococcal pili and pathogenetic potential of the organisms.

Studies on gonococcus infection. 3. Correlation of gonococcal colony morphology with infectivity for the chick embryo.

Comparable numbers of types 1, 2, 3, and 4 gonococci were placed on the intact chorioallantoic membrane of 236, 10-day old chick embryos. Types 1 and 2 organisms produced infection and could be cultured from chorioallantoic fluid 2 days later significantly more often (69%) than types 3 and 4 organisms (12%, P < 0.001). This confirms in an animal model the same correlation between colony types and infectivity observed in human volunteers and suggests that types 1 and 2 gonococci possess a fundamental virulence characteristic which is absent from types 3 and 4 organisms. Gonococcal infection of the chick embryo chorioallantoic cavity remains a useful model somewhat analogous to localized gonococcal infection in man.

Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells.

Attachment of Neisseria gonorrhoeae to amnion cells in tissue culture is facilitated if the gonococci bear pili. This has been determined by studying the number of pilated, colony type 2 gonococci associated with amnion cells after incubation in vitro as compared with the number of nonpilated, colony type 4 gonococci present with amnion cells under the same conditions. These data are supported by light microscope findings. Electron microscope studies provide visualization of fine structure of gonococcal attachment. Gonococci are also found within amnion cells in this in vitro system.

Multiple pilus motors cooperate for persistent bacterial movement in two dimensions.

In various bacterial species surface motility is mediated by cycles of type IV pilus motor elongation, adhesion, and retraction, but it is unclear whether bacterial movement follows a random walk. Here we show that the correlation time of persistent movement in Neisseria gonorrhoeae increases with the number of pili. The unbinding force of individual pili from the surface F=10 pN was considerably lower than the stalling force F>100 pN, suggesting that density, force, and adhesive properties of the pilus motor enable a tug-of-war mechanism for bacterial movement.

Association of host proteins with the broad host range filamentous phage NgoΦ6 of Neisseria gonorrhoeae.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.

Dynamics of type IV pili is controlled by switching between multiple states.

Type IV pili are major bacterial virulence factors supporting adhesion, surface motility, and gene transfer. The polymeric pilus fiber is a highly dynamic molecular machine that switches between elongation and retraction. We used laser tweezers to investigate the dynamics of individual pili of Neisseria gonorrheae at clamped forces between 8 pN and 100 pN and at varying concentration of the retraction ATPase PilT. The elongation probability of individual pili increased with increasing mechanical force. Directional switching occurred on two distinct timescales, and regular stepping was absent on a scale > 3 nm. We found that the retraction velocity is bimodal and that the bimodality depends on force and on the concentration of PilT proteins. We conclude that the pilus motor is a multistate system with at least one polymerization mode and two depolymerization modes with the dynamics fine-tuned by force and PilT concentration.

CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae.

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

How bacterial cells and colonies move on solid substrates.

Many bacteria rely on active cell appendages, such as type IV pili, to move over substrates and interact with neighboring cells. Here, we study the motion of individual cells and bacterial colonies, mediated by the collective interactions of multiple pili. It was shown experimentally that the substrate motility of Neisseria gonorrhoeae cells can be described as a persistent random walk with a persistence length that exceeds the mean pili length. Moreover, the persistence length increases for a higher number of pili per cell. With the help of a simple, tractable stochastic model, we test whether a tug of war without directional memory can explain the persistent motion of single Neisseria gonorrhoeae cells. While persistent motion of single cells indeed emerges naturally in the model, a tug of war alone is not capable of explaining the motility of microcolonies, which becomes weaker with increasing colony size. We suggest sliding friction between the microcolonies and the substrate as the missing ingredient. While such friction almost does not affect the general mechanism of single cell motility, it has a strong effect on colony motility. We validate the theoretical predictions by using a three-dimensional computational model that includes explicit details of the pili dynamics, force generation, and geometry of cells.