ABSTRACT
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.
Subject(s)
Models, Biological , Neisseria gonorrhoeae/cytology , Fimbriae, Bacterial/physiologyABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Capsid Proteins/metabolism , Host Specificity , Inovirus/metabolism , Neisseria gonorrhoeae/virology , Bacterial Outer Membrane/metabolism , Capsid Proteins/isolation & purification , Escherichia coli/virology , Haemophilus influenzae/virology , Inovirus/genetics , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/metabolism , Plasmids/genetics , Salmonella/virologyABSTRACT
Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.
Subject(s)
Bacterial Proteins/metabolism , Gonorrhea/immunology , Immune Evasion , Neisseria gonorrhoeae/pathogenicity , Bacterial Proteins/immunology , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/immunology , Fimbriae, Bacterial/immunology , Fimbriae, Bacterial/metabolism , Global Burden of Disease , Gonorrhea/epidemiology , Gonorrhea/microbiology , Humans , Incidence , Lipopolysaccharides/immunology , Lipopolysaccharides/metabolism , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/immunology , Porins/immunology , Porins/metabolismABSTRACT
The Type IV pili are displayed peritrichously on the surfaces of Neisseria gonorrhoeae cells. Here we present protocols for isolating and purifying Type IV pili and dissociating them into PilE pilin subunits. Pilus filaments are isolated from the bacterial cell surface by mechanical shearing and purified by differential precipitation and centrifugation. PilE subunits are extracted by treating the purified pili with detergent to disrupt the hydrophobic interactions holding them together in the filaments. Purified pili and pilin subunits can be used for structural, biophysical, or biochemical characterization and as antigens for antibody production.
Subject(s)
Chemical Fractionation/methods , Fimbriae Proteins/isolation & purification , Fimbriae, Bacterial/chemistry , Neisseria gonorrhoeae/cytology , Batch Cell Culture Techniques/methods , Detergents/chemistry , Fimbriae Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Neisseria gonorrhoeae/chemistryABSTRACT
The composition of Neisseria peptidoglycan has been of scientific interest for over four decades. Initial investigations focused on discovering the mechanisms causing rising rates of antibiotic resistance in N. gonorrhoeae by determining differences in peptidoglycan composition in penicillin susceptible and resistant strains. The discovery that cytotoxic peptidoglycan fragments are also released by Neisseria furthered the interest in peptidoglycan composition. This method describes the purification, enzymatic degradation, and separation of peptidoglycan fragments by high-performance liquid chromatography (HPLC). It also describes the preparation of samples so that they can be positively identified by mass spectrometry.
Subject(s)
Bacterial Proteins/isolation & purification , Neisseria gonorrhoeae/chemistry , Peptidoglycan/isolation & purification , Bacterial Proteins/chemistry , Cell Wall/chemistry , Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Neisseria gonorrhoeae/cytology , Peptidoglycan/chemistryABSTRACT
The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.
Subject(s)
Antigens, Bacterial/isolation & purification , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Vaccines/immunology , Neisseria gonorrhoeae/immunology , Secretory Vesicles/immunology , Animals , Antigens, Bacterial/immunology , Bacterial Outer Membrane/immunology , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/isolation & purification , Bacterial Vaccines/therapeutic use , Female , Filtration/instrumentation , Filtration/methods , Gonorrhea/immunology , Gonorrhea/microbiology , Gonorrhea/therapy , Humans , Immunogenicity, Vaccine , Mice , Models, Animal , Neisseria gonorrhoeae/cytology , Proteomics , Vaccination , Vagina/microbiologyABSTRACT
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.
Subject(s)
Movement , Neisseria gonorrhoeae/cytology , Fimbriae, Bacterial/metabolism , Models, Biological , Stochastic ProcessesABSTRACT
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.
Subject(s)
Fimbriae Proteins/metabolism , Neisseria gonorrhoeae/metabolism , Protein Processing, Post-Translational , Biofilms/growth & development , Glycoproteins/metabolism , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/growth & development , Neisseria gonorrhoeae/physiology , Phosphoproteins/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Cell Division/physiology , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/metabolism , Bacterial Proteins/chemistry , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Interaction Domains and Motifs , Surface Plasmon Resonance , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Membrane Transport Proteins/metabolism , Neisseria gonorrhoeae/enzymology , Neisseria meningitidis/enzymology , Neisseriaceae Infections/microbiology , Peptidoglycan/metabolism , Amino Acid Motifs , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Wall/chemistry , Cell Wall/genetics , Cell Wall/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Neisseria/classification , Neisseria/enzymology , Neisseria/growth & development , Neisseria/metabolism , Neisseria gonorrhoeae/chemistry , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/growth & development , Neisseria meningitidis/chemistry , Neisseria meningitidis/cytology , Neisseria meningitidis/growth & development , Peptidoglycan/chemistry , Peptidoglycan/toxicityABSTRACT
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.
Subject(s)
Gene Transfer, Horizontal/genetics , Models, Genetic , Neisseria gonorrhoeae/genetics , Transformation, Genetic , Genes, Bacterial/genetics , Genomic Islands/genetics , Neisseria gonorrhoeae/cytology , Nonlinear Dynamics , Stochastic ProcessesABSTRACT
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.
Subject(s)
Amidohydrolases/metabolism , Bacterial Proteins/metabolism , Metalloproteases/metabolism , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/enzymology , Peptidoglycan/metabolism , Amidohydrolases/genetics , Bacterial Proteins/genetics , Cell Division , Metalloproteases/genetics , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/metabolism , Protein BindingABSTRACT
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.
Subject(s)
Dysbiosis/diagnosis , Mucous Membrane/microbiology , Vagina/microbiology , Vaginosis, Bacterial/diagnosis , Bacterial Typing Techniques , Bacteroides/cytology , Candida albicans/cytology , Candida albicans/pathogenicity , Dysbiosis/classification , Dysbiosis/microbiology , Dysbiosis/pathology , Female , Gardnerella vaginalis/cytology , Gardnerella vaginalis/pathogenicity , Humans , Lactobacillus/cytology , Microscopy , Mucous Membrane/pathology , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/pathogenicity , Trichomonas vaginalis/cytology , Trichomonas vaginalis/pathogenicity , Vagina/pathology , Vaginosis, Bacterial/classification , Vaginosis, Bacterial/microbiology , Vaginosis, Bacterial/pathologyABSTRACT
Bacteria have long been ideal model systems for studying many biological phenomena. But when it comes to motility, we are quite often just figuring out the mechanisms underlying their ability to move in liquid or on surfaces. In the last few decades, research has emphasized the importance for bacteria to be able to adhere to and move on surfaces in order to form complex bacterial communities called biofilms. To better understand the multiple chemical and biophysical mechanisms responsible for the initial interactions of bacteria on surfaces that develop into biofilms, we present here low-cost and easy-to-implement protocols to quantitatively analyze the movement of single bacteria on surfaces by microscopy. These protocols are presented in the case of the human pathogen Neisseria gonorrhoeae that moves on surfaces solely powered by Type IV pili, motility referred to as twitching motility. These methods, however, are applicable for any motile bacteria interacting with surfaces. The precise quantification of motility coupled with genetic tools will enable us to precisely dissect the mechanisms and dynamics of bacterial surface motility which are still poorly understood.
Subject(s)
Neisseria gonorrhoeae/cytology , Humans , Imaging, Three-Dimensional , Movement , Surface PropertiesABSTRACT
In gonococci-infected transduced human urethral epithelial cells (THUEC), the role of ERK, a mitogen-activated protein kinase (MAPK), in apoptosis is unknown. We observed lowering of ERK activation in THUEC following infection with anti-apoptosis-inducing Neisseria gonorrhoeae strain CH811. An isogenic cell division mutant of this strain, Ng CJSD1 (minD deficient), which is large and abnormally shaped, reduced ERK phosphorylation levels even more than its parental strain in THUEC. This led to higher anti-apoptosis in mutant-infected cells as compared to the parental strain-infected cells. Our results suggest that N. gonorrhoeae infection reduces ERK activation in THUEC contributing to anti-apoptosis.
Subject(s)
Adenosine Triphosphatases/metabolism , Apoptosis , Bacterial Proteins/metabolism , Epithelial Cells/microbiology , Neisseria gonorrhoeae/pathogenicity , Protein Tyrosine Phosphatases/immunology , Urethral Diseases/microbiology , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/enzymology , Humans , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/enzymology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Urethral Diseases/enzymology , VirulenceABSTRACT
Polymeric filament like type IV Pilus (TFP) can transfer forces in excess of 100 pN during their retraction before stalling, powering surface translocation(twitching). Single TFP level experiments have shown remarkable nonlinearity in the retraction behavior influenced by the external load as well as levels of PilT molecular motor protein. This includes reversal of motion near stall forces when the concentration of the PilT protein is loweblack significantly. In order to explain this behavior, we analyze the coupling of TFP elasticity and interfacial behavior with PilT kinetics. We model retraction as reaction controlled and elongation as transport controlled process. The reaction rates vary with TFP deformation which is modeled as a compound elastic body consisting of multiple helical strands under axial load. Elongation is controlled by monomer transport which suffer entrapment due to excess PilT in the cell periplasm. Our analysis shows excellent agreement with a host of experimental observations and we present a possible biophysical relevance of model parameters through a mechano-chemical stall force map.
Subject(s)
Elasticity , Fimbriae, Bacterial/metabolism , Movement , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/physiology , Bacterial Proteins/metabolism , Biomechanical Phenomena , Evolution, Molecular , Kinetics , Molecular Motor Proteins/metabolism , Neisseria gonorrhoeae/metabolismABSTRACT
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.
Subject(s)
Movement , Neisseria gonorrhoeae/cytology , Biomechanical Phenomena , Fimbriae, Bacterial/metabolism , Models, Biological , ProbabilityABSTRACT
Neisseria gonorrhoeae (GC) is a strict human pathogen and the agent of the sexually transmitted disease gonorrhea. Gonococcal infections have been successfully treated with antibiotics; however, GC has repeatedly developed resistance to each new antibiotic used. Currently, third-generation cephalosporins are recommended, and resistance to these antimicrobials is emerging worldwide. Additionally, no vaccine is available to prevent GC infections. With the dire possibility of untreatable gonorrhea, there is a critical need to identify new therapeutic targets. Cell envelope and membrane vesicle proteins are key factors in pathogenesis, antibiotic resistance, biofilm formation, and general bacterial fitness. Here we describe methods for isolation and purification of GC cell envelopes and spontaneously released membrane vesicles. The isolated proteome fractions can be used in multiple downstream applications, including gel-based and gel-free quantitative proteomics, studies focused on subcellular localization of proteins, transmission electron microscopy, or strain characterization. Presented methods may be easily adapted to other bacterial species.
Subject(s)
Cell Membrane , Neisseria gonorrhoeae/cytology , Neisseria gonorrhoeae/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriological Techniques , Centrifugation/instrumentation , Centrifugation/methodsABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/metabolism , Cell Wall/metabolism , Neisseria gonorrhoeae/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/genetics , Chromatography, Liquid , Gene Knockdown Techniques , Humans , Mass Spectrometry , Neisseria gonorrhoeae/classification , Neisseria gonorrhoeae/cytology , Proteome/analysis , Proteomics/methodsABSTRACT
Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.