Commensal bacteria comprise a large part of the microbial world, playing important roles in human development, health and disease. However, little is known about the genomic content of commensals or how related they are to their pathogenic counterparts. The genus Neisseria, containing both commensal and pathogenic species, provides an excellent opportunity to study these issues. We undertook a comprehensive sequencing and analysis of human commensal and pathogenic Neisseria genomes. Commensals have an extensive repertoire of virulence alleles, a large fraction of which has been exchanged among Neisseria species. Commensals also have the genetic capacity to donate DNA to, and take up DNA from, other Neisseria. Our findings strongly suggest that commensal Neisseria serve as reservoirs of virulence alleles, and that they engage extensively in genetic exchange.

The causative agent of gonorrhea, Neisseria gonorrhoeae, bears retractable filamentous appendages called type IV pili (Tfp). Tfp are used by many pathogenic and nonpathogenic bacteria to carry out a number of vital functions, including DNA uptake, twitching motility (crawling over surfaces), and attachment to host cells. In N. gonorrhoeae, Tfp binding to epithelial cells and the mechanical forces associated with this binding stimulate signaling cascades and gene expression that enhance infection. Retraction of a single Tfp filament generates forces of 50–100 piconewtons, but nothing is known, thus far, on the retraction force ability of multiple Tfp filaments, even though each bacterium expresses multiple Tfp and multiple bacteria interact during infection. We designed a micropillar assay system to measure Tfp retraction forces. This system consists of an array of force sensors made of elastic pillars that allow quantification of retraction forces from adherent N. gonorrhoeae bacteria. Electron microscopy and fluorescence microscopy were used in combination with this novel assay to assess the structures of Tfp. We show that Tfp can form bundles, which contain up to 8–10 Tfp filaments, that act as coordinated retractable units with forces up to 10 times greater than single filament retraction forces. Furthermore, single filament retraction forces are transient, whereas bundled filaments produce retraction forces that can be sustained. Alterations of noncovalent protein–protein interactions between Tfp can inhibit both bundle formation and high-amplitude retraction forces. Retraction forces build over time through the recruitment and bundling of multiple Tfp that pull cooperatively to generate forces in the nanonewton range. We propose that Tfp retraction can be synchronized through bundling, that Tfp bundle retraction can generate forces in the nanonewton range in vivo, and that such high forces could affect infection.

Neisseria gonorrhoeae is the bacterium that causes gonorrhea, a major sexually transmitted disease and a significant cofactor for human immunodeficiency virus transmission. The retactile N. gonorrhoeae type IV pilus (Tfp) mediates twitching motility and attachment. Using live-cell microscopy, we reveal for the first time the dynamics of twitching motility by N. gonorrhoeae in its natural environment, human epithelial cells. Bacteria aggregate into microcolonies on the cell surface and induce a massive remodeling of the microvillus architecture. Surprisingly, the microcolonies are motile, and they fuse to form progressively larger structures that undergo rapid reorganization, suggesting that bacteria communicate with each other during infection. As reported, actin plaques form beneath microcolonies. Here, we show that cortical plaques comigrate with motile microcolonies. These activities are dependent on pilT, the Tfp retraction locus. Cultures infected with a pilT mutant have significantly higher numbers of apoptotic cells than cultures infected with the wild-type strain. Inducing pilT expression with isopropyl-␤-D-thiogalactopyranoside partially rescues cells from infection-induced apoptosis, demonstrating that Tfp retraction is intrinsically cytoprotective for the host. Tfp-mediated attachment is therefore a continuum of microcolony motility and force stimulation of host cell signaling, leading to a cytoprotective effect.Type IV pili (Tfp) are filamentous appendages expressed by a wide range of bacteria, including Synechocystis spp., Pseudomonas aeruginosa, Myxococcus xanthus, Xylella fastidiosa, Clostridium perfringens, enterohaemorrhagic and enteropathogenic Escherichia coli, Neisseria meningitidis, and Neisseria gonorrhoeae (29,32,37,50,54,56,61). The Tfp of several of these bacteria are known to retract, and retraction underlies twitching motility, DNA uptake, phage sensitivity, and social behavior, such as fruiting-body and biofilm formation.N. gonorrhoeae, the bacterium that causes gonorrhea, expresses multiple nonpolar retractile Tfp that promote attachment to epithelial cells (38). Retraction requires the ATPase PilT, which is proposed to disassemble the Tfp fiber (2,13,15). N. gonorrhoeae pilT null mutants express nonretractile Tfp (38,60). Cycles of Tfp extension, substrate tethering, and retraction enable N. gonorrhoeae to crawl on a glass coverslip or agar surface (twitching motility) (34). Twitching motility on epithelial cells has not been studied. Understanding N. gonorrhoeae motility behavior in this environment is important, as the bacterium infects only humans and cannot survive outside the human body.Mechanical forces of 50 to 100 pN are generated by the retraction of a Tfp fiber (24, 38). Thus, Tfp retraction allows N. gonorrhoeae to pull and exert physical stress on its substrate. Tfp retraction induces a number of responses in the infected epithelial cell. It triggers Ca 2ϩ and stress kinase signaling, cytoskeletal rearrangements, and cortical plaque formation and regulates epithelial gen...

Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced manmade devices. Type IV pili (Tfp) represent such a structure: 6-nmwide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.force polymorphism | alternate immunogenic properties

SummaryThe retractile type IV pilus participates in a number of fundamental bacterial processes, including motility, DNA transformation, fruiting body formation and attachment to host cells. Retraction of the N. gonorrhoeae type IV pilus requires a functional pilT . Retraction generates substantial force on its substrate ( > 100 pN per retraction event), and it has been speculated that epithelial cells sense and respond to these forces during infection. We provide evidence that piliated, Opa non-expressing Neisseria gonorrhoeae activates the stress-responsive PI-3 kinase/ Akt (PKB) pathway in human epithelial cells, and activation is enhanced by a functional pilT . PI-3 kinase inhibitors wortmannin and LY294002 reduce cell entry by 81% and 50%, respectively, illustrating the importance of this cascade in bacterial invasion. PI-3 kinase and its direct downstream effectors

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.

Highlights d Commensal Neisseria kill STD pathogen N. gonorrhoeae by releasing DNA into the environment d Killing requires DNA entry and recombination and a foreign DNA methylation pattern d Commensal N. elongata accelerates clearance of N. gonorrhoeae from the mouse vagina d A N. gonorrhoeae DNA uptake mutant resists this clearance

Streptococcus pyogenes, or group A Streptococcus (GAS), is a pathogen that causes a multitude of human diseases from pharyngitis to severe infections such as toxic shock syndrome and necrotizing fasciitis. One of the primary virulence factors produced by GAS is the peptide toxin streptolysin S (SLS). In addition to its well-recognized role as a cytolysin, recent evidence has indicated that SLS may influence host cell signaling pathways at sublytic concentrations during infection. We employed an antibody arraybased approach to comprehensively identify global host cell changes in human epithelial keratinocytes in response to the SLS toxin. We identified key SLS-dependent host responses, including the initiation of specific programmed cell death and inflammatory cascades with concomitant downregulation of Akt-mediated cytoprotection. Significant signaling responses identified by our array analysis were confirmed using biochemical and protein identification methods. To further demonstrate that the observed SLS-dependent host signaling changes were mediated primarily by the secreted toxin, we designed a Transwell infection system in which direct bacterial attachment to host cells was prevented, while secreted factors were allowed access to host cells. The results using this approach were consistent with our direct infection studies and reveal that SLS is a bacterial toxin that does not require bacterial attachment to host cells for activity. In light of these findings, we propose that the production of SLS by GAS during skin infection promotes invasive outcomes by triggering programmed cell death and inflammatory cascades in host cells to breach the keratinocyte barrier for dissemination into deeper tissues. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a common colonizer of the skin and mucosal surfaces of humans (1-3). GAS is typically innocuous in these locations or else leads to fairly minor and generally self-limiting infections of the skin or respiratory tract, such as impetigo or pharyngitis (1-3). In cases where an initial infection is left untreated, GAS may cause one of several severe postinfection (p.i.) sequelae, including rheumatic fever or glomerulonephritis (1-3). Furthermore, in rare cases, this exclusively human pathogen breaches the epithelial barrier and invades deeper tissues and blood, resulting in outcomes such as necrotizing fasciitis and Streptococcus toxic shock (1-3). The World Health Organization (WHO) estimates that GAS is responsible for about 18 million cases of severe postinfection sequelae and 700,000 cases of invasive disease each year (2, 4). Combined, GAS infections lead to approximately 500,000 deaths annually (2, 4).The success of GAS in causing both mild and severe infections is due largely to the myriad of secreted and surface-bound virulence factors expressed by this pathogen. One of the most potent virulence factors produced by GAS is streptolysin S (SLS), a small, ribosomally produced peptide whose mature product is predicted to be 2.7 kDa in size (5-8...