Characterization of motility and piliation in pathogenic Neisseria.

BACKGROUND: The type IV pili (Tfp) of pathogenic Neisseria (i.e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. RESULTS: In this work, we analyzed bacterial motility and monitored Tfp retraction using live-cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 µm/s, whereas N. gonorrhoeae moved with a lower speed (1.0 µm/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the -10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. CONCLUSIONS: These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Lactobacillus decelerates cervical epithelial cell cycle progression.

We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

Neisseria gonorrhoeae infection causes DNA damage and affects the expression of p21, p27 and p53 in non-tumor epithelial cells.

The constant shedding and renewal of epithelial cells maintain the protection of epithelial barriers. Interference with the processes of host cell-cycle regulation and barrier integrity permits the bacterial pathogen Neisseria gonorrhoeae to effectively colonize and invade epithelial cells. Here, we show that a gonococcal infection causes DNA damage in human non-tumor vaginal VK2/E6E7 cells with an increase of 700 DNA strand breaks per cell per hour as detected by an alkaline DNA unwinding assay. Infected cells exhibited elevated levels of DNA double-strand breaks, as indicated by a more than 50% increase in cells expressing DNA damage-response protein 53BP1-positive foci that co-localized with phosphorylated histone H2AX (γH2AX). Furthermore, infected cells abolished their expression of the tumor protein p53 and induced an increase in the expression of cyclin-dependent kinase inhibitors p21 and p27 to 2.6-fold and 4.2-fold of controls, respectively. As shown by live-cell microscopy, flow cytometry assays, and BrdU incorporation assays, gonococcal infection slowed the host cell-cycle progression mainly by impairing progression through the G2 phase. Our findings show new cellular players that are involved in the control of the human cell cycle during gonococcal infection and the potential of bacteria to cause cellular abnormalities.

N-Acyl taurines trigger insulin secretion by increasing calcium flux in pancreatic ß-cells.

Pancreatic ß-cells secrete insulin in response to various stimuli to control blood glucose levels. This insulin release is the result of a complex interplay between signaling, membrane potential and intracellular calcium levels. Various nutritional and hormonal factors are involved in regulating this process. N-Acyl taurines are a group of fatty acids which are amidated (or conjugated) to taurine and little is known about their physiological functions. In this study, treatment of pancreatic ß-cell lines (HIT-T15) and rat islet cell lines (INS-1) with N-acyl taurines (N-arachidonoyl taurine and N-oleoyl taurine), induced a high frequency of calcium oscillations in these cells. Treatment with N-arachidonoyl taurine and N-oleoyl taurine also resulted in a significant increase in insulin secretion from pancreatic ß-cell lines as determined by insulin release assay and immunofluorescence (p<0.05). Our data also show that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in insulin secretion in response to N-arachidonoyl taurine and N-oleoyl taurine treatment. However our data also suggest that receptors other than TRPV1 are involved in the insulin secretion response to treatment with N-oleoyl taurine.

Pathogenic Neisseria hitchhike on the uropod of human neutrophils.

Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.

The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.

BACKGROUND & AIMS: CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer. METHODS: Using gastric epithelial cells, we analyzed a set of H pylori strains and mutants for their ability to interact with CD46 and/or influence CD46 expression. Bacterial interaction with full-length CD46 and small CD46 peptides was evaluated by flow cytometry, fluorescence microscopy, enzyme-linked immunosorbent assay, and bacterial survival analyses. RESULTS: H pylori infection caused shedding of CD46 into the extracellular environment. A soluble form of CD46 bound to H pylori and inhibited growth, in a dose- and time-dependent manner, by interacting with urease and alkyl hydroperoxide reductase, which are essential bacterial pathogenicity-associated factors. Binding of CD46 or CD46-derived synthetic peptides blocked the urease activity and ability of bacteria to survive in acidic environments. Oral administration of one CD46 peptide eradicated H pylori from infected mice. CONCLUSIONS: CD46 is an antimicrobial agent that can eradicate H pylori. CD46 peptides might be developed to treat H pylori infection.

Neisseria gonorrhoeae infection induces altered amphiregulin processing and release.

Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections.

Adherence of clinically isolated lactobacilli to human cervical cells in competition with Neisseria gonorrhoeae.

Lactobacilli are normal inhabitants of our microbiota and are known to protect against pathogens. Neisseria gonorrhoeae is a human specific pathogenic bacterium that colonises the urogenital tract where it causes gonorrhoea. In this study we analysed early interactions between lactobacilli and gonococci and investigated how they compete for adherence to human epithelial cervical cells. We show that lactobacilli adhere at various levels and that the number of adherent bacteria does not correlate to the level of protection against gonococcal infection. Protection against gonococcal adhesion varied between Lactobacillus species. Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus reuteri were capable of reducing gonococcal adherence while Lactobacillus rhamnosus was not. Lactobacillus strains of vaginal origin had the best capacity to remain attached to the host cell during gonococcal adherence. Further, we show that gonococci and lactobacilli interact with each other with resultant lactobacilli incorporation into the gonococcal microcolony. Hence, gonococci bind to colonised lactobacilli and this complex frequently detaches from the epithelial cell surface, resulting in reduced bacterial colonisation. Also, purified gonococcal pili are capable of removing adherent lactobacilli from the cell surface. Taken together, we reveal novel data regarding gonococcal and lactobacilli competition for adherence that will benefit future gonococcal prevention and treatments.

Meningococcal outer membrane protein NhhA is essential for colonization and disease by preventing phagocytosis and complement attack.

Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide, with a rapid onset of disease and a high morbidity and mortality. NhhA is a meningococcal outer membrane protein included in the family of trimeric autotransporter adhesins. The protein binds to the extracellular matrix proteins heparan sulfate and laminin and facilitates attachment to host epithelial cells. In this study, we show that NhhA is essential for bacterial colonization of the nasopharyngeal mucosa in a murine model of meningococcal disease. Successful colonization depends on bacterial attachment but also to the capacity to overcome innate host immune responses. We found that NhhA protected bacteria from phagocytosis, which is important for the mucosal survival of bacteria. In addition, NhhA mediated extensive serum resistance that increased bacterial survival in blood and promoted lethal sepsis. The presence of NhhA protected bacteria from complement-mediated killing by preventing the deposition of the membrane attack complex. Taken together, the results of this work reveal that NhhA inhibits phagocytosis and protects bacteria against complement-mediated killing, which enhances both nasal colonization and the development of sepsis in vivo.

The ScpC protease of Streptococcus pyogenes affects the outcome of sepsis in a murine model.

The ScpC protease of Streptococcus pyogenes degrades interleukin-8 (IL-8), a chemokine that mediates neutrophil transmigration and activation. The ability to degrade IL-8 differs dramatically among clinical isolates of S. pyogenes. Bacteria expressing ScpC overcome immune clearance by preventing the recruitment of neutrophils in soft tissue infection of mice. To study the role of ScpC in streptococcal sepsis, we generated an ScpC mutant that did not degrade IL-8 and thus failed to prevent the recruitment of immune cells as well as to cause disease after soft tissue infection. In a murine model of sepsis, challenge with the ScpC mutant resulted in more severe systemic disease with higher bacteremia levels and mortality than did challenge with the wild-type strain. As expected, the blood level of KC, the murine IL-8 homologue, increased in mice infected with the ScpC mutant. However, the elevated KC levels did not influence neutrophil numbers in blood, as it did in soft tissue, indicating that additional factors contributed to neutrophil transmigration in blood. In addition, the absence of ScpC increased tumor necrosis factor, IL-6, and C5a levels in blood, which contributed to disease severity. Thus, the ScpC mutant triggers high neutrophil infiltration but not lethal outcome after soft tissue infection, whereas intravenous infection leads to highly aggressive systemic disease.

CD46 Contributes to the severity of group A streptococcal infection.

Streptococcus pyogenes (group A Streptococcus) is a human pathogen that causes a wide variety of diseases ranging from uncomplicated superficial infections to severe infections such as streptococcal toxic shock syndrome and necrotizing fasciitis. These bacteria interact with several host cell receptors, one of which is the cell surface complement regulator CD46. In this study, we demonstrate that infection of epithelial cells with S. pyogenes leads to the shedding of CD46 at the same time as the bacteria induce apoptosis and cell death. Soluble CD46 attached to the streptococcal surface, suggesting that bacteria might bind available extracellular CD46 as a strategy to survive and avoid host defenses. The protective role of human CD46 was demonstrated in ex vivo whole-blood assays showing that the growth of S. pyogenes was enhanced in blood from mice expressing human CD46. Finally, in vivo experimental infection showed that bacteremia levels, arthritis frequency, and mortality were higher in CD46 transgenic mice than in nontransgenic mice. Taken together, these results argue that bacterial exploitation of human CD46 enhances bacterial survival and represents a novel pathogenic mechanism that contributes to the severity of group A streptococcal disease.

Neisseria gonorrhoeae infection causes a G1 arrest in human epithelial cells.

Pathogenic bacteria can modulate and interfere with human cell cycle progression. Here we study the human pathogen Neisseria gonorrhoeae and its ability to influence and affect the cell cycle in two human target cell lines. We found that bacteria adhere equally well to cells synchronized into the different cell cycle phases of G1, S, and G2, but were unable to adhere to cells in M phase or G0 phase. In addition, using Western blot and/or flow cytometry analysis we demonstrate that bacterial infection for 24 h results in decreased levels of the cell cycle regulatory proteins cyclin B1, cyclin D1, and cyclin E. Further studies in N. gonorrhoeae-infected epithelial cells involving analysis of DNA content, bromodeoxyuridine (BrdU) incorporation, quantification of late mitotic cells and analysis of nuclear phenotype provide compelling evidence that a 24 h gonococcal infection arrests the cells in early G1 phase of the cell cycle. In summary, we present data showing that MS11 P+ strain of N. gonorrhoeae can down-regulate cyclins, important modulators of the cell cycle, and result in a G1 arrest.