SUMMARY Fusobacterium nucleatum is associated with colorectal cancer and promotes colonic tumor formation in preclinical models. However, fusobacteria are core members of the human oral microbiome and less prevalent in the healthy gut, raising questions about how fusobacteria localize to CRC. We identify a host polysaccharide and fusobacterial lectin that explicates fusobacteria abundance in CRC. Gal-Gal-NAc, which is overexpressed in CRC, is recognized by fusobacterial Fap2, which functions as a Gal-Gal-NAc lectin. F. nucleatum binding to clinical adenocarcinomas correlates with Gal-GalNAc expression and is reduced upon O-glycanase treatment. Clinical fusobacteria strains naturally lacking Fap2 or inactivated Fap2 mutants show reduced binding to Gal-GalNAc-expressing CRC cells and established CRCs in mice. Additionally, intravenously injected F. nucleatum localizes to mouse tumor tissues in a Fap2-dependent manner, suggesting that fusobacteria use a hematogenous route to reach colon adenocarcinomas. Thus, targeting F. nucleatum Fap2 or host epithelial Gal-GalNAc may reduce fusobacteria potentiation of CRC.

Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria.

Aims: To detect bacteriophages for Gram-positive oral pathogens in human saliva. Methods and Results: Saliva samples from 31 donors were screened for the presence of bacteriophages for Streptococcus sobrinus, Streptococcus mutans, Streptococcus salivarius, Actinomyces viscosus and Enterococcus faecalis. Bacteriophages for Enterococcus faecalis were found in seven samples. Enterococcus faecalis phages were still present in saliva re-collected from one donor one month, and one year after initial saliva collection. Conclusions:The presence and stability of the Enterococcus faecalis bacteriophages in human saliva suggests a possible role of these bacteriophages in the oral ecosystem. Significance and Impact of the Study: Phage therapy as a way to control oral bacteria might be considered.

Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath-and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 ؋ 10 4 M ؊1 min ؊1. Inhibition of the enzyme by phenylboronic acid was rapid (<1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases.

Uropathogenic Escherichia coli (UPEC) are a common cause of urinary tract infections (UTIs) in humans. While the importance of natural killer (NK) cells in innate immune protection against tumors and viral infections is well documented, their role in defense against bacterial infections is still emerging, and their involvement in UPEC-mediated UTI is practically unknown. Using a systematic mutagenesis approach, we found that UPEC adheres to NK cells primarily via its type I fimbriae and employs its hemolysinA toxin to kill NK cells. In the absence of hemolysinA, NK cells directly respond to the bacteria and secrete the cytokine TNF-α, which results in decreased bacterial numbers in vitro and reduction of bacterial burden in the infected bladders. Thus, NK cells control UPEC via TNF-α production, which UPEC counteracts by hemolysinA-mediated killing of NK cells, representing a previously unrecognized host defense and microbial counterattack mechanism in the context of UTI.