Fusobacterium nucleatum is an oral anaerobe recently found to be prevalent in human colorectal cancer (CRC) where it is associated with poor treatment outcome. In mice, hematogenous F. nucleatum can colonize CRC tissue using its lectin Fap2, which attaches to tumor-displayed Gal-GalNAc. Here, we show that Gal-GalNAc levels increase as human breast cancer progresses, and that occurrence of F. nucleatum gDNA in breast cancer samples correlates with high Gal-GalNAc levels. We demonstrate Fap2-dependent binding of the bacterium to breast cancer samples, which is inhibited by GalNAc. Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization. Inoculation with F. nucleatum suppresses accumulation of tumor infiltrating T cells and promotes tumor growth and metastatic progression, the latter two of which can be counteracted by antibiotic treatment. Thus, targeting F. nucleatum or Fap2 might be beneficial during treatment of breast cancer.

Fusobacterium nucleatumis a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment,F. nucleatumadheres 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 ofF. nucleatumto host cells. Hemagglutination of someF. nucleatumstrains 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 threefap2mutants failed to show galactose-inhibitable coaggregation withPorphyromonas gingivalisand were defective in cell binding. Afap2mutant 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.

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

Legionella pneumophila grows within cells ranging from environmental amoebae to human macrophages. In spite of this conserved strategy of pathogenesis, identification of host factors that restrict L. pneumophila intracellular replication has not been extended outside components of the mammalian innate immune response. We performed a double-stranded RNA (dsRNA) screen against more than 50% of the Drosophila melanogaster annotated open reading frames (ORFs) to identify host cell factors that restrict L. pneumophila . The majority of analyzed dsRNAs that stimulated L. pneumophila intracellular replication were directed against host proteins involved in protein synthesis or cell cycle control. Consistent with disruption of the cell cycle stimulating intracellular replication, proteins involved in translation initiation also resulted in G ₁ arrest. Stimulation of replication was dependent on the stage of cell cycle arrest, as dsRNAs causing arrest during S phase had an inhibitory effect on intracellular replication. The inhibitory effects of S phase arrest could be recapitulated in a human cell line, indicating that cell cycle control of L. pneumophila replication is evolutionarily conserved. Synchronized HeLa cell populations in S phase and challenged with L. pneumophila failed to progress through the cell cycle and were depressed for supporting intracellular replication. Poor bacterial replication in S phase was associated with loss of the vacuole membrane barrier, resulting in exposure of bacteria to the cytosol and their eventual degradation. These results are consistent with the model that S phase is inhibitory for L. pneumophila intracellular survival as a consequence of failure to maintain the integrity of the membrane surrounding intracellular bacteria. IMPORTANCE Legionella pneumophila has the ability to replicate within human macrophages and amoebal hosts. Here, we report that the host cell cycle influences L. pneumophila intracellular replication. Our data demonstrate that the G ₁ and G ₂ /M phases of the host cell cycle are permissive for bacterial replication, while S phase is toxic for the bacterium. L. pneumophila replicates poorly within host cells present in S phase. The inability of L. pneumophila to replicate relies on its failure to control the integrity of its vacuole, leading to cytosolic exposure of the bacteria and eventual degradation. The data presented here argue that growth-arrested host cells that are encountered by L. pneumophila in either the environment or within human hosts are ideal targets for intracellular replication because their transit through S phase is blocked.

Cell wall anchored virulence factors are critical for infection and colonization of the host by Gram-positive bacteria. Such proteins have an N-terminal leader sequence and a C-terminal sorting signal, composed of an LPXTG motif, a hydrophobic stretch, and a few positively charged amino acids. The sorting signal halts translocation across the membrane, allowing sortase to cleave the LPXTG motif, leading to surface anchoring. Deletion of sortase prevents the anchoring of virulence factors to the wall; the effects on bacterial physiology however, have not been thoroughly characterized. Here we show that deletion of Streptococcus pyogenes sortase A leads to accumulation of sorting intermediates, particularly at the septum, altering cellular morphology and physiology, and compromising membrane integrity. Such cells are highly sensitive to cathelicidin, and are rapidly killed in blood and plasma. These phenomena are not a loss-of-function effect caused by the absence of anchored surface proteins, but specifically result from the accumulation of sorting intermediates. Reduction in the level of sorting intermediates leads to a return of the sortase mutant to normal morphology, while expression of M protein with an altered LPXTG motif in wild type cells leads to toxicity in the host environment, similar to that observed in the sortase mutant. These unanticipated effects suggest that inhibition of sortase by small-molecule inhibitors could similarly lead to the rapid elimination of pathogens from an infected host, making such inhibitors much better anti-bacterial agents than previously believed.