PA is an opportunistic pathogen that is commonly associated with severe infection in immunocompromised hosts. Siglec-9 binds with Sias by cis interaction on the neutrophil surface, thereby reducing immunological activity. However, neutrophils bind with pathogens through trans interactions of siglec-9 with Sias. Neutrophils kill invading pathogens by NETs, along with extracellular phagocytosis. Here, we report the mode of the adsorption of Sias by PA from host serum, the interaction of PA(+Sias) with human neutrophils, and the resulting neutrophil immunological activity. The α2-3-linked sialoglycoproteins adsorbed by PA exhibited potent binding with the soluble siglec-9-Fc chimeras, CHO-siglec-9 and siglec-9 on neutrophils. The binding between PA(+Sias) and neutrophils was blocked by the synthetic sialoglycan Neu5Acα2-3Galβ1-4GlcNAc, confirming the linkage-specific, Sias-siglec-9 interaction. The PA(+Sias) and siglec-9 interaction on neutrophils reduced the level of ROS and the release of elastase, resulting in a reduction of NETs formation, demonstrating the role of the sialoglycoproteins adsorbed by PA in the weakening of neutrophil activity. The resistance of PA(+Sias) to NETs was made evident by the increased survival of PA(+Sias). Moreover, the decrease in PA(-Sias) survival demonstrated the involvement of NETs formation in the absence of the Sias-siglec-9 interaction. N-actylcysteine or sivelestat-pretreated neutrophils enhanced the survival of PA(-Sias). DNAse-pretreated neutrophils did not exhibit any NETs formation, resulting in the enhanced escape of PA(-Sias). Taken together, one of the survival mechanisms of PA(+Sias) is the diminution of innate immunity via its adsorption of sialoglycoproteins by its engagement of the inhibitory molecule siglec-9. This is possibly a general mechanism for pathogens that cannot synthesize Sias to subvert immunity.

The opportunism of Pseudomonas aeruginosa (PA) in immunocompromised hosts prompted us to explore the potential role of sialic acids (Sia) in this phenomenon. Culture of PA in the presence of exogenous Sia resulted in linkage-specific incorporation of Sia which was associated with decreased complement deposition on the bacteria. Sia acquired by PA mediated enhanced binding of bacteria to recombinant-CHO cells expressing human siglec-7 or siglec-9, as well as to human NK-cells and monocytes naturally expressing these siglecs. Therefore, Sia may be acquired by PA in the host and contribute to bacterial pathogenicity and host-cell interactions via reduction of complement deposition and siglec-dependent recognition.

Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure-activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxy-mahanine-treated cells, indicating significant contribution of the C-7-OH group. O-Methylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9-NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.