R
R. (2003). we detected for the first time PPARG on human cervical epithelium. Surprisingly, in addition to LOS sialic acid, we found that gonococcal porin (PorB) mediated binding to multiple Siglecs. PorB also bound preferentially to human Siglecs and not chimpanzee orthologs, modulating host immune reactions in a human\specific manner. Lastly, we studied the distribution of null alleles encoding an activating immune receptor. These results contribute to the understanding of the human specificity of can also infect the conjunctiva, pharynx, and rectal mucosa (Edwards & Apicella, 2004). Gonococci successfully proliferate in different host microenvironments and evade the EACC human immune system by constantly modulating their surface antigenic makeup by phase variation EACC and other mechanisms (Criss & Seifert, 2012; Edwards & Apicella, 2004; Virji, 2009). Dynamic changes in the glycan extensions of gonococcal lipooligosaccharide (LOS) are an excellent example of how evade the host immune response. The lacto\has evolved both sialic acid\dependent and sialic acid\independent interactions with human but not chimpanzee EACC Siglecs to modulate their pathogenic potential and the host inflammatory response in a species\specific manner, thus contributing to the host restriction of gonorrhea. We also explore the impact of Siglec polymorphisms in a population at high risk of gonorrhea. 2.?MATERIALS AND METHODS 2.1. Bacteria and cell lines F62 was isolated from an uncomplicated infection (Kellogg, Peacock, Deacon, Brown, & Pirkel, 1963). 15253 was isolated from a disseminated infection (O’Brien, Goldenberg, & Rice, 1983). Both strains were piliated. The mutant strains of F62 (lgtD, lgtA, lgtE, and lgtF) were constructed using plasmids and methods described previously (Gulati et al., 2015). The LOS phenotype of the mutants was EACC verified by LOS staining of protease K\treated whole\cell samples that were separated on a 12% BisCTris gel with MES running buffer. were grown overnight on chocolate agar plates with IsoVitaleX (BD Bioscience) or in GC broth supplemented with IsoVitaleX at 37C and 5% CO2. When indicated, growth media was supplemented with 30?M CMP\Neu5Ac (Nacalai USA, Inc.). Incorporation of Neu5Ac has been confirmed by loss of Erythrina Cristagalli Lectin (ECA, Vector Laboratories), which binds to the lactosamine epitope. For all binding and infection studies, bacteria were cultivated to an optical density at 600?nm equivalent to 0.4C0.6. THP\1 cells were grown in RPMI\1640 (Gibco) with 10% fetal calf serum (Gemini Bio\Products) at 37C and 5% CO2. 2.2. Siglec\Fc production Siglec\Fcs were produced as described in Padler\Karavani et al. (2014). Siglec\Fc vectors were transfected into HEK293A cells cultured in serum\free media supplemented with Nutridoma\SP (Roche). Culture supernatants were collected, and Siglec\Fcs were purified on a Sepharose Protein A Column (GE Healthcare Life Sciences). After washing with Tris\buffered saline (20?mM TrisCHCl, 150?mM NaCl, pH 8.0; TBS), Siglec\Fcs were desialylated on column by neuraminidase from Arthrobacter ureafaciens (Sigma\Aldrich) for 1?hr at room temperature. After extensive washing with TBS, Siglec\Fcs were eluted with 0.1?M glycineCHCl pH 3.0 and pH is neutralized immediately. Siglec\Fcs are concentrated by Amicon centrifugal filters (Millipore). The functionality of Siglec\Fcs has been pretested on sialoglycan arrays. 2.3. Bacteria binding assay Ninety\six\well plates were coated with 1?g/well protein A (Thermo Scientific) in 50?mM carbonate buffer pH 9.5 overnight at 4C. Wells were washed with PBS\T (0.05% Tween\20 in PBS) and blocked with 1% BSA in PBS for 1?hr at room temperature. 1?g/well Siglec\Fcs were incubated for 2?hr at room temperature. To correct for unspecific binding of Fc part, human IgG was incubated in parallel. Afterward, wells were washed with PBS\T. were pelleted, washed with HBSS, and then.