Colonization can be asymptomatic in a large number of individuals [1] and these organisms are generally referred to as carriage strains, but can also proceed to cause life-threatening infections with high morbidity and mortality in some patients or mild and banal infections in others

Colonization can be asymptomatic in a large number of individuals [1] and these organisms are generally referred to as carriage strains, but can also proceed to cause life-threatening infections with high morbidity and mortality in some patients or mild and banal infections in others. correlated to bacterial invasiveness and to variability in host cell responses via Toll-like receptor 2 (TLR2). Here, we examined whether TLR2 signaling by porins influences recovery of intracellular from epithelial cells internalization by epithelial cells. TLR2-driven intracellular signaling via ERK1/2, JNK and particularly NF-B plays a role in this process. Based on these results, it is possible that expression of porin sequence variants that strongly induce TLR2 activation may be a mechanism to enhance the invasive features of pathogenic strains. (NM) is a Gram-negative organism that colonizes the human nasopharyngeal epithelium. Colonization can be asymptomatic in a large number of individuals [1] and these organisms are generally referred to as carriage strains, but can also proceed to cause life-threatening infections with high morbidity and mortality in some patients or mild and banal infections in others. These organisms are referred to as invasive and, in some cases, hyper-invasive. Host cell invasion TSC1 is followed by bacteria dissemination into the bloodstream and penetration of the bloodCbrain barrier, the causes of sepsis and meningitis, respectively. NM express virulence factors, i.e. capsule, pili, lipo-oligosaccharide (LOS), major and minor adhesins, that promote bacterial invasion of epithelial cells by interacting with cognate host cell receptors [2, 3]. A common feature of most virulence factors is their antigenic variability and fluctuating expression levels among strains and during the bacteria life cycle (phase variability). The role of many virulence factors has been clearly defined, but invasion mechanisms independent of these have also been reported, as well as variability WAY-262611 between bacterial clones and strains, and conditions and assays [4]. Porins are antigenically variable pan-Neisserial outer membrane proteins [5] with a trimeric structure, composed of monomers with a (GC) and the commensal (NL) only express PorB. The structure of PorB has been characterized in greater detail than that of PorA [7-10]. Porins are involved in bacterial pathogenicity. NM and GC porins promote epithelial cell invasion [11-16] while NL PorB reduces it as shown in a GC mutant strain expressing NL PorB in place of GC PorB [17]. The sequence variability of PorB has been linked to the pathogenicity of hyper-invasive and invasive WAY-262611 meningococcal strains [18, 19] and to some of its host cell-associated functions (serum resistance, host cell survival, immune stimulation [20]). Critical residues in the surface-exposed loops of PorB influence organisms invasion of epithelial cells and the direct interaction of PorB with host cell receptors associated with bacterial adhesion/invasion (i.e. the laminin receptor LamR [21], the gp96 and Scavenger Receptor SREC [14]), with complement components [22] and with members of the Toll-like receptor family, specifically TLR2 and TLR1 [23]. Residues that likely mediate PorB/TLR2 interaction and subsequent host cell activation have been identified in the surface-exposed regions of loops 5 and 7 of PorB [24]. In this work, we examined the influence of PorB on internalization by epithelial cells and the contribution of PorB-induced TLR2 signaling to this process. We suggest that expression of PorB sequence variants by different strains may represent a mechanism to strengthen WAY-262611 the virulence of certain NM organisms by rendering host cells more susceptible to bacterial internalization via stimulation of TLR2. 2. Materials and Methods 2.1 Bacterial cultures NL strain Y92-1009 (ND:P1.ND,ND:F-ND:ST-3493, ST-613), NM serogroup B strain H44/76 (B:15;P1.7,16; L3,7,9, ST-32) 14 variant (lacking expression of PorA and Rmp), and the NM mutant strain expressing PorB from NL (NM-[PorB]) [24] were cultured from frozen stocks on GC agar plates containing 1% Isovitalex at 37C in a 5% CO 2 atmosphere in candle jars. The next day, colonies were resuspended in GC liquid medium containing 1% Isovitalex and grown for approx. 2-3h to exponential phase, measured spectrophotometrically by optical density at OD660. The O.D. of the cultures was adjusted to 0.2 and used as standard condition. Bacterial suspensions were appropriately diluted prior to co-incubation with BEAS-2B and HEK cells at a multiplicity of illness (MOI) of approx. 10 and 100 bacteria/cell, confirmed by viable count of the inoculum. No variations in the growth of these strains were reported. 2.2 Cell ethnicities and activation The human being bronchial epithelial cell collection, BEAS-2B cells (ATTC CRL-9609) was grown at 37C/5% CO 2 in DMEM F-12 supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin in flasks coated with 0.01 mg/ml BSA, 0.03 mg/ml bovine collagen type I and 0.01 mg/ml fibronectin to favor adherence to the plastic. HEK cells stably over-expressing TLR2 and TLR4 (TLR2 HEK cells and TLR4 HEK cells, respectively) or an empty vector (pcDNA HEK.