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Table 1. Selection of Tn-seq identified genes required for adherence of M. catarrhalis BBH18.

CSV
Figure 2. Adhesion of directed gene deletion mutants to respiratory tract epithelial cells.

Adherence of aroA, ecnAB , lgt1, and the putative lipoprotein MCR_1483 to both Detroit 562 (A) and A549 (B) cells was significantly attenuated compared to wild-type (WT). Adherence levels are expressed relative to WT (n ≥ 3) and shown as means and SEM. Statistical difference was determined with a Mann-Whitney test with * P < 0.05, ** P < 0.01, and *** P < 0.001.

https://doi.org/10.1371/journal.pone.0072193.g002

Loss of the putative outer membrane lipoprotein gene MCR_1483 and aroA resulted in the most severe attenuation of adherence, to 30-39% and 34-39% of wild-type, respectively. MCR_1483 encodes a small 48 amino acid putative lipoprotein sharing 58% identity with the LppL protein of Pseudomonas sp. GM102. In M. catarrhalis BBH18 MCR_1483 is part of a putative operon together with lysA, dapF , and xerC , whereas the representative gene cluster in Pseudomonas sp. contains two additional uncharacterized ORFs [ Ultra High Leg Strappy Vinyl Swimsuit Red Asos Shopping Online High Quality Cheap Sale New Arrival Outlet Brand New Unisex 90KBzw
]. In Pseudomonas fluorescens WCS365 this operon, more specifically xerC encoding a site specific recombinase, is required for root colonization [ 30 ]. Others postulated that in Pseudomonas sp . LppL is involved in regulation of the lysine biosynthesis gene lysA [ 29 ]. The attenuated adhesion of the aroA mutant is intriguing, especially since a direct role of this predicted cytoplasmic protein in cellular adhesion is unlikely. The aroA gene encodes a 3-phosphoshikimate 1-carboxyvinyltransferase that is involved in aromatic amino acid metabolism and generation of chorismate-derived metabolites [ 31 ]. Deletion of aro genes leads to attenuated virulence in many bacterial pathogens and often results in defective cell envelope biosynthesis [ 32 ]. It could be possible that a defective outer membrane function in M. catarrhalis BBH18Δ aroA caused in incorrect localization of “true” adhesins, thereby leading to attenuated adhesion. Interestingly, both MCR_1483 and aroA were also found to be required for complement resistance of M. catarrhalis (De Vries et al., unpublished data) and thus appear to fulfill or affect an essential virulence mechanism. More subtle effects on adherence efficiency were observed upon deletion of ecnAB and lgt1 , 59-78% and 64-80% of wild-type, respectively. In E. coli , ecnAB is reported to direct the biosynthesis of two small outer membrane lipoproteins that regulate programmed cell death [ Womens 1331 Mojave Güell Open Toe Sandals Art Best Wholesale For Sale Buy Cheap Purchase Cheap Free Shipping Cheap Best Store To Get Outlet Latest Collections XE5ux
]. Deletion of lgt1 , which encodes an α-(1,2) glucosyltransferase involved in LOS biosynthesis [ 24 ], is expected to result in a truncated LOS structure. An altered LOS structure may lead to decreased membrane stability and altered surface display of adhesins as has been shown for a LOS-deficient mutant of strain O35E [ 34 ].

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