The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Plasmid pWW115, a cloning vector for use with Moraxella catarrhalis

The plasmid shuttle vector pWW102B is able to replicate in only a modest number of Moraxella catarrhalis strains. Plasmid pWW115, a spontaneous deletion mutant of pWW102B, was shown to lack both the pACYC184-derived origin of replication and the associated chloramphenicol-resistance gene but was able to replicate in every M. catarrhalis strain tested in this study, including one strain that had been previously refractory to all types of genetic manipulations. To test the utility of this plasmid, a M. catarrhalis gene encoding the UspA2 serum-resistance factor was cloned into pWW115 and the resultant recombinant plasmid was shown to confer serum-resistance on a serum-sensitive M. catarrhalis uspA2 mutant.     

Impact of sequence diversity in the Moraxella catarrhalis UspA2/UspA2H head domain on vitronectin binding and antigenic variation

The nasopharyngeal pathogen Moraxella catarrhalis recruits vitronectin to subvert complement-mediated killing. Ubiquitous surface protein (UspA) 2 and its hybrid form UspA2H bind vitronectin at the highly diverse N-terminal head domain. Here we characterized the sequence diversity of the head domain in multiple M. catarrhalis clinical isolates (n = 51) with focus on binding of vitronectin. The head domain of the uspA2 genes from 40 isolates were clustered according to an N-terminal sequence motif of UspA2 (NTER2), i.e., NTER2A (55% of uspA2 variants), NTER2B (32.5%), NTER2C (5%), and finally a group without an NTER2 (7.5%). Isolates harbouring the uspA2H gene (n = 11) contained N-terminal GGG repeats. Vitronectin binding to isolates having UspA2 did not correlate to variation in the NTER2 motifs but occurred in UspA2H containing 6 or ≥11 of GGG repeats. Analyses of recombinant UspA2/UspA2H head domains of multiple variants showed UspA2-dependent binding to the C-terminal of vitronectin. Furthermore, polyclonal anti-UspA2 antibodies revealed that the head domain of the majority of Moraxella UspA2/2H was antigenically unrelated, whereas the full length molecules were recognized. In conclusion, the head domains of UspA2/2H have extensive sequence polymorphism without losing vitronectin-binding capacity promoting a general evasion of the host immune system.     

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.     

Ionic binding of C3 to the human pathogen Moraxella catarrhalis is a unique mechanism for combating innate immunity

Moraxella catarrhalis ubiquitous surface proteins A1 and A2 (UspA1/A2) interfere with the classical pathway of the complement system by binding C4b-binding protein. In this study we demonstrate that M. catarrhalis UspA1 and A2 noncovalently and in a dose-dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine-treated C3. In contrast, related Moraxella subspecies (n = 13) or other human pathogenic bacteria (n = 13) do not bind C3 or methylamine-treated C3. Experiments with recombinant proteins and M. catarrhalis mutants devoid of UspA1/A2 revealed that UspA1/A2 exert their actions by absorbing and neutralizing C3 from serum and restrain complement activation. UspA2 was responsible for most of the effect, and the Moraxella mutant lacking UspA2 was more sensitive to the lytic effect of human serum compared with the wild type. Interestingly, among the large number of bacteria analyzed, only M. catarrhalis has this unique ability to interfere with the innate immune system of complement by binding C3.     

The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2

Moraxella catarrhalis ubiquitous surface protein A2 (UspA2) mediates resistance to the bactericidal activity of normal human serum. In this study, an interaction between the complement fluid phase regulator of the classical pathway, C4b binding protein (C4BP), and M. catarrhalis mutants lacking UspA1 and/or UspA2 was analyzed by flow cytometry and a RIA. Two clinical isolates of M. catarrhalis expressed UspA2 at a higher density than UspA1. The UspA1 mutants showed a decreased C4BP binding (37.6% reduction), whereas the UspA2-deficient Moraxella mutants displayed a strongly reduced (94.6%) C4BP binding compared with the wild type. In addition, experiments with recombinantly expressed UspA1(50-770) and UspA2(30-539) showed that C4BP (range, 1-1000 nM) bound to the two proteins in a dose-dependent manner. The equilibrium constants (K(D)) for the UspA1(50-770) and UspA2(30-539) interactions with a single subunit of C4BP were 13 microM and 1.1 microM, respectively. The main isoform of C4BP contains seven identical alpha-chains and one beta-chain linked together with disulfide bridges, and the alpha-chains contain eight complement control protein (CCP) modules. The UspA1 and A2 bound to the alpha-chain of C4BP, and experiments with C4BP lacking CCP2, CCP5, or CCP7 showed that these three CCPs were important for the Usp binding. Importantly, C4BP bound to the surface of M. catarrhalis retained its cofactor activity as determined by analysis of C4b degradation. Taken together, M. catarrhalis interferes with the classical complement activation pathway by binding C4BP to UspA1 and UspA2.     

Effects of overproducing the universal stress protein, UspA, in Escherichia coli K-12.

A plasmid with the structural uspA gene under the control of a tac promoter was used to study the effects of altering uspA expression levels under various growth conditions. We found that increasing UspA synthesis to levels corresponding to physiologically induced levels decreased the cell growth rate in minimal medium and reduced or abolished the cells' capacity to adapt to upshift conditions. As was demonstrated by two-dimensional gel electrophoresis, increased uspA expression caused global changes in the pattern of protein synthesis. In addition, electrophoretic analysis together with V8 protease peptide mapping demonstrated that the pIs of some specific proteins became more acidic as a result of the elevation of the levels of UspA.     

卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

为制备抗卡他莫拉菌(Moraxella catarrhalis,Mc)表面蛋白UspA1胞外结构域的多克隆抗体(PcAb),对UspA1蛋白进行生物信息学分析,获取胞外结构域中抗原表位最为丰富的肽段,找到其对应的基因序列并引入大肠杆菌偏好性密码子,对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体p ET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔,经4次免疫后,用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAbIgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定,抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。     

Immune evasion of Moraxella catarrhalis involves ubiquitous surface protein A-dependent C3d binding

The complement system plays an important role in eliminating invading pathogens. Activation of complement results in C3b deposition (opsonization), phagocytosis, anaphylatoxin (C3a, C5a) release, and consequently cell lysis. Moraxella catarrhalis is a human respiratory pathogen commonly found in children with otitis media and in adults with chronic obstructive pulmonary disease. The species has evolved multiple complement evasion strategies, which among others involves the ubiquitous surface protein (Usp) family consisting of UspA1, A2, and A2 hybrid. In the present study, we found that the ability of M. catarrhalis to bind C3 correlated with UspA expression and that C3 binding contributed to serum resistance in a large number of clinical isolates. Recombinantly expressed UspA1 and A2 inhibit both the alternative and classical pathways, C3b deposition, and C3a generation when bound to the C3 molecule. We also revealed that the M. catarrhalis UspA-binding domain on C3b was located to C3d and that the major bacterial C3d-binding domains were within UspA1(299-452) and UspA2(165-318). The interaction with C3 was not species specific since UspA-expressing M. catarrhalis also bound mouse C3 that resulted in inhibition of the alternative pathway of mouse complement. Taken together, the binding of C3 to UspAs is an efficient strategy of Moraxella to block the activation of complement and to inhibit C3a-mediated inflammation.     

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (?uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ?uspA E. coli strain. The IPTG induced expression of a 56.6 kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ?uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl₂, H₂O₂, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl₂ demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.     

The bacterial universal stress protein: function and regulation

The universal stress protein A (UspA) superfamily encompasses an ancient and conserved group of proteins that are found in bacteria, Archea, fungi, flies and plants. The Escherichia coli UspA is produced in response to a large number of different environmental onslaughts and UspA is one of the most abundant proteins in growth-arrested cells. Although insights into the regulation of the E. coli uspA gene have been gained, the exact roles of the Usp proteins and Usp domains remain enigmatic; they appear, in some cases, to be linked to resistance to DNA-damaging agents and to respiratory uncouplers.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.