Development of novel isatin-nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing-bacteria.

Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a-m and 9a-c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49-7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.

Preferred conformations of lipooligosaccharides and oligosaccharides of Moraxella catarrhalis.

Moraxella catarrhalis (M. catarrhalis) is a pathogenic gram-negative bacterium that causes otitis media and sinusitis in children. Three major serotypes A, B and C are identified to account for approximately 95% of the clinical isolates. Understanding the conformational properties of different serotypes of M. catarrhalis provides insights into antigenic determinants. In this work, all-atom molecular dynamics simulations were conducted for M. catarrhalis lipooligosaccharide (LOS) bilayer systems and oligosaccharides (OS) in water solution to investigate the conformational similarities and differences of three serotypes. For up to 10 neutral monosaccharides in the core part, the conformational ensembles described by the pair-wise root mean square deviation distributions are similar among the three serotypes of either the LOS or OS. At the central ß-($1\to4$)-linkage, anti-$\psi$ conformation in conjunction with the gauche-gauche (g-) conformation of the central trisubstituted glucosyl residue is observed as the dominant conformation to sustain the structural characteristics of M. catarrhalis three types, which is further supported by calculated transglycosidic ${}^3{J}_{C,H}\Big({\psi}_H\Big)$ of serotype A in comparison to experimental data. Interestingly, the conformational variability of three serotypes is more restricted for the OS in water solution than that in the LOS bilayer systems. The LOS-LOS interactions in the bilayer systems are responsible for the increased conformational diversity despite of tight packing. Solvent-accessible surface area analysis suggests that a trisaccharide attached to the ß-($1\to 6$)-linked sugar in all three serotypes of LOS could be the common epitope and have the possibility to interact with antibodies.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

Biophysical and in silico interaction studies of aporphine alkaloids with Malonyl-CoA: ACP transacylase (FabD) from drug resistant Moraxella catarrhalis.

Malonyl-CoA:acyl carrier protein transacylase (FabD), being an essential enzyme of the FAS II pathway, is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drug-resistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the binding of McFabD with three new compounds from the class of aporphine alkaloids. ITC based binding studies have shown that apomorphine is binding to McFabD with a stronger affinity (KD = 4.87 µM) as compared to boldine (KD = 7.19 µM) and magnoflorine (KD = 11.7 µM). The possible mechanism of fluorescence quenching is found to be static with Kq values higher than 1010, which was associated with the ground state complex formation of aporphine alkaloids with McFabD. Conformational changes observed in the secondary and tertiary structure marked by the loss of helical content during the course of interactions. Molecular docking based studies have predicted the binding mode of aporphine alkaloids and it is found that these compounds are interacting in a similar fashion as known inhibitor corytuberine is interacting with McFabD. The analysis of docking poses have revealed that His 210, Leu102, Gln19, Ser101 and Arg 126 are critical residues, which may play important role in binding. The growth inhibition assay has shown that apomorphine has better MIC value (4-8 µg/ml) against Moraxella catarrhalis as compared to boldine and magnoflorine. Therefore, the current study suggests that aporphine alkaloids can act as antibacterial agents and possible target of these compounds could be FabD enzyme from the FAS II pathway, and apomorphine scaffold will be more suitable among these compounds for potential development of antibacterial agents.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Profiling the Membrane and Glycosaminoglycan-Binding Proteomes of Moraxella catarrhalis.

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.

Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry for identifying respiratory bacterial pathogens: a fast and efficient method.

Mass spectrometry has become a reference resource for identifying microorganisms in clinical microbiology services. One hundred and fifty one clinical isolates were selected from respiratory specimens routinely identified as Streptococcus pneumoniae (43), Haemophilus influenzae (64) and Moraxella catarrhalis (44). These identifications were compared with other phenotypical methods and mass spectrometry (MALDI-TOF-MS Vitek). Result discrepancies were assessed by 16S rRNA sequencing. Thirty-eight of the 43 strains of S. pneumoniae (86%) were identified as such using phenotypical methods and spectrometry. In 5 cases, MALDI-TOF identified 4 of them as Streptococcus pseudopneumoniae and 1 as S. mitis/oralis. Forty-eight of the 64 strains were identified as H. influenzae (75%) using biochemical identification systems and automated identification systems, whereas MALDI-TOF-MS Vitek identified 51 strains (79%) as such. Conventional methods and spectrometry identified all the 40 strains tested (100%) as M. catarrhalis. All strains with discrepant results were sequenced, and in all cases, the identification obtained by spectrometry was confirmed. The results obtained in this study show that mass spectrometry provides identification of these bacteria faster and in a more reliable way than those based on conventional phenotypical methods.

Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry for identifying respiratory bacterial pathogens: a fast and efficient method / La espectrometría de masas en la identificación de patógenos respiratorios bacterianos: un método rápido y eficaz

Mass spectrometry has become a reference resource for identifying microorganisms in clinical microbiology services. One hundred and fifty one clinical isolates were selected from respiratory specimens routinely identified as Streptococcus pneumoniae (43), Haemophilus influenzae (64) and Moraxella catarrhalis (44). These identifications were compared with other phenotypical methods and mass spectrometry (MALDI -TOF -MS Vitek). Result discrepancies were assessed by 16S rRNA sequencing. Thirty-eight of the 43 strains of S. pneumoniae (86%) were identified as such using phenotypical methods and spectrometry. In 5 cases, MALDI-TOF identified 4 of them as Streptococcus pseudopneumoniae and 1 as S. mitis/oralis. Forty-eight of the 64 strains were identified as H. influenzae (75%) using biochemical identification systems and automated identification systems, whereas MALDI-TOF-MS Vitek identified 51 strains (79%) as such. Conventional methods and spectrometry identified all the 40 strains tested (100%) as M. catarrhalis. All strains with discrepant results were sequenced, and in all cases, the identification obtained by spectrometry was confirmed. The results obtained in this study show that mass spectrometry provides identification of these bacteria faster and in a more reliable way than those based on conventional phenotypical methods (AU)

La espectrometría de masas se ha convertido en un recurso de referencia para la identificación de microorganismos en los servicios de Microbiología Clínica. Se estudiaron 151 aislamientos clínicos procedentes de muestras respiratorias que se identificaron rutinariamente como Streptococcus pneumoniae (43), Haemophilus influenzae (64) y Moraxella catarrhalis (44). Estos resultados se compararon con otros métodos fenotípicos y espectrometría de masas (MALDI-TOF Vitek-MS). Las discrepancias en los resultados se valoraron mediante secuenciación del ARNr 16S. Treinta y ocho de las 43 cepas de S. pneumoniae (86%) fueron identificadas como tales tanto por métodos bioquímicos como por espectrometría. En 5 casos MALDI-TOF identificó 4 como Streptococcus pseudopneumoniae y 1 como S. mitis/oralis. Cuarenta y ocho de las 64 cepas fueron identificadas como H. influenzae (75%) al utilizar galerías comerciales y sistemas automáticos, mientras que MALDI-TOF identifica como tales a 51 cepas (79%). Los métodos convencionales y la espectrometría identificaron como M. catarrhalis las 40 cepas estudiadas (100%). Todas las cepas con resultados discrepantes fueron secuenciadas, confirmándose en todos los casos la identificación obtenida por espectrometría. Los resultados obtenidos en este estudio demuestran que la espectrometría de masas ofrece una identificación de estas bacterias más rápida y fiable que la basada en métodos convencionales (AU)

An Unusual Carbohydrate Conformation is Evident in Moraxella catarrhalis Oligosaccharides.

Oligosaccharide structures derived from the lipooligosaccharide of M. catarrhalis show that the highly branched glucose-rich inner core of the oligosaccharide has an altered conformation compared to the most truncated tetra-glucose-Kdo lgt1/4Δ oligosaccharide structure. Addition of one residue each to the (1-4) and (1-6) chains to give the lgt2Δ oligosaccharide is the minimum requirement for this conformational change to occur. Extensive molecular modeling and NMR investigations have shown that the (1-3), (1-4), and (1-6) glycosidic linkages from the central α-D-Glcp have significantly altered conformational preferences between the two structures. For the lgt1/4Δ oligosaccharide the (1-3) and (1-4) linkage populates predominantly the syn minimum on the conformational free energy map and for the (1-6) linkage conformational flexibility is observed, which is supported by 1H-NMR T1 measurements. For the lgt2Δ oligosaccharide the unusual "(1-4)anti-ψ(1-6)gg" conformation, which could be confirmed by long-range NOE signals, is a dominant conformation in which the oligosaccharide is very compact with the terminal α-D-GlcNAc residue folding back towards the center of the molecule leading to an extensive intra-molecular hydrophobic interaction between the terminal residues. Comparing effective H-H distances, which were calculated for conformational sub-ensembles, with the NOE distances revealed that typically multiple conformations could be present without significantly violating the measured NOE restraints. For lgt2Δ the presence of more than one conformation is supported by the NOE data.

A pressure-driven column-based technique for the efficient extraction of DNA from respiratory samples.

Currently molecular techniques are a broadly accepted tool for diagnosis and are able to benefit patients in clinical practice. The polymerase chain reaction (PCR) has been especially incorporated into practical applications that are already in widespread use across the globe. With regard to the initial DNA extraction from clinically relevant samples, a number of commercially available kits are commonly used and are also designed to be easy to handle and less labor-intensive. In this study, the pressure system extracting DNA in column-based kit was developed, and its utility was compared with the centrifuge method using sputum from patients who were diagnosed with pneumonia. Also, due to the compact size and rapid processing time, the practical application of the pressure-based system incorporated into an automated pipetting machine was evaluated through clinical study. Our data suggests that DNA extraction by pressure was capable of serving as a substitute for the centrifuge method, and the compact and automatic nature of the pressure system device provided rapid and valuable information for clinical practice.

Ligand-bound structures of 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Moraxella catarrhalis reveal a water channel connecting to the active site for the second step of catalysis.

KdsC, the third enzyme of the 3-deoxy-D-manno-octulosonic acid (KDO) biosynthetic pathway, catalyzes a substrate-specific reaction to hydrolyze 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of KDO and phosphate. KdsC is a phosphatase that belongs to the C0 subfamily of the HAD superfamily. To understand the molecular basis for the substrate specificity of this tetrameric enzyme, the crystal structures of KdsC from Moraxella catarrhalis (Mc-KdsC) with several combinations of ligands, namely metal ion, citrate and products, were determined. Various transition states of the enzyme have been captured in these crystal forms. The ligand-free and ligand-bound crystal forms reveal that the binding of ligands does not cause any specific conformational changes in the active site. However, the electron-density maps clearly showed that the conformation of KDO as a substrate is different from the conformation adopted by KDO when it binds as a cleaved product. Furthermore, structural evidence for the existence of an intersubunit tunnel has been reported for the first time in the C0 subfamily of enzymes. A role for this tunnel in transferring water molecules from the interior of the tetrameric structure to the active-site cleft has been proposed. At the active site, water molecules are required for the formation of a water bridge that participates as a proton shuttle during the second step of the two-step phosphoryl-transfer reaction. In addition, as the KDO biosynthesis pathway is a potential antibacterial target, pharmacophore-based virtual screening was employed to identify inhibitor molecules for the Mc-KdsC enzyme.

uspA1 of Moraxella catarrhalis clinical isolates in Japan and its relationship with adherence to HEp-2 cells.

The surface protein, UspA1, of Moraxella catarrhalis is involved in adherence to human epithelial cells. We examined the expression of uspA1, and adherence to HEp-2 cells of clinical isolates. The uspA1 gene was detected in 204 of 208 isolates. The 4 uspA1-negative isolates belonged to the 16S rRNA type II with an A to G substitution at nucleotide 445 of 16S rRNA. In 13 isolates of the 16S rRNA type II, transcription of uspA1 was decreased or absent. A relationship between the extent of uspA1 transcription and adherence to HEp-2 cells was found in 5 isolates of the type I. In contrast, the 16S rRNA type II strains still had considerable adherence to HEp-2 cells. The type I uspA1 gene was expressed in Escherichia coli JM109 and the transformants adhered to HEp-2 cells at rates about 7 times higher than the host strain. These data indicated that the uspA1 was virtually ubiquitous in clinical isolates of M. catarrhalis and was responsible for adherence to HEp-2 cells of 16S rRNA type I isolates. However, the data also suggested that adherence of 16S rRNA type II strains to HEp-2 cells was attributed to factor(s) other than UspA1.

Modular arrangement of allelic variants explains the divergence in Moraxella catarrhalis UspA protein function.

Ubiquitous surface protein A molecules (UspAs) of Moraxella catarrhalis are large, nonfimbrial, autotransporter proteins that can be visualized as a "fuzzy" layer on the bacterial surface by transmission electron microscopy. Previous studies attributed a wide array of functions and binding activities to the closely related UspA1, UspA2, and/or UspA2H protein, yet the molecular and phylogenetic relationships among these activities remain largely unexplored. To address this issue, we determined the nucleotide sequence of the uspA1 genes from a variety of independent M. catarrhalis isolates and compared the deduced amino acid sequences to those of the previously characterized UspA1, UspA2, and UspA2H proteins. Rather than being conserved proteins, we observed a striking divergence of individual UspA1, UspA2, and UspA2H proteins resulting from the modular assortment of unrelated "cassettes" of peptide sequence. The exchange of certain variant cassettes correlates with strain-specific differences in UspA protein function and confers differing phenotypes upon these mucosal surface pathogens.

The Moraxella IgD-binding protein MID/Hag is an oligomeric autotransporter.

The immunoglobulin D (IgD)-binding protein MID/Hag of the human respiratory pathogen Moraxella catarrhalis is an outer membrane protein of approximately 200kDa belonging to the autotransporter family. MID also functions as an adhesin and hemagglutinin. In the present paper, the ultrastructure of MID was mapped. Using a series of Escherichia coli transformants, the last 210 aa of the C-terminal region were shown to translocate protein MID through the outer membrane suggesting that MID has a beta-barrel structure comprising of 10 transmembrane beta-sheets. Electron microscopy mapping with gold-labelled specific antibodies, and partial unravelling using guanidine hydrochloride showed that the rest of the MID protein forms an approximately 120nm long, fibrillar structure in which the individual monomers fold back on themselves to expose a globular distal domain at their tips comprising both the IgD-binding (MID962-1200) and adhesive (MID764-913) regions. This positions their N-termini close to the C-terminal membrane spanning domains. Mass measurements by scanning transmission electron microscopy (STEM) verified that the MID molecule is an oligomer.

Moraxella catarrhalis lipooligosaccharide selectively upregulates ICAM-1 expression on human monocytes and stimulates adjacent naïve monocytes to produce TNF-alpha through cellular cross-talk.

To elucidate the role of Moraxella catarrhalis lipooligosaccharide (LOS) in otitis media with effusion (OME), the effects of LOS on adhesion antigens of human monocytes were investigated. M. catarrhalis LOS selectively enhanced intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on human monocytes by significantly increasing both the surface expression intensity and the percentage of ICAM-1(+) cells. ICAM-1 upregulation on human monocytes by the LOS required surface CD14, TLR4, NF-kappaB p65 and c-Jun N-terminal kinase (JNK) activity. Our study also revealed that the LOS-induced surface ICAM-1 expression was partially mediated through a TNF-alpha dependent autocrine mechanism and could be further augmented by lipopolysaccharide-binding protein in serum. In addition, M. catarrhalis LOS also stimulated human monocytes to produce pro-inflammatory cytokines in both TLR4- and CD14-dependent pathways. Our results also indicated that enhanced surface ICAM-1 expression on monocytes may hinder their adherence to the lung epithelial monolayer. Furthermore, the LOS-activated human monocytes secreted a significantly high level of IL-8, and could stimulate adjacent naïve monocytes to produce TNF-alpha which was partially mediated via membrane ICAM-1 and IL-8/IL-8RA. These results suggest that M. catarrhalis LOS could induce excessive middle ear inflammation through a cellular cross-talk mechanism during OME.

Purification of alpha1-antichymotrypsin from human plasma with recombinant M. catarrhalis ubiquitous surface protein A1.

Alpha 1-antichymotrypsin (ACT) inhibits chymotrypsin-like enzymes, particularly neutrophil cathepsin G. Moreover, ACT in its native form suppresses chemotaxis of neutrophils and decreases neutrophil production of superoxide radicals. We recently showed that Moraxella catarrhalis ubiquitous surface protein (Usp) A1 is able to specifically bind ACT in the context of a novel virulence mechanism. In this study, we report that recombinant UspA1(557-704) coupled to CNBr-Sepharose can be used in a simple one-step purification of ACT from human plasma. UspA1(557-704)-purified ACT remains intact and active as shown by binding to M. catarrhalis and a chymotrypsin inhibition assay. The novel method for ACT isolation from plasma has important advantages in simplicity and time as compared to conventional methods.

Moraxella catarrhalis outer membrane protein CD elicits antibodies that inhibit CD binding to human mucin and enhance pulmonary clearance of M. catarrhalis in a mouse model.

The outer membrane protein CD of Moraxella catarrhalis is considered to be a potential vaccine antigen against Moraxella infection. We purified the native CD from isolate O35E, administered it to mice, and detected considerable titers of anti-CD antibodies. Anti-CD sera were cross-reactive towards six different M. catarrhalis isolates and promoted bacterial clearance of O35E in a pulmonary challenge model. To circumvent the difficulty of generating large quantities of CD from M. catarrhalis for vaccine use, the CD gene from O35E was cloned into Escherichia coli, and the recombinant CD, expressed without a signal sequence or fusion tags, represented approximately 70% of the total E. coli proteins. The recombinant CD formed inclusion bodies that were solubilized with 6 M urea and then purified by ion-exchange chromatography, a procedure that produced soluble CD of high purity and yield. Mice immunized with the purified recombinant CD had significant titers of anti-CD antibodies that were cross-reactive towards 24 different M. catarrhalis isolates. Upon challenge, these mice showed enhanced bacterial clearance of both O35E and a heterologous M. catarrhalis isolate, TTA24. In an in vitro assay, antisera to either the native or the recombinant CD inhibited the binding activity of CD to human tracheobronchial mucin in a serum concentration-dependent manner, and the extent of inhibition appeared to correlate with the corresponding anti-CD antibody titer and whole-cell enzyme-linked immunosorbent assay titer. Our results demonstrate that the recombinant CD is a promising vaccine candidate for preventing Moraxella infection.

Moraxella catarrhalis strain O35E expresses two filamentous hemagglutinin-like proteins that mediate adherence to human epithelial cells.

Two-partner secretion (TPS) systems are a family of proteins being rapidly identified and characterized in a growing number of gram-negative bacteria. TPS systems mediate the secretion of proteins, many involved in virulence traits such as hemolysis, adherence to epithelial cells, inhibition of bacterial growth, and immunomodulation of the host. A TPS system typically consists of a transporter located in the bacterial outer membrane (OM) which is responsible for the recognition and secretion of at least one large exoprotein. Two of the better-characterized TPS systems specify the Bordetella pertussis FHA and Haemophilus influenzae HMW1/HMW2 proteins. We identified three gene products of Moraxella catarrhalis strain O35E that resemble TPS proteins and designated them MhaC (transporter), MhaB1 (exoprotein), and MhaB2 (exoprotein). Western blot analysis using anti-MhaC, or antibodies reacting to both MhaB1 and MhaB2 (MhaB-reactive), revealed that these antigens are expressed in the OM of 63% of isolates tested. Mutations in the mhaC gene specifying the putative transporter of the M. catarrhalis wild-type strains O35E, O12E, and McGHS1 resulted in the absence of MhaB1/MhaB2 in the OM of mutants. These results are therefore consistent with the Mha proteins functioning as a TPS system. Furthermore, we discovered that these mhaC mutants exhibit markedly decreased binding to human epithelial cells relevant to pathogenesis by M. catarrhalis (Chang, HEp2, A549, and/or 16HBE14o(-)). Expression of O12E MhaC and MhaB1 in a nonadherent strain of Escherichia coli was found to increase the adherence of recombinant bacteria to HEp2 monolayers by sevenfold, thereby demonstrating that this M. catarrhalis TPS system directly mediates binding to human epithelial cells. The construction of isogenic mutants in the mhaB1 and mhaB2 genes of strain O35E also suggests that the MhaB proteins play distinct roles in M. catarrhalis adherence.

Characterization of the Moraxella catarrhalis opa-like protein, OlpA, reveals a phylogenetically conserved family of outer membrane proteins.

Moraxella catarrhalis is a human-restricted pathogen that can cause respiratory tract infections. In this study, we identified a previously uncharacterized 24-kDa outer membrane protein with a high degree of similarity to Neisseria Opa protein adhesins, with a predicted beta-barrel structure consisting of eight antiparallel beta-sheets with four surface-exposed loops. In striking contrast to the antigenically variable Opa proteins, the M. catarrhalis Opa-like protein (OlpA) is highly conserved and constitutively expressed, with 25 of 27 strains corresponding to a single variant. Protease treatment of intact bacteria and isolation of outer membrane vesicles confirm that the protein is surface exposed yet does not bind host cellular receptors recognized by neisserial Opa proteins. Genome-based analyses indicate that OlpA and Opa derive from a conserved family of proteins shared by a broad array of gram-negative bacteria.

Binding of vitronectin by the Moraxella catarrhalis UspA2 protein interferes with late stages of the complement cascade.

Many Moraxella catarrhalis strains are resistant to the bactericidal activity of normal human serum (NHS). The UspA2 protein of the serum-resistant strain O35E has previously been shown to be directly involved in conferring serum resistance on this strain. Testing of 11 additional serum-resistant M. catarrhalis wild-type isolates and their uspA1 and uspA2 mutants showed that the uspA1 mutants of all 11 strains were consistently serum resistant and that the uspA2 mutants of these same 11 strains were always serum sensitive. Analysis of complement deposition on four different serum-resistant M. catarrhalis strains and their serum-sensitive uspA2 mutants showed that, for three of these four strain sets, the wild-type and mutant strains bound similar amounts of early complement components. In contrast, there was a significant reduction in the amount of the polymerized C9 on the wild-type strains relative to that on the uspA2 mutants. These same three wild-type strains bound more vitronectin than did their uspA2 mutants. UspA2 proteins from these three strains, when expressed in Haemophilus influenzae, bound vitronectin and conferred serum resistance on this organism. Furthermore, vitronectin-depleted NHS exhibited bactericidal activity against these same three serum-resistant wild-type strains; addition of purified vitronectin to this serum restored serum resistance. In contrast, binding of the complement regulator C4b-binding protein by the M. catarrhalis strains used in this study was found to be highly variable and did not appear to correlate with the serum-resistant phenotype. These results indicate that binding of vitronectin by UspA2 is involved in the serum resistance of M. catarrhalis; this represents the first example of vitronectin-mediated serum resistance on a microbe.