Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Isogenic mutations in the Moraxella catarrhalis CydDC system display pleiotropic phenotypes and reveal the role of a palindrome sequence in its transcriptional regulation.

Moraxella catarrhalis is becoming an important human respiratory tract pathogen affecting significant proportions from the population. However, still little is known about its physiology and molecular regulation. To this end, the CydDC, which is a heterodimeric ATP binding cassette transporter that has been shown to contribute to the maintenance of the redox homeostasis across the periplasm in other Gram-negative bacteria, is studied here. Amino acids multiple sequence alignments indicated that M. catarrhalis CydC is different from the CydC proteins of the bacterial species in which this system has been previously studied. These findings prompted further interest in studying this system in M. catarrhalis. Isogenic mutant in the CydDC system showed suppression in growth rate, hypersensitivity to oxidative and reductive stress and increased accumulation of intracellular cysteine levels. In addition, the growth of cydC- mutant exhibited hypersensitivity to exogenous cysteine; however, it did not display a significant difference from its wild-type counterpart in the murine pulmonary clearance model. Moreover, a palindrome was detected 94bp upstream of the cydD ORF suggesting it might act as a potential regulatory element. Real-time reverse transcription-PCR analysis showed that deletion/change in the palindrome resulted into alterations in the transcription levels of cydC. A better understanding of such system and its regulation helps in developing better ways to combat M. catarrhalis infections.

ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence.

Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract.

The Vaccine Candidate Substrate Binding Protein SBP2 Plays a Key Role in Arginine Uptake, Which Is Required for Growth of Moraxella catarrhalis.

Moraxella catarrhalis is an exclusively human pathogen that is an important cause of otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. A vaccine to prevent M. catarrhalis infections would have an enormous global impact in reducing morbidity resulting from these infections. Substrate binding protein 2 (SBP2) of an ABC transporter system has recently been identified as a promising vaccine candidate antigen on the bacterial surface of M. catarrhalis. In this study, we showed that SBP1, -2, and -3 individually bind different basic amino acids with exquisite specificity. We engineered mutants that each expressed a single SBP from this gene cluster and showed in growth experiments that SBP1, -2, and -3 serve a nutritional function through acquisition of amino acids for the bacterium. SBP2 mediates uptake of arginine, a strict growth requirement of M. catarrhalis. Adherence and invasion assays demonstrated that SBP1 and SBP3 play a role in invasion of human respiratory epithelial cells, consistent with a nutritional role in intracellular survival in the human respiratory tract. This work demonstrates that the SBPs of an ABC transporter system function in the uptake of basic amino acids to support growth of M. catarrhalis. The critical role of SBP2 in arginine uptake may contribute to its potential as a vaccine antigen.

Moraxella catarrhalis AcrAB-OprM efflux pump contributes to antimicrobial resistance and is enhanced during cold shock response.

Moraxella catarrhalis is a common pathogen of the human respiratory tract. Multidrug efflux pumps play a major role in antibiotic resistance and virulence in many Gram-negative organisms. In the present study, the role of the AcrAB-OprM efflux pump in antibiotic resistance was investigated by constructing mutants that lack the acrA, acrB, and oprM genes in M. catarrhalis strain O35E. We observed a moderate (1.5-fold) decrease in the MICs of amoxicillin and cefotaxime and a marked (4.7-fold) decrease in the MICs of clarithromycin for acrA, acrB, and oprM mutants in comparison with the wild-type O35E strain. Exposure of the M. catarrhalis strains O35E and 300 to amoxicillin triggered an increased transcription of all AcrAB-OprM pump genes, and exposure of strains O35E, 300, and 415 to clarithromycin enhanced the expression of acrA and oprM mRNA. Inactivation of the AcrAB-OprM efflux pump genes demonstrated a decreased ability to invade epithelial cells compared to the parental strain, suggesting that acrA, acrB, and oprM are required for efficient invasion of human pharyngeal epithelial cells. Cold shock increases the expression of AcrAB-OprM efflux pump genes in all three M. catarrhalis strains tested. Increased expression of AcrAB-OprM pump genes after cold shock leads to a lower accumulation of Hoechst 33342 (H33342), a substrate of AcrAB-OprM efflux pumps, indicating that cold shock results in increased efflux activity. In conclusion, the AcrAB-OprM efflux pump appears to play a role in the antibiotic resistance and virulence of M. catarrhalis and is involved in the cold shock response.

A Moraxella catarrhalis two-component signal transduction system necessary for growth in liquid media affects production of two lysozyme inhibitors.

There are a paucity of data concerning gene products that could contribute to the ability of Moraxella catarrhalis to colonize the human nasopharynx. Inactivation of a gene (mesR) encoding a predicted response regulator of a two-component signal transduction system in M. catarrhalis yielded a mutant unable to grow in liquid media. This mesR mutant also exhibited increased sensitivity to certain stressors, including polymyxin B, SDS, and hydrogen peroxide. Inactivation of the gene (mesS) encoding the predicted cognate sensor (histidine) kinase yielded a mutant with the same inability to grow in liquid media as the mesR mutant. DNA microarray and real-time reverse transcriptase PCR analyses indicated that several genes previously shown to be involved in the ability of M. catarrhalis to persist in the chinchilla nasopharynx were upregulated in the mesR mutant. Two other open reading frames upregulated in the mesR mutant were shown to encode small proteins (LipA and LipB) that had amino acid sequence homology to bacterial adhesins and structural homology to bacterial lysozyme inhibitors. Inactivation of both lipA and lipB did not affect the ability of M. catarrhalis O35E to attach to a human bronchial epithelial cell line in vitro. Purified recombinant LipA and LipB fusion proteins were each shown to inhibit human lysozyme activity in vitro and in saliva. A lipA lipB deletion mutant was more sensitive than the wild-type parent strain to killing by human lysozyme in the presence of human apolactoferrin. This is the first report of the production of lysozyme inhibitors by M. catarrhalis.

Differential response of gel-forming mucins to pathogenic middle ear bacteria.

OBJECTIVE: To assess the differential response of the secretory gel forming mucins (GFM) to the most common bacterial pathogens causing otitis media, Streptococcus pneumoniae (SP), nontypeable Haemophilus influenza (NTHi), and Moraxella catarrhalis (Mcat), in a culture model of human middle ear epithelium (HMEEC). METHODS: In vitro cultured HMEEC was exposed to 5 µg/ml of bacterial whole cell lysate (WCL). RNA was extracted to generate cDNA. The expression levels of each of the targeted mucin transcripts, MUC2, MUC5AC, MUC5B and MUC19, were detected by quantitative PCR. RESULTS: The submerged HMEEC exposed to NTHi-86028NP WCL demonstrated a significant increase of MUC2, MUC5AC and MUC5B as compared to the control non-treated cells while MUC19 transcript level remained unchanged. WCL of additional major OM pathogens significantly increase the transcription of these three mucin genes as well. A combination of NTHi and SP further synergistically induced MUC2 and MUC5AC gene expression however, not all NTHi strains synergized with SP in the induction. Addition of Mcat WCL to the synergized combination of NTHi and SP did not participate in the synergistic response of mucins. CONCLUSION: The specific pathogen combinations were important in determining the degree of synergistic effects to GFM expression. The current data are substantive in guiding future work to extend our understanding of OM pathogens and GFMs.

Role of the zinc uptake ABC transporter of Moraxella catarrhalis in persistence in the respiratory tract.

Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains of M. catarrhalis tested. A mutant in which the znu gene cluster is knocked out shows markedly impaired growth compared to the wild type in medium that contains trace zinc; growth is restored to wild-type levels by supplementing medium with zinc but not with other divalent cations. Thermal-shift assays showed that the purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter of M. catarrhalis is critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir of M. catarrhalis is present in the human respiratory tract and this reservoir is important for persistence. The znu knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments with very low zinc concentrations and is critical for full virulence of M. catarrhalis in the respiratory tract in facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract.

Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions.

Iron sequestration by the human host is a first line defence against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron starvation during colonization. We determined the genetic requirements for M. catarrhalis BBH18 growth during iron starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). By subjecting a large random transposon mutant library to growth under iron-limiting conditions, mutants of the MCR_0996-rhlB-yggW operon, rnd, and MCR_0457 were negatively selected. Growth experiments using directed mutants confirmed the GAF phenotypes with ΔyggW (putative haem-shuttling protein) and ΔMCR_0457 (hypothetical protein) most severely attenuated during iron starvation, phenotypes which were restored upon genetic complementation of the deleted genes. Deletion of yggW resulted in similar attenuated phenotypes in three additional strains. Transcriptional profiles of ΔyggW and ΔMCR_0457 were highly altered with 393 and 192 differentially expressed genes respectively. In all five mutants, expression of nitrate reductase genes was increased and of nitrite reductase decreased, suggesting an impaired aerobic respiration. Alteration of iron metabolism may affect nasopharyngeal colonization as adherence of all mutants to respiratory tract epithelial cells was attenuated. In conclusion, we elucidated the genetic requirements for M. catarrhalis growth during iron starvation and characterized the roles of the identified genes in bacterial growth and host interaction.

Role of Moraxella catarrhalis outer membrane protein CD in bacterial cell morphology and autoaggregation.

Moraxella catarrhalis, an important pathogen in the human respiratory tract, causes otitis media and lower respiratory tract infections. M. catarrhalis outer membrane protein CD (OMPCD) is a major heat-modifiable OMP with demonstrable potential as a vaccine candidate. The gene encoding OMPCD of M. catarrhalis strains was subjected to nucleotide sequence analysis and then inactivated by insertional mutagenesis. The ompCD mutant strains exhibited a modest growth defect in comparison with the wild-type strains. In optical microscopy and scanning/transmission electron microscopy examinations, regarding morphology, the cell size and cell wall of the ompCD mutant strains were significantly larger and thinner, respectively, than those of the wild-type strain. Furthermore, the ompCD mutant strains exhibited significant autoaggregation and increased surface hydrophobicity, in addition to a reduction in the adherence to HEp-2 cells, compared to the wild-type strains. Strains repaired by replacing the mutated ompCD gene exhibited phenotypic characteristics very similar to those of the wild-type strains. These results indicate that M. catarrhalis OMPCD, in addition to its functions related to bacterial growth and adherence to human epithelial cells, plays a very important role in bacterial physiology and pathogenesis, including aspects such as stabilizing bacterial cell morphology and preventing autoaggregation by reducing surface hydrophobicity.

A novel group of Moraxella catarrhalis UspA proteins mediates cellular adhesion via CEACAMs and vitronectin.

Moraxella catarrhalis (Mx) is a common cause of otitis media and exacerbation of chronic obstructive pulmonary disease, an increasing worldwide problem. Surface proteins UspA1 and UspA2 of Mx bind to a number of human receptors and may function in pathogenesis. Genetic recombination events in the pathogen can generate hybrid proteins termed UspA2H. However, whether certain key functions (e.g. UspA1-specific CEACAM binding) can be exchanged between these adhesin families remains unknown. In this study, we have shown that Mx can incorporate the UspA1 CEACAM1-binding region not only into rare UspA1 proteins devoid of CEACAM-binding ability, but also into UspA2 which normally lack this capacity. Further, a screen of Mx isolates revealed the presence of novel UspA2 Variant proteins (UspA2V) in ∼14% of the CEACAM-binding population. We demonstrate that the expression of UspA2/2V with the CEACAM-binding domain enable Mx to bind both to cell surface CEACAMs and to integrins, the latter via vitronectin. Such properties of UspA2/2V have not been reported to date. The studies demonstrate that the UspA family is much more heterogeneous than previously believed and illustrate the in vivo potential for exchange of functional regions between UspA proteins which could convey novel adhesive functions whilst enhancing immune evasion.

Bacterium-generated nitric oxide hijacks host tumor necrosis factor alpha signaling and modulates the host cell cycle in vitro.

In mammalian cells, nitric oxide (NO·) is an important signal molecule with concentration-dependent and often controversial functions of promoting cell survival and inducing cell death. An inducible nitric oxide synthase (iNOS) in various mammalian cells produces higher levels of NO· from l-arginine upon infections to eliminate pathogens. In this study, we reveal novel pathogenic roles of NO· generated by bacteria in bacterium-host cell cocultures using Moraxella catarrhalis, a respiratory tract disease-causing bacterium, as a biological producer of NO·. We recently demonstrated that M. catarrhalis cells that express the nitrite reductase (AniA protein) can produce NO· by reducing nitrite. Our study suggests that, in the presence of pathophysiological levels of nitrite, this opportunistic pathogen hijacks host cell signaling and modulates host gene expression through its ability to produce NO· from nitrite. Bacterium-generated NO· significantly increases the secretion of tumor necrosis factor alpha (TNF-α) and modulates the expression of apoptotic proteins, therefore triggering host cell programmed death partially through TNF-α signaling. Furthermore, our study reveals that bacterium-generated NO· stalls host cell division and directly results in the death of dividing cells by reducing the levels of an essential regulator of cell division. This study provides unique insight into why NO· may exert more severe cytotoxic effects on fast growing cells, providing an important molecular basis for NO·-mediated pathogenesis in infections and possible therapeutic applications of NO·-releasing molecules in tumorigenesis. This study strongly suggests that bacterium-generated NO· can play important pathogenic roles during infections.

Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding.

Bacterial cell surfaces are commonly decorated with a layer formed from multiple copies of adhesin proteins whose binding interactions initiate colonization and infection processes. In this study, we investigate the physical deformability of the UspA1 adhesin protein from Moraxella catarrhalis, a causative agent of middle-ear infections in humans. UspA1 binds a range of extracellular proteins including fibronectin, and the epithelial cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Electron microscopy indicates that unliganded UspA1 is densely packed at, and extends about 800 Å from, the Moraxella surface. Using a modified atomic force microscope, we show that the adhesive properties and thickness of the UspA1 layer at the cell surface varies on addition of either fibronectin or CEACAM1. This in situ analysis is then correlated with the molecular structure of UspA1. To provide an overall model for UspA1, we have determined crystal structures for two N-terminal fragments which are then combined with a previous structure of the CEACAM1-binding site. We show that the UspA1-fibronectin complex is formed between UspA1 head region and the 13th type-III domain of fibronectin and, using X-ray scattering, that the complex involves an angular association between these two proteins. In combination with a previous study, which showed that the CEACAM1-UspA1 complex is distinctively bent in solution, we correlate these observations on isolated fragments of UspA1 with its in situ response on the cell surface. This study therefore provides a rare direct demonstration of protein conformational change at the cell surface.

Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells.

Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two-dimensional SDS-PAGE and MALDI-TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD-binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC-stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll-like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV-attributed proteins. Interestingly, OMVs modulated the pro-inflammatory response in epithelial cells, and UspA1-bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro-inflammatory response.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells.

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Moraxella catarrhalis binding to host cellular receptors is mediated by sequence-specific determinants not conserved among all UspA1 protein variants.

The Moraxella catarrhalis ubiquitous surface proteins (UspAs) are autotransporter molecules reported to interact with a variety of different host proteins and to affect processes ranging from serum resistance to cellular adhesion. The role of UspA1 as an adhesin has been confirmed with a number of different human cell types and is mediated by binding to eukaryotic proteins including carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs), fibronectin, and laminin. A distinct difference in the ability of prototypical M. catarrhalis strains to adhere to CEACAM-expressing cell lines prompted us to perform strain-specific structure-function analyses of UspA1 proteins. In this study, we characterized CEACAM binding by a diverse set of UspA1 proteins and showed that 3 out of 10 UspA1 proteins were incapable of binding CEACAM. This difference resulted from the absence of a distinct CEACAM binding motif in nonadhering strains. Our sequence analysis also revealed a single M. catarrhalis isolate that lacked the fibronectin-binding motif and was defective in adherence to Chang conjunctival epithelial cells. These results clearly demonstrate that UspA1-associated adhesive functions are not universally conserved. Instead, UspA1 proteins must be considered as variants with the potential to confer both different cell tropisms and host cell responses.

The UspA1 protein of Moraxella catarrhalis induces CEACAM-1-dependent apoptosis in alveolar epithelial cells.

Moraxella catarrhalis is a major cause of exacerbations of chronic obstructive pulmonary disease (COPD) and emphysema. M. catarrhalis-specific UspA1 and the epithelial carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) were required to induce apoptosis. M. catarrhalis-induced apoptosis was significantly enhanced in HeLa cells stably transfected with CEACAM1, compared with HeLa cells not expressing CEACAM1. Infected cells showed increased activity of caspases 3, 6, and 9 but not of caspase 8. Reduced expression of Bcl-2, translocation of Bax into the mitochondria, and cytosolic increase of apoptosis-inducing factor in M. catarrhalis-infected cells implicated the involvement of mitochondrial death pathways. In conclusion, M. catarrhalis induced apoptosis in pulmonary epithelial cells--a process that was triggered by interaction between CEACAM1 and UspA1. Thus, M. catarrhalis-induced apoptosis of pulmonary epithelial cells may contribute to the development of COPD and emphysema.

The Moraxella catarrhalis autotransporter McaP is a conserved surface protein that mediates adherence to human epithelial cells through its N-terminal passenger domain.

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.

Expression of type IV pili by Moraxella catarrhalis is essential for natural competence and is affected by iron limitation.

Type IV pili, filamentous surface appendages primarily composed of a single protein subunit termed pilin, play a crucial role in the initiation of disease by a wide range of pathogenic bacteria. Although previous electron microscopic studies suggested that pili might be present on the surface of Moraxella catarrhalis isolates, detailed molecular and phenotypic analyses of these structures have not been reported to date. We identified and cloned the M. catarrhalis genes encoding PilA, the major pilin subunit, PilQ, the outer membrane secretin through which the pilus filament is extruded, and PilT, the NTPase that mediates pilin disassembly and retraction. To initiate investigation of the role of this surface organelle in pathogenesis, isogenic pilA, pilT, and pilQ mutants were constructed in M. catarrhalis strain 7169. Comparative analyses of the wild-type 7169 strain and three isogenic pil mutants demonstrated that M. catarrhalis expresses type IV pili that are essential for natural genetic transformation. Our studies suggest type IV pilus production by M. catarrhalis is constitutive and ubiquitous, although pilin expression was demonstrated to be iron responsive and Fur regulated. These data indicate that additional studies aimed at elucidating the prevalence and role of type IV pili in the pathogenesis and host response to M. catarrhalis infections are warranted.

The Moraxella catarrhalis porin-like outer membrane protein CD is an adhesin for human lung cells.

The outer membrane protein CD (OMPCD) of Moraxella catarrhalis is an outer membrane protein with several attributes of a potential vaccine antigen. We isolated four transposon mutants of strain O35E on the basis of their reduced binding to A549 human lung cells in microcolony formation assays, and we determined that they contain a transposon in ompCD. We also found that these transposon insertions had pleiotropic effects: mutants grew slower, became serum sensitive, bound approximately 10-fold less to A549 cells, and appeared transparent when grown on solid medium. We confirmed that these various phenotypes could be attributed solely to disruption of ompCD by constructing the isogenic strain O35E.CD1. O35E-ompCD was cloned, and recombinant Escherichia coli bacteria expressing the gene product exhibited a 10-fold increase in adherence to A549 cells. This is the first report of M. catarrhalis ompCD mutants, and our findings demonstrate that this gene product is an adhesin for human lung cells.