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1.
Infect Immun ; 80(3): 982-95, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22184412

ABSTRACT

Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames (ORFs) encoding proteins that are involved in a truncated denitrification pathway or in the oxidative stress response, as well as several putative transcriptional regulators. Additionally, 200 M. catarrhalis genes were found to be downregulated when this bacterium was introduced into the nasopharynx. These downregulated genes included ORFs encoding several well-characterized M. catarrhalis surface proteins including Hag, McaP, and MchA1. Real-time reverse transcriptase PCR (RT-PCR) was utilized as a stringent control to validate the results of in vivo gene expression patterns as measured by DNA microarray analysis. Inactivation of one of the genes (MC ORF 1550) that was upregulated in vivo resulted in a decrease in the ability of M. catarrhalis to survive in the chinchilla nasopharynx over a 3-day period. This is the first evaluation of global transcriptome expression by M. catarrhalis cells in vivo.


Subject(s)
Gene Expression Regulation, Bacterial , Host-Pathogen Interactions , Moraxella catarrhalis/pathogenicity , Moraxellaceae Infections/microbiology , Nasopharynx/microbiology , Animals , Chinchilla , Disease Models, Animal , Gene Expression Profiling , Histocytochemistry , Male , Microarray Analysis , Nasopharynx/pathology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
2.
Infect Immun ; 76(11): 5322-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18678656

ABSTRACT

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.


Subject(s)
Bacterial Adhesion/genetics , Bacterial Outer Membrane Proteins/genetics , Moraxella catarrhalis/genetics , Moraxella catarrhalis/pathogenicity , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Blotting, Western , Cell Adhesion Molecules/metabolism , Cells, Cultured , Humans , Molecular Sequence Data , Moraxella catarrhalis/metabolism , Sequence Homology, Amino Acid
3.
Infect Immun ; 76(11): 5330-40, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18678659

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , DNA, Bacterial/genetics , Moraxella catarrhalis/chemistry , Moraxella catarrhalis/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Bacterial Outer Membrane Proteins/metabolism , Base Sequence , Consensus Sequence/genetics , Molecular Sequence Data , Moraxella catarrhalis/metabolism , Phenotype , Polymerase Chain Reaction , Protein Structure, Tertiary/genetics , Sequence Homology, Amino Acid
4.
J Bacteriol ; 189(1): 76-82, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17041038

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/genetics , Moraxella catarrhalis/genetics , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Genes, Bacterial , Models, Molecular , Molecular Sequence Data , Moraxella catarrhalis/chemistry , Protein Structure, Secondary , Sequence Alignment
5.
Infect Immun ; 74(3): 1588-96, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16495530

ABSTRACT

Mutant analysis was used to identify Moraxella catarrhalis gene products necessary for biofilm development in a crystal violet-based assay involving 24-well tissue culture plates. The wild-type M. catarrhalis strains that formed the most extensive biofilms in this system proved to be refractory to transposon mutagenesis, so an M. catarrhalis strain was constructed that was both able to form biofilms in vitro and amenable to transposon mutagenesis. Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; transformants able to form biofilms were identified and subjected to transposon-mediated mutagenesis. Biofilm-negative mutants of these transformants were shown to have a transposon insertion in the uspA1 gene. Nucleotide sequence analysis revealed that the biofilm-positive transformant T14 contained a hybrid O46E-O35E uspA1 gene, with the N-terminal 155 amino acids being derived from the O46E UspA1 protein. Transformant T14 was also shown to be unable to express the Hag protein, which normally extends from the surface of the M. catarrhalis cell. Introduction of a wild-type O35E hag gene into T14 eliminated its ability to form a biofilm. When the hybrid O46E-O35E uspA1 gene from T14 was used to replace the uspA1 gene of O35E, this transformant strain did not form a biofilm. However, inactivation of the hag gene did allow biofilm formation by strain O35E expressing the hybrid O46E-O35E uspA1 gene product. The Hag protein was shown to have an inhibitory or negative effect on biofilm formation by these M. catarrhalis strains in the crystal violet-based assay.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bacterial Proteins/immunology , Biofilms/growth & development , Hemagglutinins/immunology , Moraxella catarrhalis/physiology , Adhesins, Bacterial , Bacterial Adhesion/physiology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/physiology , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Cell Line , Conjunctivitis, Bacterial , Epithelial Cells , Hemagglutination Tests , Hemagglutinins/genetics , Moraxella catarrhalis/genetics , Moraxella catarrhalis/immunology
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