Darren D. Sledjeski

The Moraxella catarrhalis Porin-Like Outer Membrane Protein CD Is an Adhesin for Human Lung Cells

ABSTRACT 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 ∼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.

The Hag Protein of Moraxella catarrhalis Strain O35E Is Associated with Adherence to Human Lung and Middle Ear Cells

ABSTRACT Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8- to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues.

Moraxella catarrhalis Coaggregates with Streptococcus pyogenes and Modulates Interactions of S. pyogenes with Human Epithelial Cells

ABSTRACT The pathogens Streptococcus pyogenes and Moraxella catarrhalis colonize overlapping regions of the human nasopharynx. We have found that M. catarrhalis can dramatically increase S. pyogenes adherence to human epithelial cells and that species-specific coaggregation of these bacteria correlates with this enhanced adherence.

Use of surface-enhanced laser desorption ionization protein chip system to analyze streptococcal exotoxin B activity secreted by Streptococcus pyogenes.

Ciphergen surface-enhanced laser desorption ionization (SELDI) protein chip technology was used to analyze the secretion and autoactivation of the Streptococcus pyogenes cysteine protease SpeB. This method allowed rapid identification of both the zymogen form of the protein Mr approximately 41,000 and the fully active enzyme Mr approximately 28,500. SpeB production in culture supernatants was demonstrated to be growth-phase regulated and SpeB positive and negative variants of a blood passaged S. pyogenes isolate could readily be distinguished. In kinetic studies of the autoactivation of the zymogen form of SpeB, the sequential generation of four intermediates was detected before the accumulation of the fully active enzyme. The methods described enabled enhanced speed, use of lower sample volumes and concentrations, and a more complete molecular characterization of SpeB than allowed by existing methods of analysis using SDS-PAGE and Western immunoblotting.

Regulation of protein H expression in M1 serotype isolates of Streptococcus pyogenes

Protein H is an immunoglobulin-binding protein expressed by certain M1 serotypes of Streptococcus pyogenes. In a recent study of invasive group A isolates, it was found that none of the 16 M1 serotype isolates analyzed expressed protein H on their surface despite the presence of the protein H gene (sph) in approximately one-third of the isolates. Selection of stable protein H-expressing variants could be achieved by infection of prtH(+) non-expressing strains into a mouse skin and recovering bacteria from the spleen. This effect was independent of the transcription regulator Mga, since a similar effect was noted in an mga(-) mutant. Thus, host passage of S. pyogenes can lead to stable high level expression of Protein H.

Streptococcus pyogenes infection in mouse skin leads to a time-dependent up-regulation of protein H expression.

ABSTRACT Streptococcus pyogenes protein H (sph) is an immunoglobulin-binding protein present in the Mga regulon of certain M1 serotype isolates. Although sph is present in many strains, it is frequently not expressed. In this paper we show that protein H was highly expressed after bacteria were injected into the skin of mice and were recovered from the blood, kidney, or spleen at various times postinfection. The percentage of protein H-positive colonies increased with time, reaching 100% in the spleen and kidney within 24 to 72 h postinfection. The up-regulation of sph expression was also observed in a mga mutant.

Isolation of human plasma-inducible, growth phase- and temperature-regulated gene fusions in Streptococcus pyogenes using a Tn917–lacZ transposon

Streptococcus pyogenes is capable of causing a variety of human diseases ranging from superficial or deep tissue infections to non-infectious post-streptococcal infection sequelae. In this paper, we report the use of a Tn917-lacZ transposon to isolate random lacZ transcription fusions in the S. pyogenes chromosome. Libraries of random Tn917-lacZ mutants were generated in a representative opacity factor positive strain CS101 (M49) and an opacity factor negative strain 1881 (M1). Several different mutant phenotypes were isolated. These included: temperature-regulated promoters, growth phase/cell density-regulated promoters and a human plasma-inducible promoter. Expression of the temperature-regulated fusions was 5-10-fold higher when grown at 30 degrees C compared to growth at 37 degrees C. The growth phase-regulated fusions were induced 30-fold at late exponential phase and were repressed by a diffusible S. pyogenes factor(s). Expression of the human plasma-inducible fusion was induced 10-15-fold by human plasma or sera, 4-fold by rabbit sera and was repressed by horse and mouse sera. In addition, hemolysin negative and capsule over expression mutants were isolated. These results demonstrate the utility of Tn917-lacZ mutagenesis for the identification of S. pyogenes promoters.