Kristin A. Birkness

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a persons lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test‐negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage‐specific CD68+ epithelioid macrophages and small round CD3+ lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid‐fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead‐based assay using the Luminex 100 and were found to contain interleukin (IL)‐6, IL‐8, interferon‐γ and tumor necrosis factor‐α, cytokines known to be involved in human granuloma formation, in quantities from two‐fold to 7000‐fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Genetic transfer of antimicrobial resistance and enterotoxigenicity among Escherichia coli strains.

To understand the role of enterotoxin (Ent) plasmids in epidemics of enterotoxigenic (ET) Escherichia coli diarrhea in the United States, we studied the genetics of Ent plasmids in relation to E. coli serotypes and R plasmids. Twenty-nine ET E. coli strains, including all epidemic isolates available at the Centers for Disease Control, Atlanta, Ga. (CDC), were assessed for the ability to transfer antimicrobial resistances (if present) by conjugation, to mobilize a nonconjugative R plasmid, and to cotransfer enterotoxigenicity with R determinants. Of the 12 ET E. coli strains isolated in the United States, 5 were able to transfer R plasmids; one strain cotransferred detectable enterotoxigenicity. Another four U.S. isolates were able to mobilize plasmid DNA, but no toxin production was detected in transconjugants. Of 17 resistant ET E. coli from South Asia, 13 were able to transfer R plasmids; 5 of those 13 cotransferred detectable Ent plasmids. In all, 22 ET E. coli strains (76%) were able to initiate conjugation and genetic transfers. Six of these strains (20%) were able to cotransfer enterotoxigenicity with a conjugative R plasmid at a detectable frequency. One of the six strains transferred R and Ent determinants on a single plasmid. These data are addressed in relation to the observed immobility of Ent and R during outbreaks of ET E. coli, the efficacy of prophylactic tetracycline, and the worldwide occurrence of a limited number of ET E. coli serotypes.

Diagnostic value of plasmid analyses and assays for virulence in Yersinia enterocolitica

The possession of a 42- to 48-megadalton plasmid alone does not appear to be predictive of virulence in Yersinia species. Twelve of 100 Yersinia enterocolitica strains contained a 42 to 48-megadalton plasmid, and 4 of 30 Y. enterocolitica-like strains contained a 42- to 48-megadalton plasmid. Seven strains of Y. enterocolitica contained the 42- to 48-megadalton plasmid plus an 82-megadalton plasmid, and these were the only study strains lethal for mice. Based on restriction endonuclease digestion, the 42- to 48-megadalton plasmid DNA from these seven strains were similar and were not similar to the 42- to 48-megadalton plasmids present in the other nine strains. The ability to invade guinea pig eye tissues, calcium dependency, autoagglutination, and colonial morphology at 37 degrees C were also associated with plasmid DNA, but the relationships were either variable or not reciprocal. Neither tissue culture invasiveness nor heat-stable toxin production was associated with plasmid DNA. It was concluded that biochemical speciation and a total plasmid profile in combination with enzyme digests are predictive of virulence in Y. enterocolitica as it is measured by mouse lethality.

Nucleotide sequence of the common plasmid ofChlamydia trachomatis serovar L2: Use of compatible deletions to generate overlapping fragments

All serotypes ofChlamydia trachomatis appear to have a 7.5kbp plasmid of a generally conserved sequence which occurs in 5–10 copies per elementary body genome equivalent. The plasmid from strain L2/434/Bu was cloned and mapped for restriction enzyme sites. We determined the plasmid sequence with a family of overlapping deletions in the phagemid vector BSM13+, other recombinants created by cloning defined chlamydial restriction fragments into M13 sequencing vectors, and synthetic oligonucleotide primers. A 22-base-pair sequence, located near the uniqueSstI site of the plasmid sequence, is directly repeated four times. Although the function of this repeated sequence is uncertain, the 22-mer is in an area with an A.T-rich sequence and an open reading frame that resembles the regulatory elements and origins of replication found in other bacteria. The finding of repeated sequences on the multicopy plasmid ofC. trachomatis suggests that methods of oligonucleotide probing can be used to detect chlamydial DNA.

Plasmids and Serotypes of Hemolytic Fecal Escherichia coli

Twelve of 21 human, hemolytic, fecal isolates ofEscherichia coli produced type 1 hemolysin (HLY1), an extracellular, heat-labile molecule (alpha-hemolysin). Although no common plasmid species was apparent, 11 of 12 HLY1 strains possessed a plasmid≥60 megadaltons (Mdal); 5 of 9 strains with other hemolysins possessed a plasmid of comparable molecular mass (Fishers exact probability=0.0805). One derivative of an HLY1+strain, which contained a 125 Mdal plasmid, no longer expressed HLY1 and contained a single 102 Mdal plasmid. The presence of large plasmids of varying size and an apparent deletion mutation in HLY1 strains suggest that HLY1 determinants are located on a small, unstable genetic element. In an initial survey of 224 human fecal isolates ofE. coli, the predominant hemolytic serotype was 06:H-, and conversely most (85%) 06:H-isolates were HLY1+. Serotype appears to play an important role in HLY1 expression.

Tissue culture bilayer models to study attachment and invasion of bacterial pathogens

A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in bacterial pathogenesis. The model, incorporating epithelial and endothelial cell layers separated by a microporous membrane, makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. The model is adaptable to a variety of microbial pathogens in that any physiologically relevant eucaryotic cells can be substituted for the component layers. Examining a variety of strains ofNeisseria meningitidis we found marked differences in their ability to pass through the bilayer with ranges between 80% and <0.01% of the inoculum passing through within a 3-hour period. These differences may correlate with differences in virulence. More recently in a model developed to study the pathogenesis of lower respiratory pathogens we found that while passage ofMycobacterium avium through the bilayer differed little from strain to strain, there was a 10–40 fold difference inStreptococcus pneumoniae passage between strains causing hemorrhagic disease and strains causing pneumonia. The human cell composition and reproducibility of these systems give them advantages over animal and primary organ culture models. Additionally, their added multiple layer complexity allowing cell-to-cell communication makes them more realistic human tissue models than standard cell monolayers.