Functional glycosylphosphatidylinositol anchor signal sequences in the Pneumocystis carinii PRT1 protease family.

Pneumocystis carinii is fungus which is a frequent cause of severe pneumonia in immunocompromised individuals. The P. carinii genome contains the PRT1 subtelomeric multigene family that encodes a kexin-like serine protease which is expressed on the surface of P. carinii. Analysis of the sequence of the carboxy-terminal sequence of many copies of PRT1 showed that they contained motifs characteristic of a glycosylphosphatidylinositol (GPI) anchor signal sequence. The ability of the C-terminal sequences of PRT1 to direct the addition of a GPI anchor was tested. CD14, a GPI-anchored monocyte glycoprotein antigen, was used as the basis of a heterologous system. CD14 was truncated to remove the carboxy-terminal sequences responsible for GPI-anchor addition. Addition of carboxy-terminal sequences from PRT1 restored high-level surface expression to the truncated CD14. Further, the majority of CD14-PRT1 recombinant protein was removed from the cell membrane by treatment with GPI-specific phospholipase C. These results suggest that the carboxy-terminal residues of most of the members of the PRT1 family of proteases have the potential to form a functional GPI-attachment signal.

Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation.

Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora. alpha-Amylase, the predominant salivary enzyme in humans, binds to Streptococcus gordonii, a primary colonizer of the tooth. Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation. Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA). To study the function of this protein, an erythromycin resistance determinant [erm(AM)] was inserted within the abpA gene of S. gordonii strains Challis and FAS4 by allelic exchange, resulting in abpA mutant strains Challis-E1 and FAS4-E1. Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties. Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants. In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains. While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase. In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the abpA mutant strains grew much more poorly and produced relatively small microcolonies. Taken together, these results suggest that AbpA of S. gordonii functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by S. gordonii.

Mutualism versus independence: strategies of mixed-species oral biofilms in vitro using saliva as the sole nutrient source.

During initial dental plaque formation, the ability of a species to grow when others cannot would be advantageous, and enhanced growth through interspecies and intergeneric cooperation could be critical. These characteristics were investigated in three coaggregating early colonizers of the tooth surface (Streptococcus gordonii DL1, Streptococcus oralis 34, and Actinomyces naeslundii T14V). Area coverage and cell cluster size measurements showed that attachment of A. naeslundii and of S. gordonii to glass flowcells was enhanced by a salivary conditioning film, whereas attachment of S. oralis was hindered. Growth experiments using saliva as the sole carbon and nitrogen source showed that A. naeslundii was unable to grow either in planktonic culture or as a biofilm, whereas S. gordonii grew under both conditions. S. oralis grew planktonically, but to a much lower maximum cell density than did S. gordonii; S. oralis did not grow reproducibly as a biofilm. Thus, only S. gordonii possessed all traits advantageous for growth as a solitary and independent resident of the tooth. Two-species biofilm experiments analyzed by laser confocal microscopy showed that neither S. oralis nor A. naeslundii grew when coaggregated pairwise with S. gordonii. However, both S. oralis and A. naeslundii showed luxuriant, interdigitated growth when paired together in coaggregated microcolonies. Thus, the S. oralis-A. naeslundii pair formed a mutualistic relationship, potentially contact dependent, that allows each to grow where neither could survive alone. S. gordonii, in contrast, neither was hindered by nor benefited from the presence of either of the other strains. The formation of mutually beneficial interactions within the developing biofilm may be essential for certain initial colonizers to be retained during early plaque development, whereas other initial colonizers may be unaffected by neighboring cells on the substratum.

Retrieval of biofilms from the oral cavity.

With the use of the removable stents or bonded enamel piece models with or without a continuous bacterial layer, many in vitro or in vivo studies can be initiated. For example, studies on salivary pellicle formation, surface characteristics of biomaterials as they affect plaque development, antiplaque agents, the dynamics of adhesion of bacteria, interspecies adhesion of bacteria, the colonization of bacteria, the dynamics of bacterial growth in vivo, and the succession of growth in older supragingival plaques can be carried out.

Population structure of rat-derived Pneumocystis carinii in Danish wild rats.

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions.

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1-2 ppm) and under anaerobic conditions (

Ultrastructural and molecular characterization of Pneumocystis carinii isolated from a rhesus monkey (Macaca mulatta).

High levels of heterogeneity have been observed among isolates of Pneumocystis carinii derived from different mammalian host species. We report the characterization of P. carinii isolated from a rhesus monkey (Macaca mulatta), which was immunosuppressed as a result of infection with a chimeric simian-human immunodeficiency virus (SHIVsbg). Histopathological examination showed evidence of severe P. carinii pneumonia with a large predominance of trophozoite forms. Alveolitis consisted of typical foamy, honeycomb exudate, with only a few alveolar macrophages. The lung inflammatory response was rather moderate without type-2 pneumocyte hyperplasia or collagenosis. P. carinii organisms were sometimes observed in the bronchiolar lumen. Ultrastructurally, macaque-derived P. carinii was more similar to human- or rabbit-derived parasites than to mouse-derived P. carinii. Molecular studies were carried out on the macaque-derived P. carinii DNA at two genetic loci: the genes encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) and the mitochondrial small subunit ribosomal RNA (mt SSU rRNA). Comparison of the DNA sequences with those from P. carinii isolated from eight other host species demonstrated that the macaque-derived P. carinii was genetically distinct at both loci, and was more closely related to human-derived P. carinii than to P. carinii derived from non-primate sources. We propose that macaque-derived P. carinii be named Pneumocystis carinii f.sp. macacae.

Comparison of relative photon flux from single cells of the bioluminescent marine bacteria Vibrio fischeri and Vibrio harveyi using photon-counting microscopy.

Photon counting microscopy was used to quantify relative photon flux from 616 single sessile cells of Vibrio harveyi and 180 single sessile cells of V. fischeri. V. fischeri cells all emitted light at nearly the same level (average 15.93+/-11.48 photons/min, range 0-58.9 photons/min) when bioluminescence was induced, whereas photon flux of V. harveyi single cells was extremely variable (average 10.51+/-20.60 photons/min, range 0-211 photons/min).

Modern microscopy in biofilm research: confocal microscopy and other approaches.

Microscopy is the only technique whereby bacterial biofilms can be studied at the single-cell level in situ. Our understanding of biofilm structure, physiology and control hinges on the application of confocal scanning laser microscopy and other advanced microscopic techniques. Gene expression in four dimensions (x,y,z,t), interspecies interactions, and the role of exopolymer are being defined.

Discrimination of rat-derived Pneumocystis carinii f. sp. Carinii and Pneumocystis carinii f. sp. Ratti using the polymerase chain reaction.

The rat model of Pneumocystis carinii infection is widely used for the study of this non-culturable pathogen. Two genetically divergent <> of the organism have been detected in infected rat lungs, P. carinii formae specialis carinii and P. carinii formae specialis ratti, in some cases as a co-infection. We have developed a simple and rapid method to analyse rat-derived P. carinii samples, based on DNA amplification of a portion of the gene encoding the mitochondrial large subunit ribosomal RNA. A pair of oligonucleotide primers were designed for each special form of rat-derived P. carinii, the RC primer pair amplifying a 137 bp fragment from P. carinii f. sp. carinii DNA and the RR primer pair amplifying a 251 bp fragment from P. carinii f. sp. ratti DNA. The specificity of the primers was confirmed by sequencing the amplification products. The polymerase chain reaction (PCR) technique was consistent with, and more sensitive than, the electrophoretic karyotype method. The application of the specific PCR technique has implications for future studies on epidemiology, drug sensitivity, immunology and molecular biology of rat-derived P. carinii.

Nspl1, a new Z-band-associated protein.

Molecular characterization of a novel gene designated Neuroendocrine-Specific Protein-Like-1 (Nspl1) had revealed that this gene is expressed as two transcripts, a 1.2 kb transcript found predominantly in skeletal muscle and a 2.1 kb transcript expressed in the brain. The exceptionally high level of skeletal muscle expression prompted us to determine where the protein is localized to skeletal muscle. In vitro studies were performed using two plasmid constructs that generate full-length Nspl1 muscle-specific protein fused to the green fluorescent protein (GFP). In one construct, the GFP cDNA was fused to the N-terminus of the Nspl1 cDNA while in the second construct, the GFP cDNA was fused to the C-terminus of the Nspl1 cDNA. Transfection of either plasmid into mononucleated myoblasts showed that the Nspl1-GFP chimeric protein was associated with intermediate filaments. This was confirmed by using an antibody to stain desmin and finding that GFP-Nspl1 colocalizes with desmin. Chick primary myoblasts were transfected with the chimeric cDNAs and allowed to differentiate into mature myotubes. Results from this analysis and the use of monoclonal antibody to stain alpha-actinin, further localized the Nspl1 protein to the Z-band of mature myotubes. Confocal microscopy of the myotubes containing Nspl1-GFP demonstrates that Nspl1 is distributed continuously throughout the Z-disks.

Developmental biology of biofilms: implications for treatment and control.

Although of heterogeneous spatiotemporal and species compositions, all biofilms undergo certain common developmental events: organic molecules on the substratum can play a role in initial attachment, attached cells grow and additional cells attach from the bulk liquid. Biofilm growth is a four-dimensional (X, Y, Z and T) process similar to organ development.

Combined light microscopy and attenuated total reflection fourier transform infrared spectroscopy for integration of biofilm structure, distribution, and chemistry at solid-liquid interfaces.

Reflected differential interference contrast microscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to obtain complementary data on the structural and chemical properties of a biofilm. This information was obtained nondestructively, quasisimultaneously, and in real time, thereby permitting the verification of time-dependent relationships between the biofilm's population structure, distribution, and interfacial chemistry. The approach offers opportunities to examine these relationships on a variety of substrata in the presence of a bulk aqueous phase under controlled hydrodynamic conditions.