Louis P. Mallavia

DIFFERENTIATION OF COXIELLA BURNETII ISOLATES BY ANALYSIS OF RESTRICTION-ENDONUCLEASE-DIGESTED DNA SEPARATED BY SDS-PAGE

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Coxiella burnetii Exhibits Morphological Change and Delays Phagolysosomal Fusion after Internalization by J774A.1 Cells

ABSTRACT Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in C. burnetii survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization ofC. burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [35S]methionine-[35S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake C. burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable C. burnetii was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.

Use of pulsed field gel electrophoresis to differentiate Coxiella burnetii strains

Pulsed field gradient gel electrophoresis (PFGE) provides a powerful technique for the analysis of bacterial genomic DNA by allowing the resolution of DNA fragments as large as 9000 kilobase pairs (kbp). When isolates of Coxiella burnetii were examined using this method, the restriction enzymes Not I and Sfi I gave the fewest and most easily resolved fragments. Sfi I cuts the genome of the Priscilla isolate of C. burnetii into 15 DNA fragments ranging in size from 320 to 18 kbp, and Not I cuts the DNA of this isolate into 20 fragments from 293 to 10 kbp in size. Analysis of the undigested DNA and summing of the Sfi I restriction fragments both indicate that the C. burnetii DNA contains approximately 1600 kbp, or is about one-third the size of the DNA in Escherichia coli. Comparisons of isolates revealed that the numbers and patterns of DNA fragments observed correlate with proposed strain designations. Because PFGE allows the reproducible separation of restriction endonuclease-digested C. burnetii DNA fragments into precise bands, it greatly facilitates the selection of large DNA fragments for cloning. Bands harvested from the gel can be cloned. Clone banks are invaluable for identifying the location of specific genes and landmarks and providing material for future experiments, including DNA sequencing. Yeast artificial chromosome (YAC) cloning vectors can accept fragments as large as 500 kbp. The fragmentation patterns of C. burnetii that we have obtained with infrequent-cutting enzymes are small enough to be cloned into YAC vectors. Using a PFGE selection method means that only small libraries would have to be created and screened. Thus, the results of these experiments also demonstrate the applicability of PFGE for deriving a physical map of C. burnetii chromosomal DNA. Development of such a macrorestriction map will facilitate genetic and molecular studies with C. burnetii.

Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue

The gene encoding a protein that reacted with antibodies specific for Legionella pneumophila macrophage infectivity potentiator (LpMip) was cloned from Coxiella burnetii, the obligate intracellular rickettsia that causes Q fever in humans. Nucleotide sequencing analysis revealed an ORF encoding a gene product of 230 amino acids with a molecular mass of 25.5 kDa and a predicted pI of 10.7. The predicted amino acid sequence from the ORF shows similarity with Mip/Mip-like proteins of Legionella (46%) and Chlamydia (30%). Moreover, like LpMip, the amino acid sequence of the C terminus of this protein has over 35% identity to prokaryotic and eukaryotic FK506-binding proteins (FKBPs) that belong to a superfamily of immunophilins and are peptidyl-prolyl cis-trans isomerases (PPIases). When overproduced in Escherichia coli, the C. burnetii protein also exhibited PPIase activity. Taken together, these results demonstrate that C. burnetii encodes a Mip analogue (CbMip). A putative leader peptide at the N terminus of CbMip was detected by computer analysis. Furthermore, TnphoA mutagenesis demonstrated that in E. coli CbMip was secreted. In view of the role of Mip/Mip-like proteins in the pathogenesis of Legionella and Chlamydia, CbMip may be a C. burnetii virulence factor.

Comparison of Coxiella burnetii plasmids to homologous chromosomal sequences present in a plasmidless endocarditis-causing isolate

Coxiella burnetii is the etiological agent of human Q fever and chronic endocarditis. Different plasmids have been found in C. burnetii isolates and a correlation between disease state and plasmid type has been established. The plasmid QpRS was found in all but four of the endocarditis-causing isolates examined. These four isolates did not contain a detectable plasmid. However, when DNA from the plasmidless isolates is hybridized with 32P-labeled QpRS, homologous sequences are detected. It was hypothesized that plasmid sequences had inserted into the chromosomal DNA of the plasmidless isolate. A cosmid chromosomal gene bank was constructed from one of the plasmidless isolates and a number of clones were obtained. One clone, pEAS137, contained all of the EcoR I fragments with homology to the C. burnetii plasmids plus several non-homologous fragments. The EcoR I fragments in pEAS137 were in the same linear order as present in the chromosome of the plasmidless isolate and were shown to exist as a single contiguous sequence. This information supports the hypothesis that plasmid sequences have inserted into the chromosomal DNA and makes pEAS137 a good candidate for studying the relationships between the plasmids. Initial studies comparing pEAS137 to QpRS and QpH1 suggest that pEAS137 is more closely related to QpRS than to QpH1.

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small‐cell variant (SCV) and large‐cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low Mr was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N‐terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small‐cell‐variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted Mr of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS‐PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride‐equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double‐stranded DNA in gel mobility‐shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post‐infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.

Active Transport of Proline by Coxiella burnetii

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3.0 to 4.5, a pH range approximately that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent Km for proline transport from 45 microM to 15 microM, with the Vmax increasing from 3.6 pmol s-1 (mg dry wt)-1 to 4.8 pmol s-1 (mg dry wt)-1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. D-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.

Genetics of rickettsiae.

Classical genetic approaches useful with free-living bacteria are difficult to apply to the rickettsiae. Although rickettsial mutants have been isolated over the years, the genetic basis of these mutants is unknown, limiting their usefulness. The application of molecular biological techniques to rickettsial studies has provided the opportunity to isolate and study specific genes. Genes encoding metabolic enzymes from rickettsiae were cloned in Escherichia coli and shown to retain their regulatory properties, suggesting that recombinant DNA technology may be useful for studies of rickettsial enzymes and regulatory mechanisms. The potential use of rickettsial surface components, or virulence factors as possible antigens for protective subunit vaccines, has led to the cloning and expression in E. coli, of rickettsial chromosomal and plasmid genes encoding outer membrane proteins. The number of genes characterized in recent years has increased dramatically giving rise to an increasing source of information on rickettsial gene structure. Plasmids have only been identified in C. burnetii and possibly Rochalimaea quintana. The plasmid sequences present in C. burnetii are highly conserved suggesting that they are important to the growth and virulence of this organism. To understand the role of genes in the rickettsia-host relationship, it is critical that a genetic exchange system be developed. The recent description of transformation of R. quintana by electroporation is an important first step in this direction. The ability to introduce genetic material is necessary to address questions that cannot be resolved by studying rickettsial gene expression in E. coli.

Strategy for detection and differentiation of Coxiella burnetii strains using the polymerase chain reaction.

A method for the rapid detection of Coxiella burnetii and differentiation between strains that cause endocarditis and those that cause acute Q fever is based on the observation that the different strains contain unique plasmid sequences. This method employs the polymerase chain reaction (PCR) and requires knowledge of specific DNA sequences in the region (target) of DNA to be amplified. To detect and differentiate between C. burnetii isolates, two sets of primers are required. The first set was derived from a fragment of plasmid QpH1 which has been detected in all C. burnetii isolates. A second PCR reaction was conducted using primers specific for DNA sequences that are shared only by QpRS plasmid-containing strains of C. burnetii. The first reaction detects the presence of C. burnetii. The second PCR is necessary to determine whether the isolate contains DNA sequences associated with strains causing chronic disease. These procedures detect as few as one to ten organisms.

DNA probes for the identification of Coxiella burnetii strains.

Isolation of Coxiella Burnetii in the standard laboratory setting is hazardous; therefore most diagnoses are based on retrospective detection of a rising antibody titer to C. burnetti. As a result, this disease is usually undiagnosed or misdiagnosed. Methods for the rapid detection of C. burnetti have now been developed that utilize specific hybridization of labeled DNA probes to nucleic acid in clinical samples. One method detects the presence of C. burnetii 16S ribosomal RNA (rRNA); another uses plasmid sequences. We have developed a probe that detects C. burnetii and one that differentiates between Coxiella strains capable of causing chronic disease and those that cause the acute form. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. A third method differentiates between chronic (endocarditis-causing) strains and those that cause acute Q fever. This method uses the polymerase chain reaction (PCR), in which the target regions of DNA are amplified by iterative cycles of Taq I DNA polymerase chain extension to produce up to a 10(6) amplification of the target sequences. When Southern blotting is used in conjunction with PCR, the test detects as few as 2-9 C. burnetti cells.