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Suzana Radulovic

Publications and source records attributed to Suzana Radulovic.

10 recordsLinked to original sources
PubMed2005

Characterization of RP 333, a gene encoding CapD of Rickettsia prowazekii with UDP-glucose 4-epimerase activity.

The genetic mechanism of membrane assembly in Rickettsia prowazekii, the causative agent of epidemic typhus, is unknown. Recent progress in genomics of this small intracellular bacterium prompted us to explore membrane biogenesis as it pertains to identification and characterization of gene products that are of importance in polysaccharide biosynthesis. We chose to define the function of the R. prowazekii capD gene (RP 333) product designated as a putative epimerase. We detected a capD transcript in yolk sacs infected with R. prowazekii at ten days post-infection. The capD gene was PCR amplified and cloned into the entry vector pENTR/SD/D-TOPO and transferred to a low copy arabinose-inducible pBAD/Myc-HisA expression plasmid constructed in our laboratory. The corresponding recombinant protein (CapD) was expressed in PL2, a galE28 mutant of Escherichia coli, and has been shown to epimerize UDP-galactose to UDP-glucose without the addition of NAD+. CapD characterization is important for defense strategies targeted towards design of an R. prowazekii vaccine, generation of new antibiotics, and opening the way for research in membrane biogenesis with respect to the parasitic lifestyle of Rickettsia.

Base Sequence↗
PubMed2003

Pathogenic rickettsiae as bioterrorism agents.

The diseases caused by rickettsiae vary from mild to severe clinical presentations, with case fatality ranging from none to over 30%. The severity of rickettsial diseases has been associated with age, delayed diagnosis, hepatic and renal dysfunction, central nervous system abnormalities, and pulmonary compromise. Despite the variability in clinical presentations many pathogenic rickettsiae cause debilitating diseases, any one of which could be used as a potential biological weapon. While Rickettsia prowazekii, R. rickettsii, and Coxiella burnetii pose serious problems and are currently considered bioterrorism agents, several other species could cause havoc once intentionally released into human populations. The complicating factors include misdiagnosis due to the similarity of rickettsial-induced clinical signs to many commonly occurring infections and subsequent delayed treatment. Vigilance, preparedness, and the availability of efficacious vaccines and antibiotics are required to avert the morbidity and mortality and disturbances generated by the intentional release of pathogenic rickettsiae into large and immunologically naive human populations. This presentation reviews the rickettsial attributes that make them potential bioterrorism agents, as well as issues related to signs that would alert the responsible authorities, and the preventive measures that could reduce impact of these agents.

Animals↗
PubMed2003

Development of Rickettsia prowazekii DNA vaccine: cloning strategies.

Rickettsia prowazekii, the etiologic agent of louse-borne typhus, is listed as a category B agent under the select agent list of the United States Centers for Disease Control and Prevention. R. prowazekii was placed on the select agent list due to its potential to cause epidemic, high mortality in untreated and/or misdiagnosed cases, and ease of spread in vulnerable populations. Historically, R. prowazekii vaccines using crude antigen and/or inactivated rickettsia were partially protective but have been accompanied with undesirable toxic reactions and difficulties in standardization. The availability of the genome sequence of R. prowazekii allowed us to select genes that encode proteins with potential in immuno-protection against this human pathogen. We successfully PCR-amplified a group of genes involved in invasion (invA), cell division (fts), protein secretion (sec gene family), and virulence (ompA and ompB, virB gene family, cap and tlyA and tlyC). The generated PCR products were cloned into the Gateway cloning system and the cloned products will be introduced into Vical VR 1020-DV and VR 1012-DV DNA vaccine plasmids. Twenty-four target genes from R. prowazekii have been PCR amplified, of which fifteen have been introduced into the pENTR/SD/D-TOPO entry cloning vector.

Animals↗
PubMed2003

Subcellular localization of rickettsial invasion protein, InvA.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

Animals↗
PubMed2002

Molecular and functional analysis of the Rickettsia typhi groESL operon.

The groESL operon from an obligate, intracellular, Gram-negative bacterium Rickettsia typhi, the etiologic agent of murine typhus, was cloned and sequenced. The sequence analysis of 2229 bp of the groESL operon reveals two open reading frames of 288 nucleotides (groES) and 1653 nucleotides (groEL) separated by 20 nucleotides. The deduced amino acid sequence of R. typhi GroES and GroEL shows a high degree of identity with other bacterial GroES and GroEL. Reverse transcriptase-polymerase chain reaction and Northern blot analysis indicated that both groES and groEL are transcribed as a single mRNA. The transcriptional start point at 81 nucleotides upstream of the groES start codon was determined by primer extension. The promoter analysis shows no regulatory CIRCE element as it is known for many Gram-positive and Gram-negative bacteria. However, it contains the sequence similar to the putative sigma(70)-dependent promoter and lacks the -35 sequence of the putative sigma(32)-dependent promoter. Complementation assay by R. typhi groESL in a temperature sensitive Escherichia coli groEL mutant restored significant growth ability at non-permissive temperature.

Amino Acid Sequence↗
PubMed2002

The Rickettsia prowazekii invasion gene homolog (invA) encodes a Nudix hydrolase active on adenosine (5')-pentaphospho-(5')-adenosine.

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1). Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

Amino Acid Sequence↗
PubMed2002

Transcriptional analysis of Rickettsia prowazekii invasion gene homolog (invA) during host cell infection.

An invasion gene homolog, invA, of Rickettsia prowazekii has recently been identified to encode a member of the Nudix hydrolase subfamily which acts specifically on dinucleoside oligophosphates (Np(n)N; n >/= 5), a group of cellular signaling molecules known as alarmones. InvA is thought to enhance intracellular survival by regulating stress-induced toxic nucleotide levels during rickettsial infection. To further characterize the physiological function of InvA, the gene expression pattern during various stages of rickettsial intracellular growth was investigated. Using semiquantitative reverse transcription-PCR (RT-PCR) and real-time fluorescent probe-based quantitative RT-PCR, a differential expression profile of invA during rickettsial host cell infection was examined. The invA transcript temporarily increased during the early period of infection. Expression of rickettsial groEL, a molecular indicator of cellular stresses, was also shown to be upregulated during the early period of infection. Furthermore, invA was cotranscribed in a polycistronic message with rrp, a gene encoding the response regulator protein homolog, which is a part of a two-component signal transduction system. These results support our earlier findings that under such stress conditions dinucleoside oligophosphate pyrophosphatase may function as a buffer, enhancing rickettsial survival within the cytoplasm of a eukaryotic cell. The expression of rickettsial dinucleoside oligophosphate pyrophosphatase may be regulated by a part of the two-component signal transduction system similar to that described for response regulators in other bacterial systems.

Animals↗
PubMed2002

Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells.

The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

Animals↗
PubMed2002

Geographic association of Rickettsia felis-infected opossums with human murine typhus, Texas.

Application of molecular diagnostic technology in the past 10 years has resulted in the discovery of several new species of pathogenic rickettsiae, including Rickettsia felis. As more sequence information for rickettsial genes has become available, the data have been used to reclassify rickettsial species and to develop new diagnostic tools for analysis of mixed rickettsial pathogens. R. felis has been associated with opossums and their fleas in Texas and California. Because R. felis can cause human illness, we investigated the distribution dynamics in the murine typhus-endemic areas of these two states. The geographic distribution of R. felis-infected opossum populations in two well-established endemic foci overlaps with that of the reported human cases of murine typhus. Descriptive epidemiologic analysis of 1998 human cases in Corpus Christi, Texas, identified disease patterns consistent with studies done in the 1980s. A close geographic association of seropositive opossums (22% R. felis; 8% R. typhi) with human murine typhus cases was also observed.

Adolescent↗