The 1,995,275-bp genome ofCoxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies ofC. burnetiiresemble that ofRickettsiaeandChlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in theC. burnetiigenome. These analyses suggest that the obligate intracellular lifestyle ofC. burnetiimay be a relatively recent innovation.

Significance: Many effectors of the Coxiella burnetii Dot/Icm transporter are important for its virulence, but the lack of understanding of their biochemical activity prevents the use of them as potential therapeutic targets. Here, we found that the effector Cbu0513 (CinF) is a protein phosphatase that attacks IκBα, a key regulatory protein in NF-κB signaling. Unlike its homologs such as ST0318 from the archaeon Sulfolobus tokodaii , CinF has lost the enzymatic activity as a fructose-1,6-bisphosphate aldolase/phosphatase. Instead, it specifically targets IκBα to make it resistant to proteasome-mediated degradation in cells stimulated with NF-κB agonists. Our finding has expanded the strategy used by bacterial pathogens to inhibit host immunity, which may provide leads for the development of immune modulators for disease treatment.

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila . Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using ( i ) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and ( ii ) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila . Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii .

Escherichia coli O157:H7 strains have been implicated as etiologic agents in food-borne outbreaks of hemorrhagic colitis and the hemolytic-uremic syndrome. A prototype E. coli O157:H7 strain, designated 933, produces Shiga-like toxin I (SLT-I) and SLT-II and harbors a 60-MDa plasmid. In a previous study, streptomycin-treated mice were fed 933 together with a derivative cured of the 60-MDa plasmid (designated 933cu). Strain 933cu colonized poorly, but in approximately one-third of the animals, an isolate of 933cu was obtained from the feces that had regained the ability to colonize well. This isolate, designated 933cu-rev, killed all of the animals when fed alone to mice. In this investigation, two types of experiments were done to assess whether SLT-I, SLT-II, or both contributed to the death of mice fed 933cu-rev. (i) Mice were pretreated with monoclonal antibodies to SLT-I, SLT-II, SLT-I and SLT-II, or cholera toxin (as a control) before infection with 933cu-rev. (ii) Mice were fed either an E. coli K-12 strain carrying cloned SLT-I genes or the same K-12 strain carrying cloned SLT-II genes. The results of both types of experiments indicated that the deaths of the orally infected mice were due solely to SLT-II. Extensive histological and selected electron microscopic examinations of various tissues from the infected animals suggested that death was due to acute renal cortical tubular necrosis consistent with toxic renal damage. These data indicate a critical role for SLT-II, but not SLT-I, in renal damage associated with E. coli O157:H7 infection of streptomycin-treated mice.

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 β-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.